ZA200104653B

ZA200104653B - Treatment of infertility.

Info

Publication number: ZA200104653B
Application number: ZA200104653A
Authority: ZA
Inventors: Christian Grondahl
Original assignee: Novo Nordisk As
Priority date: 1999-02-24
Filing date: 2001-06-07
Publication date: 2002-06-07

Description

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TREATMENT OF INFERTILITY

FIELD OF THIS INVENTION

This invention relates to an improved method of in vitro fertilisation (hereinafter des- ignated IVF).

BACKGROUND OF THIS INVENTION

Since the first IVF pregnancy was delivered in 1978, this procedure has resulted in thousands of pregnancies and opened a vast new frontier of research and treatment for the infertile couples. Still, there is a significant need for improved infertility treat- ment modalities today. It is presumed that about one out of seven couples experi- ence problems with subfertility or infertility.

IVF of human oocytes has become commonly used for the treatment of female and male subfertility. The standard IVF treatment includes a long phase of hormone stimulation of the female patient, e.g. 30 days, which is initiated by suppressing the patient's own follicle stimulating hormone (hereinafter designated FSH) and luteinis- . 20 ing hormone (hereinafter designated LH) by gonadotropin releasing hormone (here- inafter designated GnRH), and this is followed by injections of exogenous gonad- . otropins, e.g. FSH and/or LH, in order to ensure development of multiple preovula- tory follicles and aspiration of multiple in vivo matured oocytes immediately before ovulation. The aspirated oocyte is subsequently fertilised in vitro and cultured, typi- cally for three days before transferral back into the uterus at the 4-8 cell stage. Con- tinuous efforts have been made to optimise and simplify this procedure. Neverthe- less, the overall pregnancy rate cannot be increased significantly over about 20% with the current treatment modalities. In a large European survey of IVF patients, it was found that 7.2 oocytes out of 11.5 aspirated oocytes per patient had undergone resumption of meiosis immediately before fertilisation, only 4.3 oocytes were fertil- ised and only 2.2 oocytes reached the 8-cell embryo stage after fertilisation and in vitro culture (ESHRE, Edinburgh, 1997).

SUBSTITUTE SHEET (RULE 26)

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Due to the very unpredictable quality of the state of the art embryos today, more than one embryo has to be transferred just to give a reasonable chance of success.

Therefore, it is common to transfer 2-3 embryos (up to 5 embryos in some countries), which carries the very large side effect of multiple pregnancies with great discomfort and risk to both patient and children. Moreover, it has been estimated that the in- creased health care expenses due to multiple birth (twins, triplets etc.) is exceeding the entire IVF expenses.

Hence, there are several disadvantages with the current treatment, the four most no- table being: 1. the risk of ovarian hyperstimulation with injecting gonadotropins which is a po- tential fatal condition that requires hospitalisation, 2. multiple pregnancies (50-1.000 times the normal frequency of twins and trip- lets, respectively), 3. the existence of considerable patient segments that do not tolerate the current method due to, e.g. polycystic ovarian syndrome and many diabetics, and 4. a potential long-term cancer risk.

Furthermore, weight gain, bloating, nausea, vomiting, labile mood and other patient discomforts together with patient reluctance to inject themselves are reported as dis- . advantages.

It is known from WO 96/00235 that certain sterol derivatives can be used for regulat- ing meiosis. An example of such a sterol is 4,4-dimethyl-5a-cholesta-8,14,24-triene- 3p-ol (hereinafter designated FF-MAS).

Herein, the term MAS compounds designates compounds which mediate the meiosis of oocytes. More specifically, MAS compounds are compounds which in the test de- scribed in Example 1 below has a percentage germinal vesicle breakdown (hereinaf- 3c terdesignated GVB) which is significantly higher than the control. Preferred MAS compounds are such having a percentage GVB of at least 50%, preferably at least 80%.

SUBSTITUTE SHEET (RULE 26)

Lo 3 .

Examples of MAS compounds are mentioned in WO 96/00235, 96/27658, 97/00884, 98/28323, 98/54965 and 98/55498, more specifically in Claim 1 thereof.

