Means and methods for in vitro fertilisation

Abstract

The present invention relates to means and methods for in vitro fertilisation, particularly to means and methods for frozen embryo transfer which comprise the use of a composition comprising an effective amount of estetrol component. The composition is intended for use prior, during and / or after embryo transfer. The use of the composition in a frozen embryo transfer fertility treatment results in favourable effects when compared to compositions used in the art to support frozen embryo transfer fertility treatments.

Description

[0001] MEANS AND METHODS FOR IN VITRO FERTILISATION

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to compositions and methods for treating infertility by frozen embryo transfer (FET). Specifically, the invention relates to compositions comprising estetrol (E4), and associated methods that increase endometrial thickness.

[0004] BACKGROUND OF THE INVENTION

[0005] Female infertility is a disorder defined by the failure to achieve a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse in women less than 35 years of age; and after 6 months of regular unprotected sexual intercourse in women 35 years of age or older (ASRM (2008). "Definitions of infertility and recurrent pregnancy loss." Fertil Steril 90(5 Suppl): S60). According to the World Health Organization, around 17.5% of the adult population, i.e., approximately 1 in 6 people worldwide, experience infertility, showing the urgent need to increase access to affordable and high-quality fertility care.

[0006] For a human pregnancy to occur, an embryo must implant in the endometrium, and for this to happen the endometrium must be in a receptive state. In humans, the “window of implantation” i.e., the time when the endometrium is most able to support trophoblast-endometrial interactions, is thought to occur during a short period of time around days 22-24 of an idealized 28-day cycle (where day 1 is the first day of menses). The endometrium becomes receptive as a result of a series of timed hormonal events during the menstrual cycle. Estrogen stimulates endometrial proliferation, resulting in an increase in endometrial thickness, and induces progesterone (P4) receptors. The exposure of the endometrium to P4 after ovulation initiates morphological and functional alterations that result in the change from a proliferative to a secretory endometrium. The epithelial glands and vasculature continue to grow and become spiral, whereas the endometrial thickness is relatively unchanged, resulting in a denser endometrium (Casper and Yanushpolsky (2016). "Optimal endometrial preparation for frozen embryo transfer cycles: window of implantation and progesterone support." Fertil Steril 105(4): 867-872.).

[0007] Assisted Reproductive Technology (ART) programs, including in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), offer infertile couples and individuals the opportunity to improve their chances of conceiving.

[0008] Embryos obtained during ART can be frozen. Frozen-thawed embryo transfer (FET) is widely used in ART treatment programs because it minimizes the risk of ovarian hyperstimulation syndrome, facilitates single embryo transfer (ET) and improves fertility and cumulative pregnancy outcomes. The synchronization of embryo and endometrium development is crucial for implantation. Endometrial preparation is of utmost importance for FET to optimize pregnancy rates. At present, the commonly used FET endometrial preparation protocols include the natural cycle (NC) and the hormone replacement treatment (HRT) cycle, with close monitoring via transvaginal ultrasonography (TVUS) and serum hormone levels. In FET cycles, a decreased trend towards pregnancy outcomes, such as implantation rate, clinical pregnancy rate and subsequent live birth rate has been shown to be associated with a thin endometrium. A typical NC involves a dominant follicle which produces estradiol (E2) leading to thickening of the endometrium (Liu et al. (2022). "Comparison of endometrial preparation protocols (natural cycle versus hormone replacement cycle) for frozen embryo transfer (COMPETE): a study protocol for a randomised controlled trial." BMJ Open 12(10): eO6398L). The diameter of the follicle is monitored by TVUS and serum luteinizing hormone (LH) levels are measured. In a modified NC, when the follicle reaches an optimal size following the LH surge, human chorionic gonadotropin (hCG) is administered to trigger ovulation. After confirmation of ovulation on TVUS, vaginal P4 is given for approximately 3 or 5 days for cleavage or blastocyst embryos respectively, and after FET, exogenous P4 support is continued for up to 10 weeks of gestation.

[0009] In HRT, in addition to use of exogenous P4 as for an NC, exogenous E2 is administered to stimulate growth of the endometrium. High doses of estrogen given to proliferate the endometrium can however lead to inhibition of follicular growth (Mumusoglu et al. (2021). "Preparation of the Endometrium for Frozen Embryo Transfer: A Systematic Review." Front Endocrinol (Lausanne) 12: 688237.). In a typical HRT cycle, oral E2 is given for approximately 10-12 days until the endometrial thickness reaches at least 7 mm, at which time vaginal P4 is added for 4 or 5 days prior to FET for cleavage or blastocyst stage embryos respectively (Liu et al. (2022) "Comparison of endometrial preparation protocols (natural cycle versus hormone replacement cycle) for frozen embryo transfer (COMPETE): a study protocol for a randomised controlled trial." BMJ Open 12(10): e063981). In cases where the endometrium remains thin despite oral E2 administration, additional vaginal E2 may be administered, and when the endometrium reaches adequate thickness, P4 is added to induce endometrial transformation. FET is performed after 5 or 6 days, and luteal support with oral E2, oral and vaginal P4 will continue for 8 weeks after the transfer if pregnancy is confirmed (Kong, Liu et al. 2022). Other protocols used in clinical practice recommend continuing E2 and P4 for up to 10 weeks of gestational age (Morch et al. (2022). "Estradiol and progesterone levels in early pregnancy after natural, estradiol + progesterone or gonadotrophin stimulated frozen embryo transfer cycle: a randomised controlled trial protocol." BMJ Open 12(7): e058468).

[0010] Literature indicates that HRT-FET increases the risk of pregnancy complications (bleeding, miscarriage, pregnancy -induced hypertension, and pre-eclampsia) (Pandey et al. (2012). "Obstetric and perinatal outcomes in singleton pregnancies resulting from IVF / ICSI: a systematic review and meta -analysis." Hum Reprod Update 18(5): 485-503.; Sazonova et al. (2012). "Obstetric outcome in singletons after in vitro fertilization with cryopreserved / thawed embryos." Hum Reprod 27(5): 1343-1350.; Roelens and Blockeel (2022). "Impact of different endometrial preparation protocols before frozen embryo transfer on pregnancy outcomes: a review." Fertil Steril 118(5): 820-827.). It has been suggested that absence of corpus luteum, which results in a lack of production of relaxin and other vasoactive substances that are important for the mother's adaptation to the pregnancy, may lead to an increased risk of abnormal maternal cardiovascular adaptation to pregnancy and subsequent increased risk of preeclampsia (Singh et al. (2020). "Frozen-thawed embryo transfer: the potential importance of the corpus luteum in preventing obstetrical complications." Fertil Steril 113(2): 252-257.; Roelens, and Blockeel (2022). "Impact of different endometrial preparation protocols before frozen embryo transfer on pregnancy outcomes: a review." Fertil Steril 118(5): 820-827.). The increased risk of detrimental maternal and perinatal outcomes following HRT-FET using estradiol valerate standard protocols was highlighted by Fan et al. (“Hormone replacement treatment regimen is associated with a higher risk of hypertensive disorders of pregnancy in women undergoing frozen-thawed embryo transfer.” Front Endocrinol (Lausanne). 2023). Fan et al. discuss that the absence of corpus luteum in HRT-FET cycles may disrupt maternal cardiovascular adaptation due to a lack of circulating vasoactive factors, contributing to the increased risk of hypertensive disorders of pregnancy.

[0011] The role of corpus luteum in spontaneous pregnancies was demonstrated to play an important role by producing crucial hormones for implantation, placentation, and pregnancy maintenance. Relaxin secreted by corpus luteum is a potent vasodilator which contributes to cardiovascular and renal adaptations seen in pregnancy (Comad KP. (2010). "Emerging role of relaxin in the maternal adaptations to normal pregnancy: implications for preeclampsia." Semin Nephrol 31:15-32.).

[0012] P4 has regulatory approval in EU and the United State (US) for supplementation of the luteal phase during ART cycles. The National Institute for Health and Care Excellence (NICE) Fertility Guideline recommends P4 for luteal phase support after IVF treatment (NICE. (2017). "Fertility problems: assessment and treatment (accessed 22 February 2024). Available at: https: / / www.nice.org.uk / guidance / cgl56.").

[0013] At present, no estrogen has been approved for use in ART cycles, although E2 is commonly used off-label to induce proliferation of the endometrium. E2 administration is continued until the endometrium reaches the target thickness (at least 7 mm on TVUS), at which time P4 is added to initiate secretory changes (El-Toukhyet al. (2008). "The relationship between endometrial thickness and outcome of medicated frozen embryo replacement cycles." Fertil Steril 89(4): 832-839.).

[0014] The use of E2 is associated with significant disadvantages, as outlined above. This underscores the need to develop approaches that address these disadvantages.

[0015] SUMMARY OF THE INVENTION

[0016] The present invention aims to provide a more effective and safer solution for women seeking treatment for infertility by FET. Based on extensive research, the ability of estetrol to optimally prepare the endometrium for FET even allowing formation of corpus luteum has been demonstrated for the first time by the inventors. Corpus luteum plays a key role in pregnancy development, and also reduces the risk of hypertensive disorders of pregnancy (e.g. pre-eclampsia). Said risk was established in women undergoing HRT-FET. It has unexpectedly been shown that estetrol supports embryo implantation and early pregnancy in FET as part of an ART treatment program. Notably, relative high ovulation rates could be observed in subjects treated with estetrol and estetrol treatment resulted in an adequate increase in endometrial thickness. In addition, estetrol treatment resulted in a LH (luteinizing hormone) peak around ovulation, but the rise is more moderate when compared to a natural cycle. This suggests that while follicular recruitment, growth and, importantly, ovulation may occur under supplementation of E4, the gonadotropin hormonal response remains milder due to the effects of estetrol on the Hypothalamic -Pituitary Axis. Under this premise, the use of estetrol instead of other estrogens used in the art, such as estradiol valerate, has the benefit of allowing physiological follicular growth and ovulation, therefore avoiding maternal and obstetric adverse effects related to the off-label administration of estradiol valerate as used in current clinical practice.

[0017] The invention therefore provides in the following aspects: Aspect 1. A composition comprising an effective amount of estetrol component for use in a Frozen Embryo Transfer (FET) fertility treatment in a subject.

[0018] Aspect 2. A composition comprising an effective amount of estetrol component for use in preparing the endometrium of a subject for Frozen Embryo Transfer (FET) for receiving and / or implanting the embryo.

[0019] Aspect 3. The composition according to aspect 1 or 2, wherein administration of said composition does not compromise LH production and / or CL formation and / or wherein LH production and / or CL formation is similar to that of similar subjects not receiving estetrol.

[0020] Aspect 4. A method of Frozen Embryo Transfer (FET) fertility treatment in a subject, wherein the method comprises a step of administering a composition comprising an effective amount of estetrol component to said subject and a step of transferring an embryo into said subject.

[0021] Aspect 5. A method of preparing the endometrium of a subject for Frozen Embryo Transfer (FET) for receiving and / or implanting the embryo, wherein the method comprises a step of administering a composition comprising an effective amount of estetrol component to said subject and a step of transferring an embryo into said subject.

[0022] Aspect 6. Use of a composition comprising an effective amount of estetrol component for the manufacture of a medicament for use in Frozen Embryo Transfer (FET) fertility treatment in a subject.

[0023] Aspect 7. Use of a composition comprising an effective amount of estetrol component for the manufacture of a medicament for preparing the endometrium of a subject for Frozen Embryo Transfer (FET) for receiving and / or implanting the embryo.

[0024] Aspect 8. Use of a composition comprising an effective amount of estetrol component in a Frozen Embryo Transfer (FET) fertility treatment in a subject.

[0025] Aspect 9. Use of a composition comprising an effective amount of estetrol component for preparing the endometrium of a subject for Frozen Embryo Transfer (FET).

[0026] Aspect 10. The composition for use according to aspect 2, the method according to aspect 5, or the use according to aspect 7 or 9, wherein preparing the endometrium of a subject for FET is bringing said endometrium into a receptive state for FET.

[0027] Aspect 11. The composition for use, the method, or the use according to any one of aspects 1 to 10, wherein the composition is for use (i.e. is administered) prior to, during, and / or after embryo transfer.

[0028] Aspect 12. The composition for use, the method, or the use according to any one of aspects 1 to 11, characterised by an increase of endometrial thickness in the subject, preferably wherein the endometrial thickness is increased compared to the endometrial thickness prior to the start of administration of the composition.

[0029] Aspect 13. The composition for use, the method, or the use according to any one of aspects 1 to 12, wherein the estetrol component is used at a daily dose which allows formation of the corpus luteum, preferably wherein the estetrol component is used at a daily dose of from about 8 mg to about 12 mg, more preferably at a daily dose of about 10 mg which allows formation of the corpus luteum. Aspect 14. The composition for use, the method, or the use according to any one of aspects 1 to 13, wherein a daily amount of estetrol component from about 5 to about 30 mg, preferably from about 10 to about 20 mg and most preferably 15 mg is administered to the subject.

[0030] Aspect 15. The composition for use, the method, or the use according to any one of aspects 1 to 14, wherein the composition comprises a daily amount of estetrol component from about 5 to about 30 mg, preferably from about 10 to about 20 mg and most preferably 15 mg.

[0031] Aspect 16. The composition for use, the method, or the use according to any one of aspects 1 to 15, wherein the estetrol component is estetrol, preferably estetrol monohydrate.

[0032] Aspect 17. The composition for use, the method, or the use according to any one of aspects 1 to 16, wherein said composition is administered for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive days.

[0033] Aspect 18. The composition for use, the method, or the use according to any one of aspects 1 to 17, wherein the estetrol component is administered from the first day of the menstmal cycle or from the second day of the menstrual cycle.

[0034] Aspect 19. The composition for use, the method, or the use according to any one of aspects 1 to 18, wherein the first day of the menstrual cycle is day one of the cycle wherein the subject experiences bleeding, distinct from spotting, for the first time in said menstrual cycle.

[0035] Aspect 20. The composition for use, the method, or the use according to any one of aspects 1 to 19, wherein an endometrial thickness of at least 7 mm is achieved after at least 14 days of estetrol component administration.

[0036] Aspect 21. The composition for use, the method, or the use according to any one of aspects 1 to 20, wherein the composition is orally administered or wherein the composition is formulated as an oral composition.

[0037] Aspect 22. The composition for use, the method, or the use according to any one of aspects 1 to 21, wherein the composition comprises the estetrol component as the sole active ingredient.

[0038] Aspect 23. The composition for use, the method, or the use according to any one of aspects, wherein the estetrol component is administered as the sole active ingredient.

