Fertilization of the mammalian egg is preceded by the maturation of the oocyte. This maturation process in mammalian oocytes includes nuclear and cytoplasmic changes. The first meiotic division from prophase I to metaphase 11 is reinitiated and completed. Only little is known about the mechanism which controls the initiation of meiosis. The presence of a diffusible meiosis regulating substance was first described by Byskov et a/. in a culture system of fetal mouse gonads [Byskov, A. G. et aL Dev Biol 52 (1976) 193-200]. A meiosis activating substance (MAS) was secreted by the fetal mouse ovary in which meiosis was ongoing, and a meiosis preventing substance (MPS) was released from the morphologically differentiated testis with resting, non-meiotic germ cells. It was suggested that the relative concentrations of MAS and MPS regulated the beginning, arrest and resumption of meiosis in the male and in the female germ cells (Byskov, A. G. et al. in The Physiology of Reproduction [eds. Knobil. E. and Neill, J. D., Raven Press, New York (1994)]. Certain sterols (T-MAS and FF-MAS) that activate oocyte meiosis have been isolated from bull testes and from human follicular fluid [Byskov, A. G. et al. Nature 374 (1995), 559-562].

In in vitro feritilization (IVF) experiments with mouse oocytes, FF-MAS has been shown to increase the fertilization rate when added to the culture medium (Hegele-Hartung, C et al., Hum Reprod 13 (suppl) : 98 (1998)). The first clinical data with human oocytes showed an improvement in nuclear and cytoplasmic maturation (Grndahl, C et al, 1999, Proceedings from The Second International Alpha Congress). Unfortunately, FF-MAS is rather labile.

Therefore, stable synthetic analogues were developed. Such compounds being known to regulate the meiosis are described in the International patent applications WO 96/00235, WO 96/27658, W097/00884, W098/28323, W098/52965 and WO 98/55498.

There is strong evidence that FF-MAS and its analogues increase fertilization rate when added to oocytes or/and IVF culture media. However, as the IVF is a rather lengthy, expensive method, many women, who undergo infertility treatment, would prefer an in vivo treatment. Therefore, it would be desirous to have a medicament for in vivo use to increase pregnancy rate. This could be done by increasing the rate of preimplantational embryo development and the implantation rate.

The present invention provides a method to increase the implantation rate of oocytes in females, preferably humans. The present invention relates to the use of FF-MAS and/or one

or more FF-MAS analogues for the preparation of a medicament that increases the implantation rate of preimplantational embryos. FF-MAS is 4,4-dimethyl-5a-cholesta-8,14,24- triene-3ß-ol. Its synthesis is described in W099/52930. FF-MAS analogues and their synthesis are described in e. g. WO 96/00235, WO 96/27658, W097/00884, W098/28323, W098/52965 and WO 98/55498. FF-MAS analogues have a percentage germinal vesicle breakdown (GVB) which is significantly higher than the control. Preferred FF-MAS analogues are such having a percentage GVB of at least 50%, preferably at least 80%. Implantation rate means the rate by which a preimplantational embryo is able to interact with the endometrium thereby giving rise to the development of a postimplantational embryo and a fetus.

When given to a woman before and during ovulation, FF-MAS and its analogues increase the implantation rate by at least 20%. More preferably, the implantation rate is increased by at least 50 %.

Due to the increase in the implantation rate, the pregnancy rate will also be higher. The medicament of this invention can therefore be used to treat females who have problems with infertility. It may also be used to increase the implantation rate in animals which is important in animal breeding.

A further object of the present invention are pharmaceutical compositions comprising FF- MAS and/or one or more FF-MAS analogues. The compositions may further comprise pharmaceutically acceptable excipients well known in the art like carriers, diluents, absorption enhancers, preservatives, buffers, agents for adjusting the osmotic pressure, tablet disintegrating agents and other ingredients which are conventionally used in the art.

Examples of solid carriers are magnesium carbonate, magnesium stearate, dextrin, lactose, sugar, talc, gelatin, pectin, tragacanth, methylcellulose, sodium carboxymethyl cellulose, low melting waxes and cacao butter.

Liquid compositions include sterile solutions, suspensions and emulsions. Such liquid compositions may be suitable for injection. The liquid compositions may contain other ingredients which are conventionally used in the art, some of which are mentioned in the list above. Further, a composition for transdermal administration of FF-MAS and/or one or more FF-MAS analogues may be provided in the form of a patch and a composition for nasal administraton may be provided in the form of a nasal spray in liquid or powder form.