In WO 95/000265, some potential meiosis regulating substances were tested on im- mature female mice. 48 hours before the test animal were killed by cervical disloca- tion, they were given a single injection of human menopausal gonadotropin contain- ing 20 IU FSH and 20 IU LH. The ovaries were removed, placed in a hypoxanthine medium and freed of extraneous tissue. Then, the oocytes were punctured out of the follicles, freed from cumulus cells and cultured in a medium containing a meiosis regulating derivative.

At present, in vitro maturation in humans has proven highly unsuccessful despite substantial interest and clinical efforts.

One object of the present invention is to treat human infertility.

Another object of the present invention is to improve the maturation of her human oocytes.

Another object of the present invention is to improve the synchrony of nu- . 20 clear, cytoplasmic and/or membranous oocyte maturation.

Another object of the present invention is to improve the fertility of oocytes. . Another object of the present invention is to improve the rate of implantation of oocytes by human in vitro maturation and fertilisation.

Another object of the present invention is to diminish the incidence of human preembryos with chromosome abnormalities (aneuploidy).

Another object of the present invention is to improve the cleavage rate of human preembryos.

Another object of the present invention is to improve the quality of human preembryos. :

SUBSTITUTE SHEET (RULE 26)

4 ad

SUMMARY OF THIS INVENTION

It has now, surprisingly, been found that the IVF treatment and the degree of side effects can be improved substantially if the woman, within a consecutive period of 30 days, avoids treatment with a hypothalamic hormone and/or a pituitary hormone or an agonist or antagonist thereof or an active derivative thereof (hereinafter this treatment is designated exogeneous stimulation) or if the exogeneous stimulation treatment of the female is only for a short period of time, e.g. less than 7 days, pref- erably less than 4 days. Using this improved method involving less or no exogene- ous stimulation, a MAS compound is used to actively mature and synchronise human oocytes in vitro, leading to fertilisation and embryo development.

Briefly, the present invention relates to a method for human in vitro fertilisation wherein a woman, within a consecutive period of 30 days, is treated with a hypotha- lamic hormone and/or a pituitary hormone or an agonist or antagonist thereof or an active derivative thereof for a period which is less than about 7 days, preferably less than about 4 days, and, thereafter, using in vitro occyte maturation wherein immature egg or eggs are retrieved from the woman and are in vitro matured in a synchronize manner using a MAS compound as defined herein. Preferred embodiments of this invention are those stated in the sub claims below. .

DETAILED DESCRIPTION OF THIS INVENTION

Referring to the female cycle, one way of performing the IVF treatment of this inven- tion is as follows:

Around days 6-9 in the cycle: Stimulation with FSH, e.g. 75-600 IU per day, prefera- bly 150-225 IU per day, e.g. for 3 days. :

SUBSTITUTE SHEET (RULE 26)

Around day 9: The eggs are retrieved from the woman using ultrasound guided aspi- ration of small to medium size follicles with a diameter of about 6-12 mm, preferably 8-10 mm.

Around day 9-11: The eggs are maturated with a MAS compound in order to stimu- late the meiosis. In this maturation step, the concentration of MAS compound may be in the range of about 0.1-100 pmol per litre, e.g. 10-20 pmol per litre. This medium may contain human serum albumin (hereinafter designated HSA), e.g. 0.8 %, and it may additionally contain some ethanol, e.g., 0.4%, which has been used to dissolve

MAS. The time for this maturation step may be in the range around 15-60 hours, e.g., about 22-40 hours, more specifically about 30-36 hours.

Around days 11-13: The eggs are fertilised in vitro.

Around days 12-16: The eggs are cultured in vitro in a suitable medium.

From the day before aspiration, the woman will receive an oestrogen, e.g. oestrogen valerate (2 x 10 mg daily). Two days later, she will also receive a progestogen, e.g.,

Progestane vagetoria, daily, which will render the lining of the uterus more prone to . 20 receive the future embryos. The duration of this treatment will be individually de- signed per patient. The doctor can chose among a variety of oestrogens and pro- . gestogens.

Around day 15-16: One or more embryos are transferred to the woman's uterus.

Hence, all in all, the complete treatment takes about 10-15 days.

Referring to the female cycle, another way of performing the IVF treatment of this in- vention is as follows: :

Around days 2-8 in the cycle: Stimulation with FSH, e.g. 75-600 IU per day, prefera- bly 150-225 IU per day, e.g. for 3 days, eventually spread over 6 days.