[0039] Aspect 24. The composition for use, the method, or the use according to any one of aspects 1 to 21, wherein the treatment additionally comprises the administration of a progestogen.

[0040] Aspect 25. The composition for use, the method, or the use according to aspect 24, wherein administration of the progestogen is for endometrial maturation.

[0041] Aspect 26. The composition for use, the method, or the use according to aspect 25, wherein the endometrial maturation is the transition from a proliferative, growing state into a prepared, secretory phase lining that is capable of receiving an embryo. Aspect 27. The composition for use, the method, or the use according to any one of aspects 24 to 26, wherein the progestogen is one or more progestogen selected from the group consisting of: progesterone, medroxyprogesterone acetate, and dydrogesterone.

[0042] Aspect 28. The composition for use, the method, or the use according to aspect 27, wherein the progestogen is micronized progesterone, micronized medroxyprogesterone acetate, and / or micronized dydrogesterone.

[0043] Aspect 29. The composition for use, the method, or the use according to any one of aspects 24 to 28, wherein a daily amount of about 50 mg to about 800 mg, preferably about 100 mg to about 400 mg, more preferably about 200 mg and even more preferably about 180 mg progesterone is administered.

[0044] Aspect 30. The composition for use, the method, or the use according to any one of aspects 24 to 29, wherein a daily amount of about 5 mg to about 100 mg, preferably about 10 mg to about 75 mg, more preferably about 40 mg and even more preferably 20 mg dydrogesterone is administered.

[0045] Aspect 31. The composition for use, the method, or the use according to any one of aspects 24 to 30, wherein the progesterone, medroxyprogesterone acetate, and / or dydrogesterone are orally administered.

[0046] Aspect 32. The composition for use, the method, or the use according to any one of aspects 24 to 30, wherein the progesterone, medroxyprogesterone acetate, and / or dydrogesterone are vaginally administered.

[0047] Aspect 33. The composition for use, the method, or the use according to aspect 32, wherein a daily amount of about 50 mg to about 800 mg, preferably about 100 mg to about 400 mg, more preferably about 180 mg progesterone is vaginally administered.

[0048] Aspect 34. The composition for use, the method, or the use according to any one of aspects 24 to 30, wherein the progesterone, medroxyprogesterone acetate, and / or dydrogesterone are administered by intramuscular injection, preferably wherein progesterone in oil is administered by intramuscular injection.

[0049] Aspect 35. The composition for use, the method, or the use according to any one of aspects 24 to 30, wherein said progestogen is administered on the same day as the estetrol component for at least 3, 4, 5, or 6 consecutive days.

[0050] Aspect 36. The composition for use, the method, or the use according to any one of aspects 24 to 30, wherein the administration of the progestogen is initiated after at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive days of estetrol component administration, preferably after 13 consecutive days of estetrol component administration.

[0051] Aspect 37. The composition for use, the method, or the use according to any one of aspects 24 to 36, wherein the progestogen is comprised in the same composition as the composition comprising the effective amount of estetrol component.

[0052] Aspect 38. The composition for use, the method, or the use according to any one of aspects 24 to 37, wherein an embryo is transferred after at least 2, 3, or 4 days of combined administration of the estetrol component and the progestogen.

[0053] Aspect 39. The composition for use, the method, or the use according to any one of aspects 1 to 38, wherein administration of the estetrol component is continued daily for up to 12 weeks post embryo transfer. Aspect 40. The composition for use, the method, or the use according to any one of aspects 1 to 39, wherein the treatment does not comprise the use of a follicle stimulating hormone (FSH) substance.

[0054] Aspect 41. A pharmaceutical kit for use in the treatment of infertility by Frozen embryo transfer (FET), said kit comprising a parenteral or oral dosage unit containing an effective amount of an estetrol component and an oral and / or vaginal dosage unit containing an effective amount of progestogen as defined in any one of the previous aspects.

[0055] Aspect 42. The pharmaceutical kit for use according to aspect 41, further comprising user instructions.

[0056] In any one of the aspects defined herein the composition may be presented as a kit-of-parts containing a packaging unit, e.g. a blister pack, containing the daily oral dosage units comprising the estetrol component, preferably the estetrol, more preferably the estetrol monohydrate, and a progesterone. The skilled person will additionally know that, within the scope of the present invention, each packaging unit, e.g. blister pack, may be numbered or otherwise marked. In a particular embodiment of the kit-of-parts, the packaging unit comprises 28 containers or a multitude of 28 containers, such as 2 to 3 times 28 containers. In a preferred embodiment, the packaging unit comprises 1 time 28 containers. Within the scope of the invention, each packaging unit may be a sealed blister pack with a cardboard, paperboard, foil plastic backing and enclosed in a suitable cover.

[0057] Also envisaged in any one of the aspects defined herein are packaging units such as bottles. The material of the bottle is not particularly limiting. In preferred embodiments, the bottle is a glass bottle characterized by a colour capable of reducing or preventing degradation of the contents of the bottle by e.g. UV light while maintaining a degree of transparency that allows for visual inspection of the contents of said bottle. Suitable colours include without limitation amber, cobalt, or vintage green.

[0058] The above and further aspects and preferred embodiments of the invention are described in the following sections and in the appended claims. The subject matter of the appended claims is hereby specifically incorporated in this specification.

[0059] BRIEF DESCRIPTION OF THE FIGURES

[0060] Figure 1. Luteinizing hormone median actual change from baseline (Efficacy population). DSG: desogestrel; E4 10 mg: estetrol monohydrate 10 mg, equivalent to estetrol 9.44 mg; E4 20 mg: estetrol monohydrate 20 mg, equivalent to estetrol 18.9 mg; P4: progesterone. Grey line: 10 mg E4. Black line: 20 mg E4. Line with triangles: 20 mg E4 / DSG. Line with circles: 20 mg E4 / P4.

[0061] Figure 2. Follicle stimulating hormone median actual change from baseline (Efficacy population). DSG, desogestrel; E4 10 mg: estetrol monohydrate 10 mg, equivalent to estetrol 9.44 mg; E4 20 mg: estetrol monohydrate 20 mg, equivalent to estetrol 18.9 mg; P4: progesterone. Grey line: 10 mg E4. Black line: 20 mg E4. Line with triangles: 20 mg E4 / DSG. Line with circles: 20 mg E4 / P4.

[0062] Figure 3. E2 median actual change from baseline (Efficacy population). DSG: desogestrel; E2, estradiol, E4 10 mg: estetrol monohydrate 10 mg, equivalent to estetrol 9.44 mg; E4 20 mg: estetrol monohydrate 20 mg, equivalent to estetrol 18.9 mg. Grey line: 10 mg E4. Black line: 20 mg E4. Line with triangles: 20 mg E4 / DSG. Figure 4. P4 median actual change from baseline (Efficacy population). DSG: desogestrel; E4 10 mg: estetrol monohydrate 10 mg, equivalent to estetrol 9.44 mg; E4 20 mg: estetrol monohydrate 20 mg, equivalent to estetrol 18.9 mg; P4: progesterone. Grey line: 10 mg E4. Black line: 20 mg E4. Line with triangles: 20 mg E4 / DSG.

[0063] Figure 5. Mean (SD) endometrial thickness during the treatment period (Efficacy population). DSG: desogestrel; E4 10 mg: estetrol monohydrate 10 mg, equivalent to estetrol 9.44 mg; E4 20 mg: estetrol monohydrate 20 mg, equivalent to estetrol 18.9 mg; P4: progesterone; SD: standard deviation. Conditions from left to right: 10 mg E4; 20 mg E4; 20 mg E4 / DSG; 20 mg E4 / P4.

[0064] Figure 6. Mean (SD) maximum endometrial thickness during the treatment period (Efficacy population). DSG: desogestrel,; E4 10 mg: estetrol monohydrate 10 mg, equivalent to estetrol 9.44 mg; E4 20 mg: estetrol monohydrate 20 mg, equivalent to estetrol 18.9 mg; P4: progesterone; SD: standard deviation. Conditions from left to right: 10 mg E4; 20 mg E4; 20 mg E4 / DSG; 20 mg E4 / P4.

[0065] Figure 7. Study Flow Chart. Line at V2 and V9: menstruation period. Star: Visit. Phone: phone call from the subject. D: day of menstruation period during pre-treatment cycle. D’: day of menstruation period during treatment cycle. EoS: end of study. EoT: end of treatment. ET: early termination. FU: follow-up. ICF: informed consent form. Max: maximum. P4: progesterone. V: visit. * After signing the ICF, the inclusion and exclusion criteria will be checked, as well as the prior medication list. If a subject uses prohibited co -medication, she will be asked to stop this medication (after consultation with her physician if needed and in agreement with the Investigator) and start a washout. Washout and all screening procedures should be completed before the start of pre-treatment cycle. **Estetrol monohydrate 5, 10, 15, 20, or 30 mg respectively, equivalent to anhydrous estetrol 4.7, 9.4, 14.2, 18.9, and 28.3 mg, respectively.

[0066] DETAILED DESCRIPTION

[0067] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0068] The terms “comprising”, “comprises” and “comprised of’ as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms also encompass “consisting of’ and “consisting essentially of’, which enjoy well-established meanings in patent terminology.

[0069] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints. This applies to numerical ranges irrespective of whether they are introduced by the expression “from ... to ...” or the expression “between... and... ” or another expression.

[0070] The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / -10% or less, preferably + / -5% or less, more preferably + / -!% or less, and still more preferably + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed. Whereas the terms “one or more” or “at least one”, such as one or more members or at least one member of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g. any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members. In another example, “one or more” or “at least one” may refer to 1, 2, 3, 4, 5, 6, 7 or more.

[0071] The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge in any country as of the priority date of any of the claims.

[0072] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings or sections of such documents herein specifically referred to are incorporated by reference.

[0073] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the invention. When specific terms are defined in connection with a particular aspect of the invention or a particular embodiment of the invention, such connotation or meaning is meant to apply throughout this specification, i.e. also in the context of other aspects or embodiments of the invention, unless otherwise defined. For example, embodiments directed to products are also applicable to corresponding features of methods and uses.

[0074] In the following passages, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0075] Reference throughout this specification to “one embodiment”, “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, alternative combinations of claimed embodiments are encompassed, as would be understood by those in the art.

[0076] Unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person. Reference is for example again made to standard handbooks as well as to the general background art referred to herein and to the further references cited therein. The term “estetrol” as used herein refers to 1,3,5 (10)-estratrien-3,15alpha,16alpha,17beta-tetrol or 15alpha- hydroxyestriol as well as hydrates of estetrol, e.g. estetrol monohydrate. “Estetrol”, or short “E4” is an estrogen steroid produced by the foetal human liver (PubChem CID: 27125). Estetrol may be described as a 3-hydroxy steroid corresponding to 17beta-estradiol wherein the 15a and 16a positions are substituted for two additional hydroxy groups. It is known that estetrol is an estrogen receptor agonist (Coelingh Bennink et al., Climacteric, 2008). The estetrol may be chemically synthetised, synthesised by the use of (mutant) recombinant enzymes, or synthesised by any combination thereof. It is therefore evident that the terms “estetrol” and “estetrol components” equally encompass further chemically modified estetrol. Estetrol may be indicated in the art by its molecular formula: C18H24O4, or by structural formula (I).

[0077] Formula (I)

[0078] It is understood that when the term “estetrol component” is mentioned throughout any section of this specification, any estetrol-containing component (i.e. compound) and / or estetrol derivative (such as an estetrol ester) is also envisaged. In the context of all embodiments described in the present disclosure, the estetrol component is preferably estetrol. More preferably, in the context of the present disclosure, a particularly preferred estetrol (component) is estetrol monohydrate. Estetrol monohydrate is a white to off-white crystalline solid that is poorly soluble in water and aqueous solutions. It is soluble in methanol, ethanol, sparingly soluble in acetone, and slightly soluble in ethyl acetate and acetonitrile. A skilled person appreciates that estetrol monohydrate corresponds to estetrol containing one molecule of water, and that the core structural formula of estetrol does not differ from Formula (I). By means of illustration and not limitation, the structural formula of estetrol monohydrate is indicated by Formula (II):

[0079] Formula (II)

[0080] Numerous references are made throughout the present specification to indicate that a certain condition in a subject is changed, i.e. treated. The terms “treatment” or “treat” are to be interpreted as both the therapeutic treatment of a symptom, disease or condition that has already developed, leading to (clinical) manifestations, as well as prophylactic or preventive measures, wherein the goal of the treatment is to prevent, lessen, or reduce the chances of incidence of an undesired affliction, such as to prevent occurrence, development and progression of blood pressure increases and / or hypertension, and (clinical) conditions that are a consequence thereof. Particularly in the context of the present invention, the term “treatment” may relate to a fertility treatment in a female subject, regardless of whether said subject has been diagnosed to have, or considered to have, fertility problems or reduced fertility when compared to a normal (i.e. average) female subject.

[0081] The term “Prevention” or “prevent” as used in the context of the invention refers to an aversion of manifestation of a condition or disease image in a subject, i.e. the establishment of preventive measures or prophylactic measures. Preventive treatment refers to treatments wherein the object is to avoid a subject’s body or an element thereof to show (worsening of) symptoms of an undesired physiological or psychological change, in the present context for example an increase of blood pressure. As used herein, the term “prevent” includes both preventing symptoms or a medical condition in general from occurring and preventing symptoms or a medical condition in general of worsening.

[0082] Without limitation, the terms "predicting" or "prediction" generally refer to a statement, declaration, indication or foretelling of a disease or condition in a subject not (yet) showing any, or a limited, clinical manifestation of said disease or condition. A prediction of a disease or condition in a subject may indicate a probability, chance or risk that the subject will develop said disease or condition, for example within a certain time period or by a certain age. Said probability, chance or risk may be indicated as any suitable qualitative or quantitative expression, wherein non-limiting examples of a quantitative expression include absolute values, ranges or statistics. Alternatively the probability, chance, or risk may be indicated relative to a suitable control subject or subject population (such as, e.g., relative to a general, normal or healthy subject or subject population). Hence, the probability, chance or risk that a subject will develop a disease or condition may be advantageously indicated as increased or decreased, or as fold-increased or fold-decreased relative to a suitable control subject or subject population. The term “prediction” of the conditions or diseases as taught herein in a subject may also particularly mean that the subject has a 'positive' prediction of such, i.e., that the subject is at risk of having such (e.g., the risk is significantly increased vis-a-vis a control subject or subject population).