The dose of FF-MAS and/or one or more FF-MAS analogues to be used will be determined by a physician and will depend among several factors on the particular compound employed

and on the route of administration. In general, the compositions of the invention are prepared by intimately bringing into association the active compound with the liquid or solid auxiliary ingredients and then, if necessary, shaping the product into the desired formulation.

Usually, not more than 1000 mg, preferably not more than 100 mg, and in some preferred instances not more than 10 mg of FF-MAS and/or one or more FF-MAS analogues is to be administered to mammals, e. g. to humans, per day. Treatment may either be continuously or intermittent. Intermittent treatment preferably starts before the ovulation and is continued until some days after ovulation.

The route of administration of FF-MAS and/or one or more FF-MAS analogues may be any route which brings the active compound (s) to its or their site of action. Examples for routes of administration are intravenous, subcutaneous, oral, intranasal or transdermal administration.

The present invention will be illustrated in detail in the following examples.

Example 1: Method used for electing MAS compounds Oocytes were obtained from immature female mice (C57BI/6J x DBA/2J F1-hybrids, Bomholtgaard, Denmark) weighing 13-16 grams, that were kept under controlled lighting and temperature. The mice received an intra-peritoneal injection of 0.2 mi gonadotropins (Gonal F, Serono, Solna, Sweden, containing 20 IU FSH, alternatively, Puregon, Organon, Swords, Ireland containing 20 IU FSH) and 48 hours later the animals were killed by cervical dislocation.

The ovaries were dissected out and the oocytes were isolated in Hx-medium (see below) under a stereo microscope by manual rupture of the follicles using a pair of 27 gauge needles. Spherical, naked oocytes (NO) displaying an intact germinal vesicle (GV) were placed in a-minimum essential medium (a-MEM without ribonucleosides, Gibco BRL, Cat. No. 22561) supplemented with 3 mM hypoxanthine (Sigma Cat. No. H-9377), 8 mg/mi Human Serum Albumin (HSA, State Serum Institute, Denmark), 0,23 mM pyrubate (Sigma, Cat. No. S-8636), 2 mM glutamin (Flow Cat. No. 16-801), 100 IU/ml penicillin and 100 IJg/ml streptomycin (Flow, Cat No. 16-700). This medium was designated Hx-medium.

The oocytes were rinsed three times in Hx-medium and cultured in 4-well multidishes (Nuncion, Denmark) in which each well contained 0.4 ml of Hx-medium and 35-45 oocytes.

One control (i. e. 35-45 oocytes cultured in Hx-medium with no addition of test compound) was always run simultaneously with the test cultures, which were made with different concentrations of the compounds to be tested.

The cultures were performed at 37 °C and 100 % humidity with 5 % C02 in air. The culture time was 22-24 hours.

By the end of the culture period, the number of oocytes with germinal vesicle (GV) or germinal vesicle breakdown (GVB) and those with polar body (PB) was counted using a stereo microscope or an inverted microscope with differential interference contrast equipment. The percentage of oocytes with GVB per total number of oocytes and the percentage of oocytes with PB per total number of oocytes was calculated in the test cultures and compared to the control culture.

%GVB= ( (number of GVB + number of PB)/total number of oocytes) x100

Example 2: Improvement of embryo implantation with FF-MAS and FF-MAS succinate in the cycling rat A total of 15 sexually mature female Wistar rats with a body weight of 200-220g and a 4-day cycle were used. The animals were divided in one control group (group 1) and two treatment groups (group 2-3) consisting of 5 animals/group, respectively.

Starting in metestrus (d1) animals were treated for eight days (d1-d8), once daily, subcutaneously with the test compounds (treatment groups) or vehicle (control group), respectively. At day 7, at proestrus, animals were mated. The success of mating was checked by the presence of sperms in the vagina at d8. At d16 animals were sacrificed. Uteri were removed and checked for the presence of implantation sides. In each animal the number of implantation sides were determined.

Control group 1 received vehicle alone [ethanol/arachisoil, 1+9 (v/v)].

Group 2 received 20 mg/kg FF-MAS, prepared in an ethanol/arachisoil vehicle.

Group 2 received 20 mg/kg FF-MAS-succinate, prepared in an ethanol/arachisoil vehicle.

The results are presented in Table 1.

Table 1: Effect of subcutaneous FF-MAS and FF-MAS-succinate treatment on implantation rate in the adult, cycling rat Group Treatment schdule Number of implantation sides[xssd] d1*-d8 d7 d16 Treatment Mating [s. c.] 1 Vehicle + 10 4 2 20 mg/kg + 15 3 FF-MAS 320 mg/kg + 12 2 FF-MAS- succinate * treatment starts at metestrus