SUBSTITUTE SHEET (RULE 26)

6 Rand

Around day 7-9: The eggs are retrieved from the woman using ultrasound guided aspiration of small to medium size follicles with a diameter of about 6-15 mm, pref- erably 8-12 mm.

Around day 7-11: The eggs are maturated with a MAS compound in order to stimu- late the meiosis. In this maturation step, the concentration of MAS compound may be in the range of about 0.01-100 umol per litre, e.g., 5-20 nmol per litre. This medium may contain human serum albumin (hereinafter designated HSA), e.g. 0.8%, and it may additionally contain some ethanol, e.g., 0.1-0.4%, which has been used to dis- solve MAS. The time for this maturation step may be in the range around 15-60 hours, e.g., about 22-40 hours, more specifically about 30-36 hours.

Around days 9-13: The eggs are fertilised in vitro. 156

Around days 10-16: The eggs are cultured in vitro in a suitable medium.

From the day before aspiration, the woman will receive an oestrogen, e.g. oestrogen valerate (2 x 10 mg daily). Two days later, she will also receive a progestogen, e.g., Progestane vagetoria, daily, which will render the lining of the uterus more prone to . receive the future embryos. The duration of this treatment will be individually de- signed per patient. The doctor can chose among a variety of oestrogens and pro- : gestogens.

Around day 13-16: One or more embryos are transferred to the woman's uterus.

Hence, all in all, the complete treatment takes about 10-15 days.

Most of the steps in the above treatment and procedure are performed in a known 3¢ manner and the remaining steps are performed in a manner known per se. More de- tails about the removal of the oocytes from follicles in the ovary, culturing of the iso- lated oocytes, the cuiture medium to be used, the fertilisation with sperm, and the

SUBSTITUTE SHEET (RULE 26)

Yt 7 - transfer of the embryo to the fallopian tube can be found in the literature, for exam- ple, in US patent specification No. 5,693,534 which is hereby incorporated by refer- ence.

According to this invention, the MAS compound is added to the culture me- dium used. In this medium, the amount of the MAS compound is in the range from about 0.01 to about 100 uM, preferably in the range from about 0.1 to about 100 uM.

The reduced risk of side effects makes the method according to the present inven- tion an attractive alternative to the current methods where GnRH is used for about 22 days and FSH is used for about 9 days before the eggs are retrieved and, thereafter, a progestogen is used for several weeks. Hence, using the treatment according to the present invention, the period in which the female patient is treated with a hypo- thalamic hormone and/or a pituitary hormone is reduced by about 80-90%. The total period of treatment by the current methods is about 4 weeks. Hence, using the treatment according to the present invention, the total period of treatment is reduced by about 50-60%.

Hypothalamic hormones are hormones present in the human hypothalamus. Pituitary hormones are hormones present in the human pituitary gland. Gonadotropic hor- . 20 mones are hormones secreted by the anterior lobe of the pituitary in vertebras and by mammalian placenta, which control the activity of gonads. Chemically, they are : glycoproteins. Examples of gonadotropic hormones are FSH, LH and chorion go- nadotropin, e.g. human chorion gonadotropin (hereinafter designated hCG). FSH stimulates growth of ovarian follicles and their oocytes in ovary and the formation of spermatozoa in testis. FSH can, e.g., be menopausal FSH or recombinant FSH. In females, LH activates the oestrogen-producing tissue of the ovaries to produce pro- gesterone, probably promotes the final stages of the development of ovarian follicles, initiates the final oocyte maturation, induces ovulation and in mammals initiates cor- pus luteum development. These hormones are known. It is obvious for the skilled art worker that, alternatively, agonists or antagonists of these hormones can be used. It is also obvious for the skilled art worker that, alternatively, active analogues of these hormones can be used. Some of these agonists, antagonists and analogues are

SUBSTITUTE SHEET (RULE 26)

8 ded known and other can be prepared by process known per se. Examples of such known processes are chemical synthesis and genetic engineering.