[0083] The terms "diagnosing" or "diagnosis" are indicative for a process of recognizing, deciding on or concluding on a disease or condition in a subject on the basis of symptoms and signs and / or from results of various diagnostic procedures (such as, for example, from knowing the presence, absence and / or quantity of one or more biomarkers of or clinical symptoms characteristic for the diagnosed disease or condition). “Diagnosis of’ the diseases or conditions as taught herein in a subject may particularly mean that the subject has such disease or condition. A subject may be diagnosed as not having such despite displaying one or more conventional symptoms or signs reminiscent of such. In the present context, a subject may be diagnosed to have fertility problems.

[0084] A skilled person is aware that terms such as “quantity”, “amount” and “level” are synonyms and have a well- defined meaning in the art. The terms as used herein may particularly refer to an absolute quantification of a molecule such as a steroid, in (a sample taken from) a subject, or to a relative quantification of a molecule or analyte in a sample, i.e., relative to another value such as relative to a reference value as taught herein, or to a range of values indicating a base-line of a certain parameter. These values or ranges of values may be obtained from one single subject or from a group of subjects (i.e. at least two subjects). Alternatively, the terms “quantity”, “amount”, and “dose” or “dosage” may be used herein to quantify the presence of a certain substance or molecule such as an estetrol component in the composition subject of the invention.

[0085] Unexpectedly, the inventors have found that estetrol is particularly well suited to optimally prepare the endometrium for FET even at doses allowing formation of corpus luteum. This is a particularly interesting finding since corpus luteum is known to play a key role in pregnancy development, and also reduces the risk of hypertensive disorders of pregnancy (e.g. pre-eclampsia). Moreover, it has unexpectedly been shown that estetrol supports embryo implantation and early pregnancy in FET as part of an ART treatment program. It should also be borne in mind that hormonal responses using estetrol are generally milder due to the effects of estetrol on the body's regulatory systems. At certain doses, it has been observed that in the majority of individuals exposed to estetrol, the negative feedback on the hypothalamus typically induced by estrogen supplementation remains minimal, resulting in only a modest suppression of FSH and LH levels, which nevertheless remain below the thresholds observed during a physiological natural cycle. Moreover, the use of estetrol instead of currently estrogens used in the art such as estradiol has the further benefit that a follicle stimulating hormone (FSH) substance is not needed to achieve the desired follicular growth.

[0086] A first aspect of the invention is therefore directed to a composition comprising an effective amount of estetrol component for use in a Frozen Embryo Transfer (FET) fertility treatment in a subject. Thus, the invention equally provides a method of Frozen Embryo Transfer (FET) fertility treatment in a subject, wherein the method comprises a step of administering a composition comprising an effective amount of estetrol component to said subject and a step of transferring an embryo into said subject. Alternatively worded, the invention envisages the use of a composition comprising an effective amount of estetrol component for the manufacture of a medicament for use in Frozen Embryo Transfer (FET) fertility treatment in a subject. Yet alternatively worded, the invention hence provides the use of a composition comprising an effective amount of estetrol component in a Frozen Embryo Transfer (FET) fertility treatment in a subject.

[0087] The term “Fertility treatment” as used herein refers to a range of medical interventions aiming to assist and / or treat people who are having difficulties or are considered to have difficulties to conceive or maintain a pregnancy. Different fertility treatments may address different fertility problems and range from simple lifestyle changes and medications to more complex assisted reproductive technologies (ART). ART are a group of medical procedures aiming to achieve pregnancy in a subject. ART is primarily directed to managing fertility issues, which may involve the manipulation of egg cells, sperm cells, embryos, or a combination thereof to increase the chances of pregnancy. ART are typically used when other fertility treatments, such as medications or lifestyle changes, have not resulted in pregnancy in the subject. Known ART include without limitation In Vitro Fertilization (IVF), Intracytoplasmic Sperm Injection (ICSI), FET, the use of donor egg cells and / or donor sperm cells, gestational surrogacy, and assisted hatching.

[0088] The term “Frozen embryo transfer”, abbreviated throughout the present disclosure as “FET” is a process known to a person of ordinary skill in reproductive technology and treatments. FET is used in assisted reproductive technology (ART) and comprises thawing and transferring an embryo produced at an earlier point in time which has been cryopreserved (frozen) into the uterus of a subject. FET is routinely used in IVF treatments, wherein embryos are fertilized in vitro and subsequently frozen for future use. FET is performed after the embryos have been frozen, often due to a prior IVF cycle where multiple embryos were fertilized. In brief, the general FET procedures comprises the steps of preparing the uterus to receive the embryo such as by hormone treatment to thicken the uterine lining, resulting in a (more) receptive state for implantation. On the day of transfer, one or more embryo’s are thawed and placed into the utems of the subject, typically by means of a thin catheter. Optionally, after about 10 to 14 days a blood test can be conducted to determine whether implantation of the embryo was successful and resulted in pregnancy. FET offers flexibility to the subject in terms of timing and providing multiple attempts to become pregnant without the need to undergo new IVF cycles. Furthermore, it has been described in the art that delayed FET transfer of the embryo may reduce complications in subjects at risk of ovarian hyperstimulation syndrome (OHSS). It is to be understood that reference throughout the present disclosure to “FET fertility treatment” not only encompasses the actual step of embryo transfer but also any steps in preparation of the embryo transfer and post-embryo transfer treatment, unless specified explicitly otherwise.

[0089] The subject referred to herein is a female subject, preferably a female human subject, most preferably a female human subject of reproductive age. The reproductive age for women indicates the span of years during which a woman is biologically capable of conceiving and bearing children. This period is influenced by various physiological, genetic, and environmental factors. Optionally, the subject is between 18 and 60 years old, preferably between 21 and 50 years old, more preferably between 25 and 45 years old, more preferably between 30 and 40 years old. Optionally, the subject is a subject that has actively yet unsuccessfully attempted to become pregnant for at least about 6 months, preferably for at least about 12 months, preferably for at least about 24 months. Optionally, the subject is a subject that has actively yet unsuccessfully attempted to become pregnant by means of one or more FET fertility treatments, preferably wherein said one or more FET fertility treatments comprised the use of an estradiol-comprising composition. Optionally, the subject is a subject that has actively yet unsuccessfully attempted to become pregnant for at least about 6 months, preferably for at least about 12 months, preferably for at least about 24 months, by means of one or more FET fertility treatments that comprised the use of an estradiol-comprising composition. Optionally, the subject is a subject that has actively yet unsuccessfully attempted to become pregnant after at least 1, 2, 3, 4, 5, or 6 FET fertility treatments that comprised the use of an estradiol-comprising composition. Optionally, the subject is a subject that has failed to become pregnant after at least 1, 2, 3, 4, 5, or 6 IVF treatments. Optionally, the subject is a female subject that has a suspected or confirmed genetic and / or familial predisposition for having reduced fertility. Optionally, the subject is a female subject that wishes to become pregnant from a male subject that has a suspected or confirmed genetic and / or familial predisposition for having reduced fertility.

[0090] A further aspect of the invention is directed to a composition comprising an effective amount of estetrol component for use in preparing the endometrium of a subject for Frozen Embryo Transfer (FET) for receiving and / or implanting the embryo. Thus, the invention equally provides in a method of preparing the endometrium of a subject for Frozen Embryo Transfer (FET) for receiving and / or implanting the embryo, wherein the method comprises a step of administering a composition comprising an effective amount of estetrol component to said subject and a step of transferring an embryo into said subject. Alternatively worded, the invention envisages the use of a composition comprising an effective amount of estetrol component for the manufacture of a medicament for preparing the endometrium of a subject for Frozen Embryo Transfer (FET) for receiving and / or implanting the embryo. Yet alternatively worded, the invention provides the use of a composition comprising an effective amount of estetrol component for preparing the endometrium of a subject for Frozen Embryo Transfer (FET) for receiving and / or implanting the embryo.

[0091] The composition or composition for use described throughout the present disclosure at least comprises an effective amount of an estetrol component. Therefore, expressions such as “an effective amount” and “a pharmaceutically effective amount” refers to an amount necessary to obtain a physiological effect and may indicate a therapeutically effective amount and / or a prophy lactically effective amount. The physiological effect may be achieved by a single dose or by multiple doses. A “therapeutically effective amount” or “therapeutically effective dose” indicates an amount of estetrol component that when administered brings about a clinical positive response with respect to fertility treatment of a subject afflicted by suboptimal conceiving chances. Similarly, a “prophylactically effective amount” or “prophylactically effective dose” refers to an amount of estetrol component that inhibits or delays the onset of clinical manifestation of condition as being sought by a researcher, veterinarian, medical doctor or other clinician. In all embodiments described throughout the present disclosure, the effective amount may be considered to act therapeutically (i.e. treatment or assisting in treatment of fertility problems) or prophylactically (i.e. preventing rejection of the embryo after transfer to the subject). A skilled person is aware that terms such as “quantity”, “amount” and “level” are synonyms and have a well-defined meaning in the art and appreciates that these may particularly refer to an absolute quantification of an estetrol component which is considered an effective amount for the applications described herein. Preferably, an effective amount of an estetrol component according to the present invention is an amount, which leads to an endometrial thickness of at least 7 mm after 14 days of administration of said estetrol component.

[0092] The term “endometrium” is well known to a skilled person and refers to the inner lining of the uterus, a tissue in the female reproductive system. The endometrium plays a crucial role in the menstrual cycle and pregnancy by providing a suitable environment for embryo implantation and supporting foetal development if pregnancy occurs. In ART, several parameters of the endometrium such as but not limited to the endometrial thickness and endometrial quality are monitored to ensure the uterus is prepared for (i.e. in a receptive state for) embryo implantation, as these factors significantly influence the likelihood of successful conception. The endometrium essentially consists of a functional layer (i.e. the stratum functionalis) which is the top layer that thickens, sheds, and regenerates with each menstrual cycle, and the basal layer (i.e. the stratum basalis) which is the stable bottom layer that is responsible for regenerating the functional layer after menstruation.

[0093] In some embodiments of the compositions for use, methods, and uses described herein, the composition described herein has as effect that when administered said composition prepares the endometrium for FET. A skilled person appreciates that expressions such as but not limited to “preparing the endometrium for FET” entails that said endometrium is brought into a receptive state for FET. The receptive state of the endometrium, also indicated throughout the art as the window of implantation, is a defined period during the menstrual cycle when the endometrial lining is most prepared to allow an embryo to implant successfully. This state is crucial for the initiation of pregnancy and is influenced by hormonal changes, such as changes in estrogen and progesterone levels. In a natural cycle, after ovulation progesterone levels increase and transform endometrial cells to promote an environment suitable for embryo attachment and nourishment. Estrogens play a role earlier in the cycle by stimulating the initial thickening and growth of the endometrial lining. For treatments such as IVF and, more specifically, FET, timing the embryo transfer to coincide with this receptive phase significantly improves the chances of successful implantation and pregnancy. The receptive state of the endometrium corresponds to structural changes such as an increase in endometrial thickness to from about 7 mm to 14 mm, increased vascularisation, and a so-called increase in “sponginess”. In addition, glands within the endometrium secrete nutrient-rich substances to accommodate the embryo, including but not limited to proteins, growth factors, and lipids. Medical professionals may evaluate and / or monitor changes in the endometrium using different procedures such as but not limited to endometrial receptivity analysis (ERA), hormonal monitoring, and ultrasound measurements. Each of these procedures aid to define the optimal timing for IVF, FET, or other ART procedures. Optionally, the composition prepares the endometrium for FET after at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive administration days.

[0094] Each of the compositions for use, methods, and uses described herein aim to minimize the chance of rejection of the embryo in FET procedures and hence maximize the chance of embryo implantation in FET procedures. Hence, in a further aspect, the invention is directed to a composition comprising an effective amount of estetrol component for use in increasing the chances of successful embryo implantation in a FET fertility treatment in a subject. Thus, the invention equally provides a method of increasing the chances of successful embryo implantation in a FET fertility treatment in a subject, wherein the method comprises a step of administering a composition comprising an effective amount of estetrol component to said subject and a step of transferring an embryo into said subject. Alternatively worded, the invention provides in use of a composition comprising an effective amount of estetrol component for the manufacture of a medicament for use in increasing the chances of successful embryo implantation in a FET fertility treatment in a subject. Yet alternatively worded, the invention provides in use of a composition comprising an effective amount of estetrol component for increasing the chances of successful embryo implantation in a FET fertility treatment in a subject. In certain embodiments, the chance of successful embryo implantation in a FET fertility treatment in a subject is increased by administration of the composition described herein by at least about 5%, preferably by at least about 10%, preferably by at least about 15%, preferably by at least about 20%, preferably by at least about 25%, preferably by at least about 30%, preferably by at least about 35%, preferably by at least about 40%, preferably by at least about 50%, preferably by at least about 60%, preferably by at least about 75%, preferably by at least about 90% when compared to a FET procedure that does not comprise administration of the composition comprising an effective amount of estetrol component as described herein. In further embodiments, the chance of successful embryo implantation in a FET fertility treatment in a subject is increased by administration of the composition described herein by at least about 5%, preferably by at least about 10%, preferably by at least about 15%, preferably by at least about 20%, preferably by at least about 25%, preferably by at least about 30%, preferably by at least about 35%, preferably by at least about 40%, preferably by at least about 50%, preferably by at least about 60%, preferably by at least about 75%, preferably by at least about 90% when compared to a FET procedure that comprises administration of a composition comprising an effective amount of an estrogen that is not an estetrol component. In further embodiments, the chance of successful embryo implantation in a FET fertility treatment in a subject is increased by administration of the composition described herein by at least about 5%, preferably by at least about 10%, preferably by at least about 15%, preferably by at least about 20%, preferably by at least about 25%, preferably by at least about 30%, preferably by at least about 35%, preferably by at least about 40%, preferably by at least about 50%, preferably by at least about 60%, preferably by at least about 75%, preferably by at least about 90% when compared to a FET procedure that comprises administration of a composition comprising an effective amount of estradiol.

[0095] In some embodiments of the compositions for use, methods, and uses described herein, the composition comprising the estetrol component is administered at an effective daily amount wherein an ovulation rate of at least about 30% amongst treated subjects is maintained, preferably wherein an ovulation rate of at least about 40% amongst treated subjects is maintained, preferably wherein an ovulation rate of at least about 50% amongst treated subjects is maintained, preferably wherein an ovulation rate of at least about 60% amongst treated subjects is maintained. With regard to the term “treated subjects”, for the purposes of this embodiments the treatment subjects are characterized by an age of between 18 years and 40 years, who are willing to use a barrier method of contraception during the total study period, who experienced ovulation in the pre -treatment cycle < day 24 (±1) after start of menses, with subsequent progesterone concentration of > 16 nmol / L and with next menstruation > 6 days after ovulation, having a Body Mass Index from about 18 kg / m2to about 30 kg / m2, have a good physical and mental health, and that have both ovaries visible upon ultrasonography examination. In certain embodiments, the composition comprising the estetrol component is administered at an effective daily amount wherein the increase in luteinizing hormone is increased more moderately when compared to the increase of luteinizing hormone in a natural cycle of the treated subject.

[0096] In certain embodiments of the compositions for use, methods, and uses described herein, the chance of embryo rejection in a FET fertility treatment in a subject is decreased by administration of the composition described herein by at least about 5%, preferably by at least about 10%, preferably by at least about 15%, preferably by at least about 20%, preferably by at least about 25%, preferably by at least about 30%, preferably by at least about 35%, preferably by at least about 40%, preferably by at least about 50%, preferably by at least about 60%, preferably by at least about 75%, preferably by at least about 90% when compared to a FET procedure that does not comprise administration of the composition comprising an effective amount of estetrol component as described herein. In further embodiments, the chance of embryo rejection in a FET fertility treatment in a subject is decreased by administration of the composition described herein by at least about 5%, preferably by at least about 10%, preferably by at least about 15%, preferably by at least about 20%, preferably by at least about 25%, preferably by at least about 30%, preferably by at least about 35%, preferably by at least about 40%, preferably by at least about 50%, preferably by at least about 60%, preferably by at least about 75%, preferably by at least about 90% when compared to a FET procedure that comprises administration of a composition comprising an effective amount of an estrogen that is not an estetrol component. In further embodiments, the chance of embryo rejection in a FET fertility treatment in a subject is decreased by administration of the composition described herein by at least about 5%, preferably by at least about 10%, preferably by at least about 15%, preferably by at least about 20%, preferably by at least about 25%, preferably by at least about 30%, preferably by at least about 35%, preferably by at least about 40%, preferably by at least about 50%, preferably by at least about 60%, preferably by at least about 75%, preferably by at least about 90% when compared to a FET procedure that comprises administration of a composition comprising an effective amount of estradiol.

[0097] In some embodiments of the compositions for use, methods, and uses described herein, the composition described throughout the present disclosure is administered to the subject prior to, during, and / or after embryo transfer. While in each of these timepoints the composition comprises at least an effective amount of an estetrol component, the dosage may be adjusted depending on the point in time of the FET procedure the composition is administered. In addition, in certain instances the estetrol component may be the sole (i.e. only) active pharmaceutical ingredient comprised in the composition, or even the sole (i.e. only) active pharmaceutical ingredient administered to the subject. In other instances, the composition may comprise at least one further pharmaceutical ingredient in addition to the estetrol component, such as but not limited to a progestogen, or the at least one further pharmaceutical ingredient such as but not limited to a progestogen may be administered in a further composition or even by a different mode of administration than the estetrol component.

[0098] In some embodiments of the compositions for use, methods, and uses described herein, the administration of the composition described herein results in (i.e. has as effect) that the endometrial thickness of the subject is increased, preferably wherein the endometrial thickness of the subject is increased when compared to the endometrial thickness prior to the start of administration of the composition. In certain embodiments, the endometrial thickness is increased by at least about 1 mm, preferably by at least about 2 mm, preferably by at least about 3 mm, preferably by at least about 4 mm, preferably by at least about 5 mm, preferably by at least about 6 mm, preferably by at least about 7 mm, preferably by at least about 8 mm, preferably by at least about 9 mm, preferably by at least about 10 mm, preferably by at least about 11 mm, preferably by at least about 12 mm, preferably by at least about 13 mm, preferably by at least about 14 mm, wherein optionally said endometrial thickness increase occurs between day 3 and day 21 of administering the composition described herein. In certain embodiments, the endometrial thickness after treatment is between about 7 mm and about 14 mm, optionally between about 8 mm and about 13 mm, optionally between 9 mm and about 12 mm, optionally between about 10 mm and about 11 mm. In preferred embodiments, administration of the composition described herein results in an endometrial thickness of from about 7 mm to about 14 mm.

[0099] Preferably, the compositions for use, methods, and uses described herein aim to provide the subject with a daily amount of estetrol component that still allows the formation of the corpus luteum, such as for example a daily amount of from about 6 mg to about 14 mg, preferably from about 8 mg to about 12 mg, most preferably a daily amount of about 10 mg. In further embodiments, the compositions for use, methods, and uses described herein aim to provide the subject with a daily amount of estetrol component that still allows the formation of the corpus luteum while resulting in an endometrial thickness in the subject of from about 7 mm to about 14 mm, such as for example a daily amount of from about 6 mg to about 14 mg, preferably from about 8 mg to about 12 mg, most preferably a daily amount of about 10 mg.

[0100] The term “corpus luteum” as used herein is to be interpreted according to its broadest interpretation in the art and therefore refers to a temporary endocrine structure in female ovaries which develops from an ovarian follicle during the luteal phase of the menstrual cycle following the release of a secondary oocyte from the follicle during ovulation. A new corpus luteum develops each menstrual cycle. The corpus luteum plays a critical role in the early stages of pregnancy by producing essential hormones that prepare and maintain the uterine lining (endometrium) for embryo implantation and support early foetal development. The corpus luteum produces progesterone, estradiol, relaxin, and inhibin A. Upon implantation of an embryo, the syncytiotrophoblast (the multinucleate the epithelial covering of the highly vascular embryonic placental villi, which invades the wall of the utems to establish nutrient circulation between the embryo and the mother) releases human chorionic gonadotropin (hCG) which signals the corpus luteum to continue producing progesterone beyond the normal period in case of no implantation of about 10 to 14 days until the placenta is fully developed and takes over progesterone production (around about 10 to 12 weeks of the pregnancy). As described in the background section, the corpus luteum in spontaneous pregnancies was demonstrated to play an important role by producing crucial hormones for implantation, placentation, and pregnancy maintenance. Relaxin secreted by corpus luteum is a potent vasodilator which contributes to cardiovascular and renal adaptations seen in pregnancy. Hence, the effect of the composition described herein of allowing formation of the corpus luteum is that favorable conditions for embryo implantation and pregnancy are obtained. Optionally, relaxin is increased upon use of the composition described herein prior, during, and / or after FET in the subject by at least about 10%, preferably at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% increased when compared to a subject that has not received an estrogen-comprising composition prior, during, and / or after FET, or preferably when compared to a subject that has received an estradiol-comprising composition prior, during, and / or after FET. Optionally, relaxin is increased upon use of the composition described herein prior, during, and / or after FET in the subject by at least about 1.5 fold, preferably at least about 2 fold, at least about 5 fold, preferably at least about 10 fold when compared to a subject that has not received an estrogen-comprising composition prior, during, and / or after FET, or preferably when compared to a subject that has received an estradiol-comprising composition prior, during, and / or after FET.

[0101] In embodiments, the compositions for use, methods, and uses described herein aim to increase production of the luteinizing hormone and / or stimulation of corpus luteum (trans)formation. Hence, in certain aspects the invention is directed to a composition comprising an effective amount of estetrol component for use in stimulating Luteinizing Hormone (LH) production and / or corpus luteum formation during Frozen Embryo Transfer (FET). Hence, the invention equally provides a method of stimulating Luteinizing Hormone (LH) production and / or corpus luteum formation during Frozen Embryo Transfer (FET), wherein the method comprises a step of administering a composition comprising an effective amount of estetrol component to said subject and a step of transferring an embryo into said subject. Alternatively worded, the invention envisages the use of a composition comprising an effective amount of estetrol component for the manufacture of a medicament for stimulating Luteinizing Hormone (LH) production and / or corpus luteum formation during Frozen Embryo Transfer (FET). Yet alternatively worded, the invention provides a use of a composition comprising an effective amount of estetrol component for stimulating Luteinizing Hormone (LH) production and / or corpus luteum formation during Frozen Embryo Transfer (FET).

[0102] The term “luteinizing hormone” or “LH” as used herein is to be interpreted according to its broadest interpretation in the art and therefore refers to a hormone produced by gonadotropic cells in the anterior pituitary gland. The production of LH is regulated by gonadotropin-releasing hormone from the hypothalamus. In females, an acute rise of LH known as an LH surge, triggers ovulation and transformation of the ruptured follicle into a corpus luteum. The corpus luteum then produces progesterone, a hormone crucial for maintaining the uterine lining and supporting early pregnancy. LH helps maintain the corpus luteum, ensuring it continues to produce progesterone until the placenta takes over this role if pregnancy occurs.

[0103] In embodiments of the compositions for use, methods, and uses described herein, wherein a progestogen is administered during treatment of the subject with the composition described herein or embodiments wherein a progestogen is comprised in the composition described herein, the progestogen may cause, or assist with, endometrial maturation. A skilled person appreciates that “endometrial maturation” as used herein indicates a transition from a proliferative, growing state of the endometrium into a prepared, secretory phase lining. A skilled person also appreciates that this prepared secretory phase lining is capable of (i.e. configured to, receptible to) receive an embryo and allows implantation of the embryo. It has been described in the art that endometrial maturation is regulated by hormones such as estrogen and progesterone and is a process that occurs in two main phases. In a first phase prior to ovulation (i.e. the proliferative phase), the endometrium thickens due to cellular division which leads to a build up in tissue to anticipate a possible pregnancy. In a second phase after ovulation (i.e. the secretory phase) the progesterone produced by the corpus luteum stimulated further maturation wherein the glands in the endometrium produce nutrient-rich secretions that create an optimal environment for a fertilized egg cell.

[0104] In certain embodiments of the compositions for use, methods, and uses described herein, the composition described herein is administered such that the subject receives an effective amount of estetrol component. In preferred embodiments, the composition described herein is administered such that the subject receives from about 1 mg to about 40 mg of estetrol component, preferably from about 5 mg to about 30 mg of estetrol component, preferably from about 5 mg to about 25 mg of estetrol component, preferably from about 10 mg to about 25 mg of estetrol component, preferably from about 10 mg to about 20 mg of estetrol component, preferably from about 15 mg to about 20 mg of estetrol component, preferably about 15 mg or about 20 mg of estetrol component, more preferably about 15 mg (such as from 14.5 mg to 15.5 mg) of estetrol component. In further embodiments, the composition described herein comprises an effective amount of estetrol component. In preferred embodiments, the composition described herein comprises from about 1 mg to about 40 mg of estetrol component, preferably from about 5 mg to about 30 mg of estetrol component, preferably from about 5 mg to about 25 mg of estetrol component, preferably from about 10 mg to about 25 mg of estetrol component, preferably from about 10 mg to about 20 mg of estetrol component, preferably from about 15 mg to about 20 mg of estetrol component, preferably about 15 mg or about 20 mg of estetrol component, more preferably about 15 mg (such as from 14.5 mg to 15.5 mg) of estetrol component. In yet further embodiments, the composition described herein comprises a daily effective amount of estetrol component. In preferred embodiments, the composition described herein comprises a daily amount from about 1 mg to about 40 mg of estetrol component, preferably a daily amount from about 5 mg to about 30 mg of estetrol component, preferably a daily amount from about 5 mg to about 25 mg of estetrol component, preferably a daily amount from about 10 mg to about 25 mg of estetrol component, preferably a daily amount from about 10 mg to about 20 mg of estetrol component, preferably a daily amount from about 15 mg to about 20 mg of estetrol component, preferably a daily amount of about 15 mg or about 20 mg of estetrol component, more preferably about 15 mg (such as from 14.5 mg to 15.5 mg) of estetrol component.

[0105] In certain embodiments, the composition described herein is administered such that the subject receives an effective amount of estetrol, estetrol monohydrate, or an ester of estetrol. In preferred embodiments, the composition described herein is administered such that the subject receives from about 1 mg to about 40 mg of estetrol, estetrol monohydrate, or an ester of estetrol, preferably from about 5 mg to about 30 mg of estetrol, estetrol monohydrate, or an ester of estetrol, preferably from about 5 mg to about 25 mg of estetrol, estetrol monohydrate, or an ester of estetrol, preferably from about 10 mg to about 25 mg of estetrol, estetrol monohydrate, or an ester of estetrol, preferably from about 10 mg to about 20 mg of estetrol, estetrol monohydrate, or an ester of estetrol, preferably from about 15 mg to about 20 mg of estetrol, estetrol monohydrate, or an ester of estetrol, preferably about 15 mg or about 20 mg of estetrol, estetrol monohydrate, or an ester of estetrol, more preferably about 15 mg (such as from 14.5 mg to 15.5 mg) of estetrol, estetrol monohydrate, or an ester of estetrol. In further embodiments, the composition described herein comprises an effective amount of estetrol, estetrol monohydrate, or an ester of estetrol. In preferred embodiments, the composition described herein comprises from about 1 mg to about 40 mg of estetrol, estetrol monohydrate, or an ester of estetrol, preferably from about 5 mg to about 30 mg of estetrol, estetrol monohydrate, or an ester of estetrol, preferably from about 5 mg to about 25 mg of estetrol, estetrol monohydrate, or an ester of estetrol, preferably from about 10 mg to about 25 mg of estetrol, estetrol monohydrate, or an ester of estetrol, preferably from about 10 mg to about 20 mg of estetrol, estetrol monohydrate, or an ester of estetrol, preferably from about 15 mg to about 20 mg of estetrol, estetrol monohydrate, or an ester of estetrol, preferably about 15 mg or about 20 mg of estetrol, estetrol monohydrate, or an ester of estetrol, more preferably about 15 mg (such as from 14.5 mg to 15.5 mg) of estetrol, estetrol monohydrate, or an ester of estetrol. In yet further embodiments, the composition described herein comprises a daily effective amount of estetrol, estetrol monohydrate, or an ester of estetrol. In preferred embodiments, the composition described herein comprises a daily amount from about 1 mg to about 40 mg of estetrol, estetrol monohydrate, or an ester of estetrol, preferably a daily amount from about 5 mg to about 30 mg of estetrol, estetrol monohydrate, or an ester of estetrol, preferably a daily amount from about 5 mg to about 25 mg of estetrol, estetrol monohydrate, or an ester of estetrol, preferably a daily amount from about 10 mg to about 25 mg of estetrol, estetrol monohydrate, or an ester of estetrol, preferably a daily amount from about 10 mg to about 20 mg of estetrol, estetrol monohydrate, or an ester of estetrol, preferably a daily amount from about 15 mg to about 20 mg of estetrol, estetrol monohydrate, or an ester of estetrol, preferably a daily amount of about 15 mg or about 20 mg of estetrol, estetrol monohydrate, or an ester of estetrol, more preferably about 15 mg (such as from 14.5 mg to 15.5 mg) of estetrol, estetrol monohydrate, or an ester of estetrol. Preferably, the composition described herein comprises a daily amount of about 4.7, 9.4, 14.2, 18.9, or 28.3 mg of estetrol.