In a preferred embodiment, the present invention relates to a method or use wherein the period in which said woman is treated with a hypothalamic hormone and/or a pi- tuitary hormone or an agonist or antagonist thereof or an active derivative thereof is 0 (zero) days.

In a further preferred embodiment, the present invention relates to a method or use wherein the woman is treated for infertility, and/or for improving the maturation of her oocytes, and/or for improving the synchrony of nuclear, cytoplasmic and/or membra- nous oocyte maturation, and/or for improving the fertility of her oocytes, and/or for improving the rate of implantation of her oocytes by human in vitro maturation and fertilisation.

In a further preferred embodiment, the present invention relates to a method or use wherein the consecutive period is one menstrual cycle.

In a further preferred embodiment, the present invention relates to a method or use wherein the hormones are gonadotropic releasing hormones or an agonist or an- : tagonist thereof or analogues thereof or gonadotropic hormones or an agonist or an- tagonist thereof or analogues thereof.

In a further preferred embadiment, the present invention relates to a method or use wherein the gonadotropic hormone is FSH or an agonist or antagonist thereof or analogues thereof.

In a further preferred embodiment, the present invention relates to a method or use wherein the period in which the female patient is freated with FSH or an agonist or 3¢ antagonist thereof or analogues thereof, prior to the retrieval of the egg, is less than 7 days, preferably less than 4 days, and is at least 1 day.

SUBSTITUTE SHEET (RULE 26)

bd 9 - in a further preferred embodiment, the present invention relates to a method or use wherein the period in which the female patient is treated with FSH or an agonist or antagonist thereof or analogues thereof is 2, 3 or 4 days.

In a further preferred embodiment, the present invention relates to a method or use wherein no chorion gonadotropin, e.g. human chorion gonadotropin or an agonist or antagonist thereof or analogues thereof is used.

In a further preferred embodiment, the present invention relates to a method or use wherein no gonadotropic releasing hormone, e.g. GnRH, or an agonist or antagonist thereof or analogues thereof is used.

In a further preferred embodiment, the present invention relates to a method or use wherein the dosage of MAS compound is in the range from about 0.01 pM per litre to about 100 uM per litre, preferably in the range from about 0.1 uM per litre to about 100 uM per litre. in a further preferred embodiment, the present invention relates to a method or use wherein the MAS compound is one of the compounds mentioned in WO 96/00235, . 20 96/27658, 97/00884, 98/28323, 98/54965 and 98/55498, more specifically com- pounds mentioned in Claim 1 thereof.

In a further preferred embodiment, the present invention relates to a method or use wherein the MAS compound is FF-MAS.

Additionally, the present invention relates to the use of a hypothalamic hormone and/or a pituitary hormone or an agonist or antagonist thereof or an active derivative thereof in the manufacture of a hormone product which is to be administered to a woman who, within a consecutive period of 30 days, is treated with a hypothalamic hormone and/or a pituitary hormone or an agonist or antagonist thereof or an active derivative thereof for a period which is less than about 7 days, preferably less than about 4 days, and from whom, immediately after said period, one or more oocytes

SUBSTITUTE SHEET (RULE 26)

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Ras are aspirated, where after said oocyte(s) is/are cultivated in a convenient medium containing a MAS compound as defined herein, where after said oocyte(s) is/are fer- tilised with human sperm, and, where after, the resulting embryo(s) is/are transferred to a woman.

Additionally, the present invention relates to use of a hypothalamic hormone and/or a pituitary hormone or an agonist or antagonist thereof or an active derivative thereof and of a MAS compound for the manufacture of a medicament for the treatment of human in vitro fertilisation wherein a hypothalamic hormone and/or a pituitary hor- 10 mone or an agonist or antagonist thereof or an active derivative thereof is, within a consecutive period of 30 days, used to treat a women for a period which is less than about 7 days, preferably less than about 4 days, and, thereafter, the MAS compound is used in an in vitro oocyte maturation of the egg or eggs retrieved from this woman.

Additionally, the present invention relates to a pharmaceutically kit in unit dosage form for use by in vitro fertilisation comprising 1-8 separate unit dosages, said kit comprising less than 7, preferably less than 4, and at least 1 separate dosage units for sequential daily administration of a hypothalamic hormone and/or a pituitary hor- mone or an agonist or antagonist thereof or an active derivative thereof for sequen- tial daily administration and 1 dosage units of a MAS compound. This kit may have - the preferred features described above.