[0106] A skilled person readily appreciates that the term “daily” corresponds to about once every 24 hours. Hence when the expression “daily amount” is used herein said expression corresponds to an amount that describes the total amount of each day (i.e. of about 24 hours), and when the expression “daily administered” is used herein said expression corresponds to an administration about once every 24 hours. It is equally appreciated by a skilled person that to reach a daily amount, said daily amount can be reached by a once daily administration of a composition comprising the daily amount, or that said daily amount can be reached by more than one administration of a composition cumulatively arrive at the daily amount.

[0107] Optionally, the composition described herein is administered for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,

[0108] 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 consecutive days. Optionally, the composition described herein is administered once daily for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,

[0109] 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 consecutive days. In further embodiments wherein the composition described herein is administered for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 consecutive days the composition comprises a constant amount of estetrol component. In alternative embodiments wherein the composition described herein is administered for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 consecutive days the composition comprises a variable amount of estetrol component. In each of the embodiments described herein the composition may be administered for from 1 to 30, 2 to 29, 3 to 28, 4 to 27, 5 to 26, 6 to 25, 7 to 24, 8 to 23, 9 to 22, 10 to 21, 11 to 20, 12 to 19, 13 to 18, 14 to 17, 15 to 16 consecutive days.

[0110] Preferably, the composition described herein is administered from the first day of the menstrual cycle. Alternatively, the composition described herein is administered from the second day of the menstrual cycle. A skilled person appreciates that the first day of the menstrual cycle is the first day wherein the subject experiences bleeding and / or spotting, preferably bleeding distinct from spotting, for the first time in that menstrual cycle. In certain embodiments, the composition described herein is administered within 48 hours, preferably within 36 hours, preferably within 24 hours, preferably within 12 hours from the onset of bleeding and / or spotting, preferably bleeding distinct from spotting, in the menstrual cycle. In certain embodiments, the composition described herein is administered within 48 hours, preferably within 36 hours, preferably within 24 hours, preferably within 12 hours from the onset of bleeding and / or spotting, preferably bleeding distinct from spotting, in the menstrual cycle. The terms “bleeding” and “spotting” are to be interpreted according to their well-accepted meaning in the art. Therefore, the term “bleeding” as used herein refers to vaginal blood loss that requires the use of sanitary protection with a tampon, pad or panty liner; and “spotting” as used herein refers to minimal vaginal blood loss that does not require the new use of sanitary protection, including pantyliners. Accordingly, a “bleeding / spotting episode” is indicative for one or more consecutive bleeding / spotting days bounded on either end by two bleeding / spotting free days.

[0111] The composition described herein is used in a FET context to increase endometrial thickness and bring the endometrium into a receptive state for accepting an embryo and increase the chances for successful implantation thereof. In certain embodiments, administration of the composition leads to an endometrial thickness of at least about 7 mm is achieved. Optionally, administration of the composition results in an endometrial thickness of at least about 8 mm, preferably at least about 9 mm, preferably at least about 10 mm, preferably at least about 11 mm, preferably at least about 12 mm, preferably at least about 13 mm, preferably at least about 14 mm is achieved. Optionally, administration of the composition results in an endometrial thickness of at least about 7 mm is achieved after at least 3 days, preferably after at least 4 days, preferably after at least 5 days, preferably after at least 6 days, preferably after at least 7 days, preferably after at least 8 days, preferably after at least 9 days, preferably after at least 10 days, preferably after at least 11 days, preferably after at least 12 days, preferably after at least 13 days administration of the composition (which may be administered for a non-limited number of additional days after the indicated amount of days).

[0112] In preferred embodiments, the composition is orally administered and / or the composition is formulated as an oral composition (i.e. an oral dosage unit). The composition described herein is particularly suited for formulation as an oral dosage unit with oral ingestion as the envisaged mode of administration. After oral ingestion, the active ingredient, i.e. the estetrol component, is subjected to the first-pass effect. However, equally envisaged are dosage units formulated towards alternative administration methods such as but not limited to sublingual, buccal, or sublabial dosage units, that are intended to avoid the first -pass effect. The composition described herein may therefore be suitably or even specifically manufactured for sublingual, buccal, and / or sublabial administration. The dosage unit may be able to rapidly release the estetrol component when contacted with an aqueous solvent such as saliva. Hence, in such embodiments the composition described herein is an orodispersible composition which releases at least about 50%, preferably at least about 60%, more preferably at least about 70%, yet more preferably at least about 80%, most preferably more than about 80% of the estetrol and / or drospirenone within about 5 minutes, preferably within about 3 minutes, more preferably within about 2.5 minutes, more preferably within about 90 seconds, most preferably within about 90 seconds. In further embodiments, the composition described herein rapidly disintegrates in the oral cavity when it comes into contact with saliva and disperses the estetrol and / or progestogen into the saliva so it may be absorbed through the mucosal lining of the oral cavity. A skilled person is aware of methods to determine the release rate of a pharmaceutically active ingredient such as estetrol and progestogen from a dosage unit. Non-limiting standardized tests generally accepted in the field include the disintegration test according to Ph Eur. 2.9.1 (“Disintegration of tablets and capsules”) and USP <701> (“Disintegration”), for example using water as the disintegration medium.

[0113] The term “sublingual” as used herein refers to the pharmacological route of administration by which the estetrol and / or drospirenone (comprised in the composition) diffuse into the blood through tissues under the tongue.

[0114] The term “buccal” as used herein refers to the pharmacological route of administration by which the estetrol and / or drospirenone (comprised in the composition) diffuse into the blood through tissues of the buccal vestibule, the area inside the mouth between the lining of cheek (the buccal mucosa) and the teeth / gums.

[0115] The term “sublabial” as used herein refers to the pharmacological route of administration by which the estetrol and / or drospirenone (comprised in the composition) are placed between the lip and the gingiva.

[0116] The composition as described herein, particularly upon formulation as an oral dosage unit but not limited to those embodiments, may comprise one or more suitable excipients. The term “excipient” may be a ''carrier' ’ indicative for any solvent, diluent, buffer (including but not limited to neutral buffered saline, phosphate buffered saline, or optionally Tris-HCl, acetate or phosphate buffers), solubiliser (including but not limited to Tween 80 or Polysorbate 80), colloid, dispersion medium, vehicle, filler, chelating agent (including but not limited to EDTA or glutathione), amino acid, protein, disintegrant, binder, lubricant, wetting agent, stabiliser, emulsifier, sweetener, colorant, flavoring, aromatiser, thickener, any agent suitable to achieve a depot effect, coating, antifungal agent, any preservative (including but not limited to Thimerosal™ or thiomersal, benzalkonium chloride, or benzyl alcohol), antioxidant (including but not limited to ascorbic acid, sodium metabisulfite), tonicity controlling agent, absorption delaying agent, adjuvant, bulking agent (including but not limited to lactose, mannitol) and any other ingredient that may influence any parameter or characteristic of the oral dosage unit. A skilled person understands that one or more excipients may be used in the composition or oral dosage unit on condition that the one or more excipient is compatible with the pharmaceutical ingredients (i.e. in the context of the present disclosure at least estetrol and drospirenone) and that a pharmaceutically acceptable formulation is obtained.

[0117] In certain embodiments, said excipient may be an active pharmaceutical ingredient excipient, binder excipient, carrier excipient, co-processed excipient, coating system excipient, controlled release excipient, diluent excipient, disintegrant excipient, dry powder inhalation excipient, effervescent system excipient, emulsifier excipient, lipid excipient, lubricant excipient, modified release excipient, penetration enhancer excipient, permeation enhancer excipient, pH modifier excipient, plasticiser excipient, preservative excipient, preservative excipient, solubiliser excipient, solvent excipient, sustained release excipient, sweetener excipient, taste making excipient, thickener excipient, viscosity modifier excipient, filler excipient, compaction excipient, dry granulation excipient, hot melt extrusion excipient, wet granulation excipient, rapid release agent excipient, increased bioavailability excipient, dispersion excipient, solubility enhancement excipient, stabilizer excipient, capsule filling excipient, or any combination hereof. A skilled person is aware that use of such media and agents for pharmaceutical active substances is common practice and incorporation of these excipients is hence well known in the art. It is evident that all of the used ingredients should be non-toxic in the concentration contained in the final pharmaceutical composition and should not negatively interfere with the activity of the one or more pharmaceutically active ingredients, in this context at least the estetrol component.

[0118] The composition described herein may alternatively be described as a solid or semi solid dosage unit such as a tablet, a capsule, a cachet, a pellet, a pill, powder, granules, or any combination thereof. For example, the composition described herein may be a tablet comprising the estetrol component-containing composition. The term "solid or semi-solid dosage unit" also encompasses capsules that contain a liquid, e.g. an oil, in which the estetrol component may be dissolved or dispersed.

[0119] Tablets and equivalent solid and semi-solid dosage units can suitably contain materials such as binders (e.g. hydroxypropylmethyl cellulose, polyvinyl pyrrolidone (povidone, PVP), other cellulosic materials and starch), diluents (e.g. lactose (monohydrate) and other sugars, starch (e.g. maize starch), dicalcium phosphate and cellulosic materials), disintegrating agents (e.g. starch polymers and cellulosic materials (e.g. sodium starch glycolate) and lubricating agents (e.g., (magnesium) stearates and talc). These tablets and equivalent solid dosage units may be prepared by any suitable means, which have been described in detail in the art (e.g. Kaur, Int Res J Pharm, 2012). Non-limiting examples of processing methods of the estetrol component when manufacturing the dosage unit include wet granulation, e.g. using an aqueous solution or an organic solution, direct compression, 3D printing, or by coating carrier particles with the estetrol component using an organic or inorganic solvent.

[0120] In certain embodiments, the composition described herein is formulated as a tablet comprising in addition to estetrol a fdler, a superdisintegrant, a binder and disintegrant, a further binder, a lubricant, and optionally a progestogen such as progesterone, medroxyprogesterone acetate, or dydrogesterone. In preferred embodiments, the composition is comprised in a tablet comprising an estetrol component such as estetrol, lactose, sodium starch glycolate, maize / com starch, povidone, and magnesium stearate. Preferably, the composition described herein is formulated as a tablet comprising estetrol monohydrate, lactose monohydrate, sodium starch glycolate type A, maize starch, povidone K30, magnesium stearate, and optionally a progestogen such as progesterone, medroxyprogesterone acetate, or dydrogesterone. Optionally, the tablet is coated with a coating agent. In further optional embodiments, the coating agent comprises hypromellose, hydroxypropylcellulose, titanium dioxide, red iron oxide, hydrogenated cottonseed oil, and talc. By means of illustration and not limitation, a suitable coating agent is AquaPolish Pink 044.08 MS. A skilled person further appreciates that any excipients present in any dosage unit such as an oral dosage unit should adhere to pharmaceutical grade industry quality standards such as Ph. Eur. and USP-NF.

[0121] In certain embodiments, the composition comprising an effective amount of estetrol component is administered to the subject orally. In alternative embodiments, the composition comprising an effective amount of estetrol component is administered to the subject vaginally. In yet alternative embodiments, a composition comprising an effective amount of estetrol component is administered to the subject both orally and vaginally. In further embodiments, a first composition comprising an effective amount of estetrol component is administered to the subject orally and a second composition comprising an effective amount of estetrol component is administered to the subject vaginally. In further embodiments, the first composition is administered prior to administration of the second composition. In alternative embodiments, the first composition is administered concomitantly to administration of the second composition. In yet alternative embodiments, the first composition is administered after administration of the second composition.

[0122] By means of illustration and not limitation, an oral dosage unit comprising the composition described herein may be manufactured by a process involving wet granulation, e.g. using an aqueous solution or an organic solution, direct compression, 3D printing, or by coating carrier particles with the estetrol component using an organic or inorganic solvent. A skilled person appreciates that a wet granulation process may suitable comprise the successive steps of: dispensing and sieving of the active ingredient(s) and excipients, blending the sieved materials in a processor, granulation, screening (i.e. further sieving) of the granules, and one or more blending steps of the sieved granules with one or more further excipients. Afterwards, if desired in view of the final dosage unit the granules may be compressed into for example a tablet, optionally involving a coating step of said tablet.

[0123] In certain embodiments, a progestogen is administered to the subject either in the same composition as the estetrol component-comprising composition or by another means. Optionally, the progestogen is a progestin. The terms “progestogen”, “progestogen”, “gestagen”, or “gestogen” and derived hereof “progestogenic compounds” as used both herein and in the art refer to any molecule that produces effects similar to those of the natural female sex hormone progesterone in the body of a subject. Progestagens are considered to be agonists of the progesterone receptors and their functions have been thoroughly examined in the art (inter alia discussed in Kuhl, Pharmacology of estrogens and progestagens: influence of different routes of administration, Climacteric, 2005). Progestins are a subgroup of progestagens that comprise synthetic progestagens. While the above terms may be used interchangeably in the art, there is a general understanding that when progestin is mentioned, synthetic progestagens are meant. Examples of progestins suitable for the embodiments described herein that comprise the use of a progestogen are: levonoigestrel, norgestimate, norethisterone, dydrogesterone, drospirenone, 3-beta- hydroxydesogestrel, 3-ketodesogestrel, 17-deacetylnorgestimate, 19-norprogesterone, acetoxypregnenolone, allylestrenol, amgestone, chlormadinone, cyproterone, demegestone, desogestrel, dienogest, dihydrogesterone, dimethisterone, ethisterone, ethynodiol diacetate, fluorogestone acetate, gastrinone, gestodene, gestrinone, hydroxymethylprogesterone, hydroxyprogesterone, lynestrenol, mecirogestone, medroxyprogesterone, megestrol, melengestrol, nomegestrol, norethindrone, norethynodrel, norgestrel (including d-norgestrel, and dl- norgestrel), norgestrienone, normethisterone, progesterone, quingestanol, (17 alpha)-17-hydroxy-ll-methylene- 19-norpregna-4, 15-dien-20-yn-3-one, tibolone, trimegestone, algestone-acetophenide, nestorone, promegestone, 17-hydroxyprogesterone esters, 19-nor-17hydroxyprogesterone, 17alpha-ethynyltestosterone, 17alpha-ethynil- 19-nortestosterone, d-17beta-acetoxy-13beta-ethyl-17alpha-ethynylgon-4-en-3-one oxime, 6beta, 7beta;15beta,16beta-dimethylene-3-oxo-17-pregna-4,9(ll)-diene-21, 17beta-carbolactone or tanaproget and precursors of these compounds that are capable of liberating these progestagens in vivo. In certain embodiments wherein a progestogen is administered to the subject, either in the same composition as the estetrol component-comprising composition or by another means, the progestogen may be selected from the group consisting of progesterone, medroxyprogesterone acetate, and dydrogesterone.

[0124] “Progesterone” (commonly abbreviated as “P4”; PubChem CID 5994) is an endogenous steroid and progestogen sex hormone involved in the menstrual cycle, pregnancy, and embryogenesis of women and constitutes the major progestogen in the body. Progesterone is a well-documented substance and has been used in the art for indications including but not limited to contraception, female hormone replacement therapy, and feminizing hormone therapy. Progesterone may be indicated in the art by reference to its structural formula C21H30O2, or by the structural formula (III):

[0125] Formula (III)

[0126] It is understood that when the term “progesterone” is used herein, any progesterone derivatives are also envisaged.

[0127] The term “medroxyprogesterone acetate” (PubChem CID 6279) refers to an acetate ester resulting from the formal condensation of the 17alpha-hydroxy group of medroxyprogesterone with the carboxy group of acetic acid, medroxyprogesterone acetate is a widely used progestin in menopausal hormone therapy and in progestogen-only birth control. It is a steroid ester, an acetate ester, a 20 -oxo steroid, a 3-oxo-Delta(4) steroid and a corticosteroid and is functionally related to medroxyprogesterone. Medroxyprogesterone acetate may be indicated in the art by reference to its structural formula C24H34O4, or by the structural formula (IV):

[0128] Formula (IV) It is understood that when the term “medroxyprogesterone acetate” is used herein, any medroxyprogesterone acetate derivatives are also envisaged.

[0129] The progestin “dydrogesterone” (PubChem CID 9051), interchangeably indicated in the art such as “isopregnenone” and “didrogesteron”, has been used for numerous medical indications including dysfunctional bleeding, infertility, dysmenorrhea, endometriosis, and menopause hormone therapy. Dydrogesterone may be indicated in the art by reference to its structural formula C21H28O2 or by the structural formula (V):

[0130] It is understood that when the term “dydrogesterone” is used herein, any dydrogesterone derivatives are also envisaged.

[0131] Optionally, the progestogen may be a micronized progestogen, such as but not limited to micronized progesterone, micronized medroxyprogesterone acetate, and / or micronized dydrogesterone. The term “micronized” as used throughout the present disclosure is to be interpreted according to its commonly accepted interpretation in the art and therefore indicates particles (in the present context progestogen particles) that have an average diameter of from about 1 pm to about 100 pm, preferably from about 2 pm to about 75 pm, more preferably from about 5 pm to about 50 pm. Optionally, the micronized progestogen has an average size (i.e. average diameter) of less than about 100 pm, preferably less than about 75 pm, preferably less than about 50 pm, preferably less than about 30 pm, preferably less than about 25 pm, preferably less than about 20 pm, preferably less than about 15 pm, preferably less than about 10 pm, preferably less than about 5 pm, preferably less than about 2.5 pm.

[0132] The method of administration of the progesterone is not particularly limiting for the invention. Hence, by means of illustration and not limitation progesterone, medroxyprogesterone acetate, dydrogesterone, and any of their micronized forms may be administered orally, vaginally, or by means of intramuscular injection. By means of illustration and not limitation, upon using intramuscular injection as mode of administration of the progestogen, a “progestogen-in-oil” formulation may be used, for example a progesterone -in-oil formulation.

[0133] In certain embodiments wherein progesterone is administered, preferably orally administered, together with the composition comprising the estetrol component or wherein progesterone is administered as part of the composition, preferably the oral composition, comprising the estetrol component, a daily amount of from about 25 mg to about 1000 mg, preferably a daily amount of from about 50 mg to about 800 mg, preferably a daily amount of from about 75 mg to about 600 mg, preferably a daily amount of from about 100 mg to about 400 mg, preferably a daily amount of from about 150 mg to about 300 mg, preferably a daily amount of about 200 mg, more preferably a daily amount of about 180 mg progesterone or micronized progesterone may be used.

[0134] In certain embodiments wherein progesterone is administered, preferably vaginally, together with the composition comprising the estetrol component or wherein progesterone is administered as part of the composition, preferably vaginal composition, comprising the estetrol component, a daily amount of from about 25 mg to about 1000 mg, preferably a daily amount of from about 50 mg to about 800 mg, preferably a daily amount of from about 75 mg to about 600 mg, preferably a daily amount of from about 100 mg to about 400 mg, preferably a daily amount of from about 150 mg to about 300 mg, preferably a daily amount of about 180 mg progesterone or micronized progesterone may be used.

[0135] In certain embodiments wherein dydrogesterone is administered together with the composition comprising the estetrol component or wherein dydrogesterone is administered as part of the composition comprising the estetrol component, a daily amount of from about 5 mg to about 100 mg, preferably a daily amount of from about 10 mg to about 75 mg, preferably a daily amount of from about 15 mg to about 50 mg, preferably a daily amount of from about 25 mg to about 75 mg, preferably a daily amount of about 40 mg, more preferably a daily amount of about 20 mg dydrogesterone or micronized dydrogesterone may be used.

[0136] Also envisaged is the combined use of more than one progestogen with the composition comprising the estetrol component. Hence, administration of any combination of effective amounts of progesterone, medroxyprogesterone acetate, dydrogesterone, micronized progesterone, micronized medroxyprogesterone acetate, and / or micronized dydrogesterone is envisaged by the invention. By means of illustration and not limitation, progesterone and dydrogesterone may be orally administered on the same day. When P4 blood values are not increasing as desired after administration of a certain progestogen, combined administration of progesterone and dydrogesterone to adapt luteal phase support may be considered. In these cases, additional vaginal or intramuscular administration of a progestogen is considered. Preferably, dydrogesterone is administered vaginally or intramuscularly on the same day as oral progesterone.

[0137] As indicated above, the progestogen may administered on the same days, preferably on the same time points as the estetrol component. In further embodiments, the progestogen may be administered on the same days, preferably on the same time points as the estetrol component for at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive days.

[0138] Optionally, the administration of the progestogen may be initiated (i.e. started) after at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive days of estetrol component administration, preferably after about 13 consecutive days of estetrol component administration.

[0139] In certain embodiments, the embryo is transferred after at least 1, 2, 3, 4, 5, or 6 days of administration of the estetrol component-comprising composition. In further embodiment, the embryo is transferred after at least 1, 2, 3, 4, 5, or 6 days of combined administration of the estetrol component and the progestogen. A skilled person appreciates that in the context of the present invention, the expression “transfer of the embryo” indicates the introduction of a thawed or thawing embryo that was frozen in an earlier point in time to the uterine lining (i.e. the endometrial tissue) of the subject. It is evident that further manipulations may be conducted to the embryo before introduction to the uterine lining (i.e. the endometrial tissue) of the subject.

[0140] Also envisaged by the invention is the administration and / or continued administration of the composition comprising an effective amount of estetrol component after embryo transfer. This administration or continued administration (i.e. if the composition comprising the estetrol component was already administered prior to and / or during embryo transfer) can be for up to at least 1 week, preferably for up to at least 2 weeks, preferably for up to at least 3 weeks, preferably for up to at least 4 weeks, preferably for up to at least 5 weeks, preferably for up to at least 6 weeks, preferably for up to at least 7 weeks, preferably for up to at least 8 weeks, preferably for up to at least 9 weeks, preferably for up to at least 10 weeks, preferably for up to at least 11 weeks, preferably for up to at least 12 weeks after (i.e. post) embryo transfer.

[0141] In certain embodiments, the composition comprising an effective amount of estetrol component further comprises a FSH substance. In alternative embodiments, the composition comprising an effective amount of estetrol component does not comprise a FSH substance. In further embodiments, no FSH substance is administered to the subject during the period wherein the composition subject of the invention is administered. By means of illustration and not limitation, FSH substances commonly used in the art comprise urinary FSH (uFSH) and recombinant FSH (recFSH). Hence, in preferred embodiments the composition comprising an effective amount of estetrol component does not comprise uFSH or recFSH.

[0142] The role of FSH (Follicle Stimulating Hormone) is well known in the art and its role in the female reproductive system has been described at numerous occasions (e.g. in Orlowski and Sarao, StatPearls Publishing, 2024). Optionally, the follicular growth upon use prior, during, and / or after FET in a subject of the composition described herein is at least about 10%, preferably at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% increased when compared to a subject that has not received an estrogen-comprising composition prior, during, and / or after FET, or preferably when compared to a subject that has received an estradiol -comprising composition prior, during, and / or after FET. Optionally, the follicular growth upon use of the composition described herein prior to, during, and / or after FET is increased by at least about 1.5 fold, preferably at least about 2 fold, at least about 5 fold, preferably at least about 10 fold when compared to a subject that has not received an estrogen-comprising composition prior, during, and / or after FET, or preferably when compared to a subject that has received an estradiol-comprising composition prior, during, and / or after FET.

[0143] A further aspect of the invention is directed to a pharmaceutical kit for use in the treatment of infertility by frozen embryo transfer (FET) wherein said kit comprises a parenteral or oral dosage unit containing an effective amount of an estetrol component and an oral and / or vaginal dosage unit containing an effective amount of progestogen. Preferably, the kit further comprises user instructions. Preferably, the kit further comprises means to manipulate and / or thaw a frozen embryo. In preferred embodiments, the kit comprises an effective amount of estetrol, estetrol monohydrate, or an ester of estetrol and / or an effective amount of progesterone, medroxyprogesterone acetate, dydrogesterone, or any combination thereof.

[0144] A further aspect of the invention is directed to packaging units comprising the composition described herein. The packaging units may comprise at least 14, preferably at least 21, even more preferably at least 28, containers for holding separately packaged and individually removable composition described herein, wherein each container comprises at least one composition described herein. Optionally, each of the containers for holding one or more compositions described herein is individually visually arranged to present a recommended order of administration.

[0145] The skilled person appreciates that within the scope of the present invention, each packaging unit, e.g. blister pack, may be numbered or otherwise marked. The packaging units may be provided in any suitable packaging means known in the art, non-limiting examples being troches, sachets, pouches, bottles, fdms, sprays, microcapsules, implants, rods or blister packs.

[0146] By means of illustration and not limitation, each packaging unit may be a sealed blister pack with a cardboard, paperboard, foil plastic backing and enclosed in a suitable cover.

[0147] Also envisaged in any one of the aspects defined herein are packaging units such as bottles. The material of the bottle is not particularly limiting. In preferred embodiments, the bottle is a glass bottle characterized by a color capable of reducing or preventing degradation of the contents of the bottle by e.g. UV light while maintaining a degree of transparency that allows for visual inspection of the contents of said bottle. Suitable colors include without limitation amber, cobalt, or vintage green.

[0148] In a particular embodiment of the invention the packaging unit comprises 28 containers or a multiple of 28 containers, such as 2, 3, 6, or more multiples of 28 containers. In a preferred embodiment, the composition described herein can be supplied in (plastic) blister packs. In a preferred embodiment, the composition described herein is supplied in cardboard cartons containing 1, 3 or up to 6 blister packs.

[0149] The packaging unit disclosed herein may be a “compliance package”. As known from the art, “compliance packages” are packaging units of variable sizes and formats that, next to providing a suitable storage means for one or more medicaments, aim to provide assistance and / or guidance to a subject to comply with an intended period of administration. As a non-limiting example, the packaging unit may be provided with numerical indications and / or symbols that allow the subject to keep track of for example said subject’s menstrual cycle. In an alternative non-limiting example, the packaging unit may comprise means to send an electronic signal to a subject when a predefined time of administration (i.e. a certain day) is reached and the dosage unit for that time point is still contained in the packaging unit. In those examples, the electronic signal may be sent to a data storage means and / or sent to a user-defined electronic device, smartphones and smart wear begin illustrative examples hereof. In certain embodiments, distinct portions of the packaging unit(s) provide a different sensory trigger to a subject, non-limiting examples being distinct colors or roughness.

[0150] While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing description. Accordingly, it is intended to embrace all such alternatives, modifications, and variations as follows in the spirit and broad scope of the appended claims. The herein disclosed aspects and embodiments of the invention are further supported by the following non-limiting examples. The following specific experimental examples are provided in support of the claimed invention but are not to be seen as limiting the scope of the invention. EXAMPLES

[0151] Example 1: Clinical study to investigate the effect of different doses of E4 on endometrial thickness, ovarian suppression and the mean follicular diameter of the largest follicle in each ovary

[0152] This was a clinical study performed in healthy women of child-bearing potential to study the effect of E4 10 mg and 20 mg as monohydrate alone or combined with either progesterone (P4) or desogestrel (DSG) by daily oral administration for 28 days on ovarian suppression, the mean follicular diameter of the largest follicle in each ovary (mean of two directions) and the endometrial thickness. The latter was measured by TVUS during the pre- treatment period until ovulation, during the study treatment period, and at the follow-up visit. At each TVUS, endometrial thickness was measured.

[0153] MAIN CRITERIA FOR INCLUSION

[0154] • At least 18 years and not older than 40 years of age

[0155] • Willing to use a barrier method of contraception during the total study period.

[0156] • Ovulation in the pre-treatment cycle < day 24 (±1) after start of menses, with subsequent progesterone (P) concentration of > 16 nmol / L and with next menstruation > 6 days after ovulation

[0157] • Body Mass Index > 18 and < 30 kg / m2

[0158] • Good physical and mental health

[0159] • Both ovaries visible upon ultrasonography

[0160] • Willing to give informed consent in writing

[0161] MAIN CRITERIA FOR EXCLUSION

[0162] • Clinically significant abnormal results of hematology, biochemistry, urinalysis, or ECG

[0163] • Clinically significant abnormalities of the uterus and / or ovaries

[0164] • Clinically relevant abnormal cervical cytology < 1 year before study start

[0165] • Known or suspected breast cancer or a history of breast cancer

[0166] • Known or suspected pregnancy

[0167] • Lactation

[0168] • Status post-partum or post-abortion < 2 months before study start

[0169] • Previous use of depot progestogen preparations in the last 6 months

[0170] • Use of sex steroids other than the medication of study

[0171] • Contraindications for contraceptive steroids

[0172] Data were collected to evaluate the following variables:

[0173] • Follicle development and endometrial thickness were measured by transvaginal ultrasounds

[0174] • Hypothalamic -pituitary -ovarian (HPO) function was evaluated by measuring of LH, FSH, E2, P, and SHBG

[0175] • Safety and tolerability was evaluated by recording hematology, biochemistry, urinalysis, physical, gynecological and breast examinations, blood pressure (BP), heart rate (HR), body weight (BW), and objective and subjective side effects. A questionnaire was completed by the subjects about well-being during study medication use. STATISTICAL METHODS

[0176] No formal statistical testing was performed. Analysis of data is based on descriptive statistics.