The present invention is further illustrated by the following examples, which, how- ever, are not to be construed as limiting. The features disclosed in the foregoing de- scription, in the following examples and in the claims may, both separately and in any combination thereof, be material for realising the invention in diverse forms thereof.

Example 1

Method used for electing MAS compounds

SUBSTITUTE SHEET (RULE 26)

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Oocytes were obtained from immature female mice (C57BL/6J x DBA/2J F1, Bom- holtgaard, Denmark) weighing 13-16 grams, that were kept under controlled tem- perature (20-22 °C), light (lights on 06.00-18.00) and relative humidity (50-70%). The mice received an intra-peritoneal injection of 0.2 ml gonadotropins (Gonal-F, Serono) containing 20 IU FSH and 48 hours later the animals were killed by cervical disloca- tion. The ovaries were dissected out and the oocytes were isolated in Hx-medium (see below) under a stereomicroscope by manual rupture of the follicles using a pair of 27 gauge needles. Spherical oocytes displaying an intact germinal vesicle (here- inafter designated GV) were divided in cumulus enclosed oocytes (hereinafter desig- nated CEO) and naked oocytes (hereinafter designated NO) and placed in o- minimum essential medium (a-MEM without ribonucleosides, Gibco BRL, Cat. No. 22561) supplemented with 3 mg/ml bovine serum albumin (BSA, Sigma Cat. No. A- 7030), 5 mg/ml human serum albumin (HSA, Statens Seruminstitut, Denmark), 0.23mM pyruvate (Sigma, Cat. No S-8636), 2 mM glutamine (Flow Cat. No. 16-801), 100 IU/ml penicillin and 100 pg/ml streptomycin (Flow, Cat No. 16-700). This medium was supplemented with 3 mM hypoxanthine (Sigma Cat. No. H-9377) and desig- nated Hx-medium. The oocytes were rinsed three times in Hx-medium and oocytes of uniform size were divided into groups of CEO and NO. CEO and NO were cultured in 4-well multidishes (Nuncion, Denmark) in which each well contained 0.4 ml of Hx- . 20 medium and the compound to be tested in a concentration of 10 uM. One control well (i.e., 35-45 oocytes cultured in identical medium with no addition of test com- pound) was always cultured simultaneously with 3 test wells (35-45 oocytes per well supplemented with test compound). The oocytes were cultured in a humidified at- mosphere of 5% CO, in air for 24 hours at 37°C. By the end of the culture period, the number of oocytes with GV, GVB and polar bodies (hereinafter designated PB), re- spectively, were counted using a stereo microscope (Wildt, Leica MZ 12). The per- centage of GVB, defined as percentage of oocytes undergoing GVB per total number of oocytes in that well, was calculated as: % GVB = ((number of GVB + number of PB)/ total number of oocytes) X 100.

SUBSTITUTE SHEET (RULE 26)

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Example 2

Patients for in vitro fertilisation (IVF) normally undergo a long (4 weeks) gonadotropin based protocol that leads to the aspiration of in vivo matured oocytes. These oocytes are subsequently fertilised in vitro and replaced as 4-8 cell embryos to the uterus of the patient by a cervical catheter.

In vitro maturation with FF-MAS

Procedure

All IVF patients can potentially receive this treatment, age range 20 to 45 year with or without displaying Polycystic ovarian syndrome (PCO) and with or without a regular cycle. In the case of irregular cycle or amemorhea (no cyclic activity) this procedure could be preceded with oral contraceptive for various lengths (1-10 month) and with- drawn upon initiation of the following procedure. In the beginning (day 1-6, prefera- bly day 3-6) of the cycle (day 1 = 1° day of menses), the patient will be clinically ex- amined and may or may not receive a small priming FSH stimulus individually de- signed for each patient (length: 1-7 days, doses: 75 IU to 750 |U, preferentially 3-4 days with doses 150 to 300 IU recombinant or urinary based FSH) with or without the use of GNRH antagonist and with or without hCG. Small to medium size follicles : (size: 4 to 16 mm, preferential 8 to 12 mm follicles) will be aspirated under ultrasound guidance using a low/reduced suction pressure and specially designed (more rigid) : needles. The aspirated fluid will be searched for cumulus oocytes complexes (COC) and once identified under the stereomicroscope (with or without the use of embryo fiiters), the COC will be placed in culture. A wide variety of oocyte culture media or media components known to the skilled worker can be used, however the oocytes will be induced to resume meiotic maturation by exposure to FF-MAS. Human serum albumin (HSA) may or may not be added to the medium. If added, it can be in a con- centration of 0.1 to 100 mg/ml, preferentially 5 to 15 mg/ml or 0.5 to 1.5 % (vol- ume/volume. The formulation of FF-MAS may be in the form of an ethanol stock so- lution, DMSO or other organic solvent solution or it may be in form of FF-MAS/HSA dry coated wells ready to use just by adding the appropriate culture medium. The du-