[0177] Efficacy was evaluated by measurements of follicle development, endometrial thickness, laboratory assessments, and the PK analysis of E4.

[0178] Safety was evaluated by analysing data collected on hematology, biochemistry, urinalysis, physical, gynecological and breast examination, BP, HR, BW, and objective and subjective side effects.

[0179] RESULTS

[0180] Ovulation rate

[0181] A high ovulation rate (0.60) was observed in the E4 10 mg treatment group. The ovulation rate was lower in the E4 20 mg treatment group (0.30, Table 1).

[0182] Table 1. Ovulation rate (Efficacy population)

[0183] E4: estetrol monohydrate; E4 10 mg: estetrol monohydrate 10 mg, equivalent to estetrol 9.44 mg; E4 20 mg: estetrol monohydrate 20 mg, equivalent to estetrol 18.9 mg.aOvulation rate: number of ovulations per treatment group divided by the number of subjects in the Efficacy population.

[0184] The distribution of the Hoogland scores across the treatment groups is presented in Table 2. In 68.6% of the efficacy population, the Hoogland scores could be determined. The overall Hoogland score was higher in the E4 10 mg treatment group than in the E4 20 mg treatment group, with scores ranging from “ 1 : no activity” to “6: ovulation” in both groups.

[0185] Table 2. Distribution of Hoogland scores across the treatment groups (Efficacy population)

[0186] E4: estetrol monohydrate; E4 10 mg: estetrol monohydrate 10 mg, equivalent to estetrol 9.44 mg; E4 20 mg: estetrol monohydrate 20 mg, equivalent to estetrol 18.9 mg; FLS: follicle-like structure; LUF: luteinized unruptured follicle.

[0187] In Figure 1, the median actual change from baseline of luteinizing hormone (LH) levels is presented. The E4 -only groups showed an increase from baseline at each time point.

[0188] In Figure 2, the median actual change from baseline of the FSH levels is presented. The E4-only groups showed an increase from baseline at every time point, except for the E4 10 mg group on day 27.

[0189] In Figure 3, the median actual change from baseline of the E2 levels is presented.

[0190] In Figure 4, the median actual change from baseline of the P4 levels is presented.

[0191] Endometrial thickness

[0192] The mean endometrial thickness was higher in the E4 20 mg group than in the E4 10 mg group. In both of these groups, the maximum value of the mean endometrial thickness was observed on Day 21. On Day 21, the endometrium in the E4 10 mg group was 8.18 mm (i.e., an increase of 3.22 mm compared with Day 3) and in the E4 20 mg group, the thickness was 9.55 mm (i.e., an increase of 4.10 mm (Figure 5)). In terms of requirements for frozen embryo transfer (FET), estrogen supplementation aims to achieve an endometrial thickness of at least 7-8 mm by cycle day 14+ / -1 to optimize implantation rates. Both, E4 10 and 20 mg align well with these requirements, as the 7 mm threshold is achieved and surpassed by cycle day 15.

[0193] The mean of the individual maximum endometrial thickness measured during each treatment cycle was also higher in the E4 20 mg than in the E4 10 mg group (a mean thickness of 9.44 mm in the E4 10 mg group and of 11.03 mm in the E4 20 mg group) (Figure 6). RESULTS SUMMARY

[0194] • Both the ovulation rate and the follicular development according to the Hoogland scoring decreased with increasing dose in the E4-only groups.

[0195] • The mean maximum follicle size increased during treatment in the E4-only groups.

[0196] • The maximum endometrial thickness increased with increasing dose in the E4-only groups.

[0197] • In the E4-only groups LH concentrations increased more than in the 20 mg E4 / P4 group.

[0198] • For the E4-only groups, a relatively stable change in FSH from baseline with only a slight increase was observed, staying mostly below 2 U / L.

[0199] • In the E4-only groups E2 increased compared to the day 3 level, the increase being more pronounced in the 10 mg than in the 20 mg E4 group.

[0200] • The P levels increased from day 15 onwards in the E4-only groups in subjects who ovulated.

[0201] • The SHBG concentrations increased in all treatment groups. SHBG increased dose -dependently in the E4-only groups.

[0202] • A monotherapy of 10 mg or 20 mg E4 did not notably affect the SHBG concentration after the first 2 (±1) treatment days.

[0203] CONCLUSIONS

[0204] Both the ovulation rate and the follicular development according to the Hoogland scoring decreased with increasing dose in the E4-only groups. The maximum endometrial thickness increased with increasing dose in the E4-only groups.

[0205] Both the 10 mg and 20 mg E4-only groups show an LH peak around ovulation (Days 12-15), but the rise is moderate compared to a natural cycle, wherein the LH surge is typically much higher (10-75 IU / L). This suggests that while ovulation may occur, the hormonal response is milder due to the effects of E4 on the body's regulatory systems.

[0206] The FSH concentrations showed a small increase compared to the treatment day 3 level. In the E4-only groups, E2 increased compared to the treatment day 3 level, the increase being more pronounced in the 10 mg than in the 20 mg E4 group. The P4 levels increased from day 15 onwards in the E4-only groups in subjects who ovulated.

[0207] Estetrol was well tolerated and there was no evidence of any safety problem.

[0208] Example 2: A prospective, randomized, double blind, five-arm, dose finding Phase 2 study to evaluate the effect of different doses of estetrol (E4) on endometrial thickening in healthy reproductive age women

[0209] STUDY DESIGN

[0210] This is a prospective, randomized, double blind, five -arm, dose finding Phase 2 study in healthy reproductive age women. In total approximately 75 subjects (15 subjects per treatment arm) will be randomized into 5 treatment arms: estetrol monohydrate 5, 10, 15, 20 and 30 mg (E4 5 mg, E4 10 mg, E4 15 mg, E4 20 mg and E4 30 mg, respectively), equivalent to anhydrous estetrol 4.72, 9.44, 14.2, 18.9 and 28.3 mg, respectively. Subjects will receive the assigned E4 dose, orally, once a day, for 28 consecutive days. Starting from Day 14, P4 200 mg, orally, once a day will be started and continued for 15 consecutive days for endometrial maturation. Each subject will undergo a screening period, a pre-treatment cycle, a treatment period and a follow-up period. In case of current or recent use of hormonal contraception, the subjects will follow a washout before the start of the pre- treatment cycle. The study schedules are summarized in Figure 7.

[0211] STUDY OBJECTIVES

[0212] Primary objective:

[0213] To evaluate the effect of different doses of E4 on endometrial thickness to determine the E4 minimal effective dose (MED) to achieve an endometrial thickness of at least 7 mm after 14 days of treatment in healthy women of reproductive age.

[0214] Secondary objectives:

[0215] 1. To evaluate the effect of E4 on endometrial receptivity in healthy reproductive age women

[0216] 2. To evaluate the effect of E4 on ovulation.

[0217] 3. To evaluate the safety and tolerability of E4.

[0218] STUDY ENDPOINTS

[0219] Primary efficacy endpoint for primary objective:

[0220] Endometrial thickness >7 mm measured by transvaginal ultrasound (TVUS) at Day (D’)14 of menstrual cycle (of the treatment cycle).

[0221] Efficacy endpoints for secondary objective #1:

[0222] 1.1 Endometrial thickness measured by TVUS at D’20±lday of menstrual cycle.

[0223] 1.2 Endometrial volume measured by 3-dimension TVUS at D’20±lday of menstrual cycle.

[0224] 1.3 Endometrial pattern (triple-line) measured by TVUS at D’ 14±lday of menstrual cycle.

[0225] 1.4 Endometrial pattern (“luteinized endometrium”) measured by TVUS at D’20±lday of menstrual cycle

[0226] Efficacy endpoints for secondary objective #2:

[0227] 2.1 Follicle growth measured by TVUS at D3, D7, Dll, D14, D17, D20 and D28 of pre-treatment cycle and at D’3, D’7, D’ll, D’ 14, D’17, D’20 and D’28 of treatment cycle.

[0228] 2.2 Estradiol (E2), P4, follicle stimulating hormone (FSH) and luteinizing hormone (LH) plasma levels at D3, D7, Dll, D14, D17, D20 and D28 of pre-treatment cycle and at D’3, D’7, D’ ll, D’ 14, D’ 17, D’20 and D’28 of treatment cycle.

[0229] 2.3 Hoogland Score at D3, D7, Dll, D14, D17, D20 and D28 of pre -treatment cycle and at D’3, D’7, D’ ll, D’ 14, D’ 17, D’20 and D’28 of treatment cycle (9).

[0230] Safety endpoints for secondary objective #3 : 3.1 Frequency of treatment emergent adverse events (TEAEs) (including treatment -emergent serious adverse events [SAEs]).

[0231] 3.2 Vital signs and routine clinical laboratory tests results (haematology and chemistry).

[0232] SUBJECTS POPULATION

[0233] Description:

[0234] Healthy women of reproductive age, >18 to <45 years of age at randomization / treatment allocation, with regular menstrual cycle.

[0235] Number of subjects enrolled:

[0236] Approximately 75 subjects (15 subjects per treatment arm).

[0237] STUDY PRODUCTS

[0238] Investigational Medicinal Product (IMP):

[0239] The following doses of IMP will be administered orally once daily, at approximately the same time each day for 28 consecutive days:

[0240] Arm 1: estetrol monohydrate 5 mg (E4 5 mg), equivalent to anhydrous estetrol 4.72 mg

[0241] Arm 2: estetrol monohydrate 10 mg (E4 10 mg), equivalent to anhydrous estetrol 9.44 mg

[0242] Arm 3: estetrol monohydrate 15 mg (E4 15 mg), equivalent to anhydrous estetrol 14.2 mg

[0243] Arm 4: estetrol monohydrate 20 mg (E420 mg), equivalent to anhydrous estetrol 18.9 mg

[0244] Arm 5: estetrol monohydrate 30 mg (E4 30 mg), equivalent to anhydrous estetrol 28.3 mg

[0245] Auxiliary Medicinal Product (AxMP): P4 200 mg, orally, once daily in the evening at approximately the same time each day, from D’ 14, for 15 consecutive days.

[0246] TREATMENT AND STUDY DURATION

[0247] Treatment Duration:

[0248] The total treatment duration (E4+P4) for each subject will be 1 cycle (28 days) consisting of 14 days of treatment with E4 alone followed by 15 days of combined treatment with E4 and P4 (from D’ 14 evening to D’28).

[0249] Study Duration:

[0250] Individual subject participation may be up to 120 days.

[0251] This includes:

[0252] A screening period of up to 50 days (including a washout period of up to maximum 28 days),

[0253] A pre-treatment cycle of maximum 35 days,

[0254] A treatment period of 28 days,

[0255] A follow-up period of 7 days. INCLUSION CRITERIA

[0256] Subjects who meet all the following criteria are eligible for the study: Women aged >18 to <45 years at randomization / treatment allocation, who are not receiving any hormonal treatment, have a regular menstmal cycle and have a BMI >18.0 kg / m2and <30.0 kg / m2.

[0257] EXCLUSION CRITERIA

[0258] Subjects who have any of the following during the screening visit are not eligible for enrolment in this study: “Menstmal cycle length (after washout period, if applicable) shorter than 21 days or longer than 35 days, uterine abnormalities (e.g. fibroid tumors, endometrial polyps, etc.), history of surgical uterine curettage or any uterine surgery ,and acute or chronic pelvic inflammatory disease, endometriosis, and usual contra-indications to estrogen and progestogen use.”

[0259] VISIT SCHEDULE

[0260] The study schedules are summarized in Figure 7.

[0261] The study will include 16 visits that will consist of the following:

[0262] Screening visit: Visit (V)l

[0263] The screening visit will be conducted maximum 50 days before the start of the pre -treatment cycle. At this visit, the study will be explained to the subject and the subject information letter and informed consent form (ICF) will be provided to the subject. After signing the ICF, the inclusion and exclusion criteria will be checked, as well as the prior medication list. If a subject uses a prohibited co -medication, she will be asked to stop this medication and to start a washout after consultation with her physician if needed and in agreement with the Investigator. In case prohibited medication cannot be stopped, the subject will be considered as a screen failure. All screening procedures should be completed before the start of the pre-treatment cycle. During the screening visit the subject will be instmcted to call the study site on the first day of her first menstruation (DI) following the completion of the screening period / washout period. During this call the next visit will be scheduled (D3 evaluation visit of pre- treatment cycle).

[0264] Pre-treatment cycle evaluation visits:

[0265] V2 (D3+1 day), V3 (D7±l day), V4 (Dll±l day), V5 (D14+1 day), V6 (D17±l day), V7 (D20+lday), V8 (D28±l day)

[0266] At pre-treatment cycle evaluation D28 visit (V8), the subject will be randomized for the treatment group and will receive the study medication. The subject will be instmcted to call the study site on the first day of her next menstruation (D’ l) following the pre-treatment cycle (the site should be available for this call during weekend and official public holidays). During this call the final eligibility of the subject will be confirmed based on the length of her pre-treatment cycle. After eligibility is confirmed, the subject will be given the instruction to start the study medication (the IMP should be started not later than on the second day of menstmal cycle) and D’3 (Baseline) treatment cycle evaluation visit (V9) will be planned.

[0267] Treatment cycle evaluation visits: V9 (D’3+lday, Baseline visit), V10 (D’7±l day), Vll (D’ll±l day), V12 (D’14+1 day), V13 (D’17±l day), V14 (D’20±l day), V15 (D’28±lday)

[0268] At the D’ 14 treatment cycle evaluation visit (VI 0), the subjects will receive a blister containing P4200 mg tablets. They will be instmcted to start the intake in the evening and to continue the intake of both E4 and P4 orally daily for 14 days.

[0269] The D’28 end of treatment (EoT) visit (V15) will be planned.