SUBSTITUTE SHEET (RULE 26)

fad 13 - ration of in vitro maturation may vary from 4 to 60 hours, preferentially 30 to 40 hours. The concentration of FF-MAS may vary from 0.01 uM to 100 uM, preferably from 0.1 uM to 100 uM, more preferred from 5 uM to 30 uM, even more preferred from 10 uM to 30 uM. Following in vitro maturation, the oocytes may be fertilised by conventional IVF or by intracytoplasmatic sperm injection (hereinafter designated

ICSI) or by future appropriate fertilisation methods leading to fertilised zygotes and the developing embryo may be transferred on day 1 to day 6 after fertilisation, pref- erentially on day 2 to 3, either as single egg transfer or multiple egg transfer. The pa- tient can receive progesterone and/or oestrogen therapy before and after the transfer in individually designed protocols to prime and sustain appropriate receptive endo- metrial lineage.

Compared with the know procedures, better results were obtained using the above procedure.

Example 3

Use of FF-MAS for in vitro maturation of immature human oocytes . 20 The female patient was started on a brief ovarian stimulation with recombinant FSH with an average daily dose of 225 on day 2 in the cycle and continued for a total of three times on alternating days, i.e., 2", 4", and 6" day in the cycle. At least 3 folli- cles of 10 mm or more on day 7 lead to aspiration of immature follicles in the size be- tween 8-12 mm. Follicles were aspirated and immature (GV stage) cumulus enclosed oocytes were cultured in oocyte culture system containing a standard in vitro culture (IVC) media (IVF 20 (which is available from Scandinavian IVF Science AB, Gothen- burg, Sweden)) additionally containing human serum albumin (0.8%) and FF-MAS (5 uM). All oocytes were cultured under normal conditions at 37°C in the incubator.

Each oocyte was cultured in one well in a four-chamber culture dish as culture media system. The duration of exposure to the culture media with treatment was 30 hours before ICSI or in vitro fertilization was performed. Preembryos were evaluated for cleavage stage and fragmentation / morphology at 1, 2 and 3 days post ICSI/IVF. Af-

SUBSTITUTE SHEET (RULE 26)

14 a ter 3 days of culture, a selection of the best preembryos, typically two preembryos, were replaced to the female patient.

Compared with the know procedures, similar clinical outcome was obtained.

However, in this example, compared with the known procedures, a reduced hormone exposure was used and, consequently, a reduced side effect profile was obtained here.

Example 4

Using the procedure described in Example 3 with the proviso that in stead of using

FF-MAS in a concentration of 5 uM, FF-MAS was used in a concentration of 20 uM, similar clinical outcome was obtained in this procedure as was obtained with the known procedures. However, in this example, compared with the known procedures, a reduced hormone exposure was used and, consequently, a reduced side effect profile was obtained here.

SUBSTITUTE SHEET (RULE 26)

Claims

yo 15 - CLAIMS

1. A method for human in vitro fertilisation wherein a woman, within a consecutive period of 30 days, is treated with a hypothalamic hormone and/or a pituitary hor- mone or an agonist or antagonist thereof or an active derivative thereof for a pe- riod which is less than about 7 days, preferably less than about 4 days, and, thereafter, using in vitro oocyte maturation wherein egg or eggs are retrieved from the woman and are matured using a MAS compound as defined herein.

2. The use of a hypothalamic hormone and/or a pituitary hormone or an agonist or antagonist thereof or an active derivative thereof in the manufacture of a hormone product which is to be administered to a woman who, within a consecutive period of 30 days, is treated with a hypothalamic hormone and/or a pituitary hormone or an agonist or antagonist thereof or an active derivative thereof for a period which is less than about 7 days, preferably less than about 4 days, and from whom, im- mediately after said period, one or more oocytes are aspirated, where after said oocyte(s) is/are cultivated in a convenient medium containing a MAS compound as defined herein, where after said oocyte(s) is/are fertilised with human sperm, and, where after, the resulting embryo(s) is/are transferred to a woman.

3. Use of a hypothalamic hormone and/or a pituitary hormone or an agonist or an- tagonist thereof or an active derivative thereof and of a MAS compound for the manufacture of a medicament for the treatment of human in vitro fertilisation wherein a hypothalamic hormone and/or a pituitary hormone or an agonist or an- tagonist thereof or an active derivative thereof is, within a consecutive period of days, used to treat a women for a period which is less than about 7 days, preferably less than about 4 days, and, thereafter, the MAS compound is used in an in vitro oocyte maturation of the egg or eggs retrieved from this woman. 30 4. A method or use according to any one of the preceding clams wherein said woman is treated for infertility, and/or for improving the maturation of her oocytes, and/or for improving the synchrony of nuclear, cytoplasmic and/or membranous oocyte maturation, and/or for improving the fertility of her oocytes, and/or for im- SUBSTITUTE SHEET (RULE 26)

16 Re proving the rate of implantation of her oocytes by human in vitro maturation and fertilisation.

5. A method or use according to any one of the preceding clams wherein the period in which said woman is treated with a hypothalamic hormone and/or a pituitary hormone or an agonist or antagonist thereof or an active derivative thereof is 0 (zero) days.

6. A method or use according to the preceding claim wherein the consecutive period is one menstrual cycle.

7. A method or use according to any one of the preceding claims wherein the hor- mones are gonadotropic releasing hormones or an agonist or antagonist thereof or analogues thereof or gonadotropic hormones or an agonist or antagonist thereof or analogues thereof.

8. A method or use according to the preceding claim wherein the gonadotropic hor- mone is FSH or an agonist or antagonist thereof or analogues thereof.

9. A method or use according to the preceding claim wherein the period in which the : female patient is treated with FSH or an agonist or antagonist thereof or ana- logues thereof, prior to the retrieval of the egg, is less than 7 days, preferably less than 4 days, and is at least 1 day.

10. A method or use according to the preceding claim wherein the period in which the female patient is treated with FSH or an agonist or antagonist thereof or ana- logues thereof is 2, 3 or 4 days.

11. A method or use according to any one of the preceding claims wherein no chorion gonadotropin, e.g. human chorion gonadotropin or an agonist or antago- nist thereof or analogues thereof is used. SUBSTITUTE SHEET (RULE 26)

pa 17 -

12. A method or use according to the previous claim wherein no gonadotropic re- leasing hormone, e.g. GnRH, or an agonist or antagonist thereof or analogues thereof is used.

13. A method or use according to any one of the previous claims wherein the dos- age of MAS compound is in the range from about 0.01 to about 100 uM, prefera- bly in the range from about 0.1 to about 100 uM.

14. A method or use according to any one of the previous claims, wherein the MAS compound is one of the compounds mentioned in WO 96/00235, 96/27658, 97/00884, 98/28323, 98/54965 and 98/55498, more specifically compounds men- tioned in Claim 1 thereof.

15. A method or use according to the previous claim wherein the MAS compound is FF-MAS.

16. A pharmaceutically kit in unit dosage form for use by in vitro fertilisation compris- ing 1-8 separate unit dosages, said kit comprising less than 7, preferably less than 4, and at least 1 separate dosage units for sequential daily administration of

. 20 a hypothalamic hormone and/or a pituitary hormone or an agonist or antagonist thereof or an active derivative thereof for sequential daily administration and 1 : dosage units of a MAS compound.

17. A kit according to the previous claim having the preferred features described in any one of the above subclaims.

18. Any novel feature or combination of features described herein. SUBSTITUTE SHEET (RULE 26)