[0270] End of study (EoS) / early termination (ET) visits: VI 6

[0271] The EoS visit will take place at the end of the follow-up period: 7(+2) days after EoT visit.

[0272] STATISTICAL METHODS

[0273] • Sample size determination: to be determined

[0274] • Statistical populations: details will be provided in the study protocol.

[0275] • Efficacy variables: Endometrial thickness, endometrial pattern (triple line), endometrial volume, size of the dominant follicle, E2, P4, FSH and LH plasma levels, Hoogland Score.

[0276] • Analysis of primary efficacy variables: Paired t-test if data are normally distributed or Wilcoxon test if data are non-normally distributed.

[0277] • Analysis of secondary efficacy variables: Paired t-test if data are normally distributed or Wilcoxon test if data are non-normally distributed.

[0278] • Safety parameters: adverse events (AEs), SAEs, hematology, biochemistry, urinalysis, vital signs.

[0279] • Analysis of safety parameters: for quantitative variables, paired t-test if data are normally distributed or Wilcoxon test if data are non-normally distributed.

[0280] • Handling of dropouts or missing data: details will be provided in the study protocol.

[0281] Example 3: Open label, single arm study in in vitro fertilization (IVF) population with frozen-thawed embryo transfer

[0282] STUDY RATIONALE

[0283] The preparation of the endometrium is a crucial step for successful embryo implantation during a frozen embryo transfer (FET) cycle. FET protocols include regular monitoring of endometrial thickness progression as it determines the doses and the timing for the administration of hormonal medications as well as the optimum window for the embryo transfer. In FET cycles, a decreased trend towards pregnancy outcomes, such as implantation rate, clinical pregnancy rate and subsequent live birth rate has been shown to be associated with a thin endometrium (thickness <7 mm).

[0284] Commonly applied hormone replacement therapy (HRT)-FET cycles are associated with an increased risk of hypertensive disorders of pregnancy (HDP), which include conditions like preeclampsia. This is due to several factors related to both the use of exogenous estrogen and the absence of a functional corpus luteum.

[0285] Elevated Estrogen in HRT-FET: o Exogenous estrogen, administered during HRT-FET cycles, disrupts the natural vascular remodelling of uterine spiral arteries, which is critical for healthy placental development and maternal cardiovascular adaptation. Proper vascular remodelling is essential for ensuring sufficient blood flow between the mother and the developing foetus.

[0286] • Absence of Corpus Luteum: o In natural cycles (NCs), the corpus luteum (formed after ovulation) produces progesterone and supports vascular remodelling. In HRT-FET cycles, the absence of the corpus luteum results in impaired vascular remodelling, which can contribute to a higher risk of HDP.

[0287] • Increased Risk of Hypertensive Disorders of Pregnancy (HDP) : o Women undergoing HRT-FET are at a higher risk of HDP, including preeclampsia, compared to those who conceived in natural cycles. One reason for this is the premature rise in estradiol, which can suppress the invasion of extravillous trophoblasts (cells responsible for anchoring the placenta to the uterus). This suppression negatively impacts placental development and increases the likelihood of HDP.

[0288] The following studies have been considered to elaborate on the above:

[0289] Fan et al. Hormone replacement treatment regimen is associated with a higher risk of hypertensive disorders of pregnancy in women undergoing frozen-thawed embryo transfer. Front Endocrinol (Lausanne). 2023.

[0290] Highlights the higher risks associated with HRT-FET protocols, particularly concerning HDP and caesarean delivery rates, emphasizing the need for careful consideration in clinical decisions.

[0291] It is explained that the absence of a functional corpus luteum leads to a lack of certain vasoactive factors, which are crucial for maternal cardiovascular adaptation during pregnancy. This absence contributes to an increased risk of HDP.

[0292] NC-FET and HRT-FET compare as follows in terms of singleton pregnancy outcomes:

[0293] • Hypertensive Disorders of Pregnancy (HDP): The incidence of HDP is significantly higher in the HRT-FET group (6.21%) compared to the NC-FET group (4.09%) with a p-value of 0.017, indicating a statistically significant difference.

[0294] • Caesarean Section: Caesarean deliveries are notably higher in the HRT-FET group (78.47%) compared to NC-FET (66.81%), with an odds ratio (OR) of 1.70, signifying a substantial increased likelihood.

[0295] • Preterm Birth and Extremely Preterm Birth: Both preterm birth (9.26% vs. 6.51%) and extremely preterm birth (1.18% vs. 0.38%) are more frequent in HRT-FET, with significant p- values (0.001 and 0.010, respectively).

[0296] Dalsgaard et al. Impact of frozen thawed embryo transfer in hormone substituted cycles on thrombotic risk markers. Thromb Res. 2022.

[0297] Explains the impact of HRT-FET cycles on thrombotic risk markers and thus risks linked to thrombotic events in HRT-FET cycles. Specifically, it compares thrombin generation between AC-FET (artificial cycle using estrogen substitution) and NC-FET (natural cycle), emphasizing the need for careful monitoring and possible interventions.

[0298] 1. AC-FET (E2 substitution): o There is a significant increase in thrombin generation (measured as nM / min) in AC- FET cycles compared to baseline (p < 0.0001) and compared to NC-FET (p = 0.001). o Markers affected include:

[0299] • Thrombin potential (ETP): Altered thrombotic activity increases.

[0300] • Fibrin: Impairs fibrinolysis (the breakdown of clots).

[0301] • Time to peak: Faster thrombin generation leads to quicker clot formation.

[0302] 2. Clinical Consequences: o Increased risk of thromboembolic events (VTE): Due to the pro -thrombotic state created in AC-FET cycles. o Pro-thrombotic state: The altered thrombin activity before pregnancy predisposes to clot formation. o Increased risk of Hypertensive Disorders of Pregnancy (HDP) : This is likely due to the baseline thrombotic conditions present before pregnancy.

[0303] Tao et al. Risks of Placenta Previa and Hypertensive Disorders of Pregnancy Are Associated With Endometrial Preparation Methods in Frozen-Thawed Embryo Transfers. Front Med (Lausanne). 2021

[0304] Shows that HRT-FET is linked to an increased risk of adverse obstetrical outcomes. The comparison is made between HRT-FET and ovarian stimulation Fresh ET (OS-FET) meaning cycles where the embryo is transferred as fresh.

[0305] In terms of consequences from HRT, they highlight the following:

[0306] 1. Caesarean Section Rates: o HRT-FET cycles had a higher rate of caesarean sections (84.3%) compared to OS-FET cycles (76.3%) with a statistically significant p-value (< 0.001).

[0307] 2. Pregnancy Outcomes: o Live Birth Rate (LBR): HRT-FET group had a lower LBR (76%) compared to OS-FET (80%) with a significant difference (p < 0.001). o Miscarriage Rates: Higher in the HRT-FET group (21.3%) compared to the OS-FET group (17.7%) (p < 0.001). o Birth Weight: Lower birth weights were observed in the HRT-FET group (2982.3 g) versus OS-FET (3025.0 g), with a significant p-value (< 0.001). o Small-for-Gestational-Age: Higher rates in OS-FET (8.7%) compared to HRT-FET (7.2%) (p < 0.001).

[0308] 3. Hypertensive Disorders of Pregnancy (HDP): o HDP incidence was significantly higher in the HRT-FET group (5.3%) compared to the OS-FET group (3.5%), with a p-value < 0.001.

[0309] Explanation of the above:

[0310] • The supra-physiological levels of estrogen and progesterone (E + P) in HRT may negatively affect placental development and endometrial receptivity.

[0311] • It may also influence trophoblast invasion and vascular remodeling, both critical for normal placentation and pregnancy outcomes.

[0312] It is expected that one or more of these disadvantages associated with HRT can be improved when E4 is used in the treatment of infertility by Frozen embryo transfer (FET) instead of estradiol or, more in particular, E2V.

[0313] DOSES

[0314] Subjects will receive the assigned E4 dose as determined in the study described in Example 2, orally, once a day, for at least 16 consecutive days. Starting from Day 14, P4 200 mg, orally, once a day will be started and continued for at least 3 consecutive days for endometrial maturation.

[0315] TREATMENT DURATION

[0316] 2 weeks before embryo transfer (ET), which can be prolonged up to 12 weeks after ET in accordance with assisted reproductive technology (ART) clinical practices

[0317] FOLLOW UP PERIOD

[0318] At least 1 week after the last dose of study medication

[0319] KEY INCLUSION CRITERIA

[0320] Women for whom HRT-FET is indicated.

[0321] AGE

[0322] >18 to <35years of age

[0323] KEY EXCLUSION CRITERIA

[0324] Conditions that may interfere with IVF success include fibroid tumours, ovarian dysfunction, abnormal hormone levels, and uterine abnormalities.

[0325] PRIMARY OBJECTIVE

[0326] To evaluate the effect of E4 on endometrial thickness in women undergoing an HRT-FET cycle. ENDPOINTS

[0327] Endometrial thickness measured by TVUS of at least >7 mm at Day 14 of menstrual cycle.

[0328] SECONDARY OBJECTIVES

[0329] 1. To evaluate the effect of E4 on the development of early pregnancy.

[0330] 2. To evaluate the effect of E4 on implantation rate.

[0331] 3. To evaluate the safety and tolerability of E4.

[0332] ENDPOINTS

[0333] Efficacy endpoints for secondary objective #1:

[0334] CPR confirmed by:

[0335] • hCG blood level at Day 10 after FET

[0336] • Presence of foetal sac in the uterus and foetal heartbeats detected on TVUS performed at 5 weeks of gestation.

[0337] Efficacy endpoint for secondary objective #2:

[0338] Embryo implantation rate defined as percentage of embryos that were transferred and developed at least to the stage of foetal heart activity documented by TVUS at 5 weeks of gestation.

[0339] Safety endpoints for secondary objective #3 :

[0340] • Frequency of TEAEs (including treatment- emergent SAEs)

[0341] • Vital signs and routine clinical laboratory tests results (haematology and chemistry)

[0342] EXPECTED OUTCOME

[0343] The use of E4 in the treatment of infertility by Frozen embryo transfer (FET) will have the following advantages compared to commonly applied hormone replacement therapy (HRT)-FET cycles with E2 or, more in particular, E2V and may even favorably compare to ovarian stimulation (OS)-FET in terms of the following:

[0344] Reduced risk of placenta previa and hypertensive disorders of pregnancy (HDP), which include conditions like preeclampsia;

[0345] Lower levels of estrogen and preferably no premature rise in estradiol;

[0346] Presence of corpus luteum;

[0347] Lower cesarian delivery rate;

[0348] Higher cumulative pregnancy / implantation rate;

[0349] Lower incidence of preterm and extremely preterm birth:

[0350] Lower impact on thrombotic risk markers such as thrombin generation, thrombin potential, fibrin and time to peak and thus lower risk of thrombotic events (venous thromboembolism (VTE)); thrombin activity before pregnancy is hardly influenced and the pro -thrombotic state is less pronounced leading to less predisposition to clot formation;

[0351] Higher live birth rate; Lower miscarriage rate;

[0352] Higher birth weight;

[0353] Lower rates of small-for-gestational-age.

Claims

43CLAIMS1. A composition comprising an effective amount of estetrol component for use in a Frozen Embryo Transfer (FET) fertility treatment in a subject, wherein the estetrol component is used to prepare the endometrium for receiving and / or implanting the embryo.

2. A method of Frozen Embryo Transfer (FET) fertility treatment in a subject, wherein the method comprises a step of administering a composition comprising an effective amount of estetrol component to said subject and a step of transferring an embryo into said subject, wherein the estetrol component is used to prepare the endometrium for receiving and / or implanting the embryo.

3. Use of a composition comprising an effective amount of estetrol component for the manufacture of a medicament for use in Frozen Embryo Transfer (FET) fertility treatment in a subject, wherein the estetrol component is used to prepare the endometrium for receiving and / or implanting the embryo.

4. Use of a composition comprising an effective amount of estetrol component in a Frozen Embryo Transfer (FET) fertility treatment in a subject, wherein the estetrol component is used to prepare the endometrium for receiving and / or implanting the embryo.

5. The composition for use according to claim 1, the method according to claim 2, or the use according to claim 3 or 4, wherein preparing the endometrium of a subject for receiving and / or implanting the embryo is bringing said endometrium into a receptive state for receiving and / or implanting the embryo.

6. The composition for use, the method, or the use according to any one of claims 1 to 5, wherein a daily amount of estetrol component from about 5 to about 30 mg, preferably from about 10 to about 20 mg and most preferably 15 mg is administered to the subject.

7. The composition for use, the method, or the use according to any one of claims 1 to 6, wherein the estetrol component is estetrol, preferably estetrol monohydrate.

8. The composition for use, the method, or the use according to any one of claims 1 to 7, wherein said composition is administered for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive days.

9. The composition for use, the method, or the use according to any one of claims 1 to 8, wherein the estetrol component is administered from the first day of the menstrual cycle or from the second day of the menstrual cycle.

10. The composition for use, the method, or the use according to any one of claims 1 to 9, wherein the composition is orally administered or wherein the composition is formulated as an oral composition.

11. The composition for use, the method, or the use according to any one of claims 1 to 10, wherein the treatment additionally comprises the administration of one or more progestogens, preferably selected from the group consisting of: progesterone, medroxyprogesterone acetate, and dydrogesterone.

12. The composition for use, the method, or the use according to claim 11, wherein the progesterone, medroxyprogesterone acetate, and / or dydrogesterone are orally administered, vaginally administered, or administered by intramuscular injection.4413. The composition for use, the method, or the use according to claim 11 or 12, wherein said progestogen is administered on the same day as the estetrol component for at least 3, 4, 5, or 6 consecutive days.

14. The composition for use, the method, or the use according to any one of claims 11 to 13, wherein the administration of the progestogen is initiated after at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive days of estetrol component administration, preferably after 13 consecutive days of estetrol component administration.

15. The composition for use, the method, or the use according to any one of claims 11 to 14, wherein an embryo is transferred after at least 2, 3, or 4 days of combined administration of the estetrol component and the progestogen.

16. The composition for use, the method, or the use according to any one of claims 11 to 15, wherein administration of the estetrol component is continued daily for up to 12 weeks post embryo transfer.

17. A pharmaceutical kit for use in the treatment of infertility by Frozen embryo transfer (FET) as defined in any one of claims 1 to 16, said kit comprising a parenteral or oral dosage unit containing an effective amount of an estetrol component and an oral and / or vaginal dosage unit containing an effective amount of progestogen.

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