HK1067865B - Use of hcg and lh in controlled ovarian hyperstimulation - Google Patents

Description

Use of hCG and LH in controlled ovarian hyperstimulation

Technical Field

The present invention relates to the field of in vitro and in vivo Assisted Reproductive Technology (ART), in particular Controlled Ovarian Hyperstimulation (COH) using gonadotropins.

Background

Treatment of infertility using Assisted Reproductive Technologies (ART), such as In Vitro Fertilization (IVF) or IVF in combination with intracytoplasmic sperm injection (ICSI) and Embryo Transfer (ET), requires COH to increase the number of oocytes⁽¹⁾. Standard protocol for COH⁽²⁾Comprising a down-regulation phase in which endogenous Luteinising Hormone (LH) is suppressed by administration of a gonadotropin releasing hormone (GnRH) agonist, followed by a stimulation phase in which Follicle Stimulating Hormone (FSH) is administered daily, typically at about 150 IU/day, to induce follicular development (folliculogenesis). Other molecules with FSH activity may also be used. Another option is to start stimulation after natural or induced menstruation, usually starting at about day 6 or 7 of FSH administration, to prevent the occurrence of LH-surges by administration of GnRH antagonists. In the technical scheme of the auxiliary reproductive technology of the T-T. Ovulation is triggered by the administration of a single bolus (single bolus) of hCG (5-10,000IU) when at least 3 follicles > 16mm (one of which is 18 mm). The time to recover oocytes was determined 36-38 hours after hCG injection.

The rationale behind the use of GnRH agonists and antagonists of the present invention is to prevent the onset of activity at inappropriate timesLH surge leading to premature ovulation and luteinization of follicles⁽³⁾. GnRH agonist therapy has become widely used clinically. It has been found that a prolonged treatment regimen (i.e. a regimen which first initiates ovulation induction or which is initiated before in the luteal phase of the cycle) makes it easier to schedule the patient for administration, with higher follicular yields and better overall clinical outcome⁽⁴⁾. The use of GnRH antagonists is relatively new clinically, but similar beneficial effects are expected, with the advantage of shorter treatment cycles.

By prolonging the time of administration of a GnRH agonist or GnRH antagonist, endogenous LH is greatly inhibited. The situation contradictory to the development of the mortise and tenon is different from the natural period. The natural cycle LH levels slowly increased, peaking in the middle of the day.

The role of LH and Chorionic Gonadotropin (CG) in ovulation induction and assisted reproductive technologies has been explored. As is well known and recognized in the art: techniques and methods for inducing ovulation are distinct from the COH regimen, although both may involve administration of FSH.

Hillier et al demonstrated that very low levels of LH are sufficient to generate follicles⁽⁵⁾。

Esposito et al investigated the role of endogenous LH in ART cycles stimulated with rFSH. They concluded that: the estradiol level of follicular fluid, oocyte productivity and fertilization are all improved when the serum LH concentration is higher than 0.5-1.0IU/L⁽⁶⁾。

WO 00/67778(Applied Research System) discloses the use of LH in the stimulatory phase to induce folliculogenesis, in particular to promote the development of single dominant follicles, in the induction of ovulation.

The European recombinant human LH research group reported that rhLH (75 and 225 IU/day) was administered to women with hypogonadotropic hypogonadism to support rhFSH-induced follicular development with an increased number of follicles and an increased diameter of follicles compared to a control group receiving rhFSH only⁽⁷⁾。

Filicori et al investigated the role of low doses of hCG as a replacement for LH in controlled ovarian hyperstimulation (Filicori et al; J.Clin.Endocrinol.Metab.841999; 2659-2663). Administration of hCG (50 IUhCG/day) was started simultaneously with administration of FSH and hCG was administered daily continuously until ovulation was initiated with a bolus of hCG. The number of small (< 10 mm), medium (10-14 mm) and large (> 14 mm) follicles obtained in the hCG-treated group was similar to that obtained in the FSH-only group, but the cumulative dose of FSH and the stimulation time of FSH were decreased in the hCG-treated group.

Messsinis et al reported ovulation induction in anovulatory women (world health organization group I) using a regimen of daily doses of hMG (75 IU each of FSH and LH) during the stimulatory phase and single or multiple doses of hCG during the luteal phase. As a result, it was found that the pregnancy rate of patients receiving multiple doses of hCG in the luteal phase was significantly increased as compared with the control group receiving only a single dose of hCG for inducing/inducing ovulation⁽⁸⁾。

Proper follicular development is, of course, a fundamental condition for the success of assisted reproductive technologies. However, there are some cases where ovulation and fertilization are completed, but improper implantation of the embryo leads to failure to conceive. Other cases are spontaneous preterm birth (abortion) in the first three months of pregnancy. Both of these problems are related to the condition of the endometrium, which is quite sensitive to hormone levels. It follows that even if follicular development, ovulation and fertilization all occur, successful pregnancy is not guaranteed, as implantation and premature birth problems are often encountered.

The problem of easy abortion or failure to bed in some patients is eventually solved, but this requires repetition of multiple ART cycles with consequent negative physiological and psychological effects on the patient. For other patients, these problems are a permanent obstacle to their inability to become pregnant.

Therefore, there is a need to provide methods for increasing implantation rates and decreasing early yields, particularly in conjunction with COH.

Disclosure of Invention

It is an object of the present invention to provide an improved method for administering gonadotropins for COH, thereby increasing implantation rates and decreasing fluid yields.

It is another object of the present invention to provide a method for providing multiple oocytes for recovery of ART.

It is a further object of the invention to provide a method of increasing pregnancy rates in patients who do not undergo COH, such as patients following the natural ovulation cycle or who have received ovulation induction, for example by increasing implantation rates and/or decreasing miscarriage rates.

In a first aspect the invention provides the use of human chorionic gonadotropin (hCG) or an analogue thereof in the manufacture of a medicament for promoting implantation and/or reducing miscarriage rates of an embryo in a human patient, said medicament being for administration prior to ovulation or ovulation triggering.

In a second aspect the invention provides the use of hCG or an analogue thereof, for promoting implantation and/or reducing miscarriage rates of an embryo in a human patient, said hCG or an analogue thereof being administered prior to ovulation or ovulation triggering.

A third aspect of the invention provides the use of hCG or an analogue thereof in the manufacture of a medicament for use in conjunction with COH in a human patient using FSH or an analogue thereof for aiding implantation and/or reducing miscarriage rates, wherein the medicament is to be administered starting before day 10 after commencing FSH treatment.

In a fourth aspect the invention provides the use of hCG or an analogue thereof for use in conjunction with Controlled Ovarian Hyperstimulation (COH) in a human patient using FSH or an analogue thereof for aiding implantation and/or reducing miscarriage rates, wherein administration of the hCG or an analogue thereof is commenced before day 10 after commencing FSH treatment.

A fifth aspect of the invention provides a pharmaceutical composition for aiding implantation of an embryo and/or reducing miscarriage rates, optionally preferably in combination with COH, said pharmaceutical composition comprising 25-1000IU hCG or an analogue thereof per dose.

A sixth aspect of the invention provides the use of human luteinising hormone (hLH) or an analogue thereof in the manufacture of a medicament for use in combination with Controlled Ovarian Hyperstimulation (COH) in a human patient using FSH or an analogue thereof for aiding implantation and/or reducing miscarriage rates, wherein the medicament is to be administered initially before day 10 after commencing FSH treatment, preferably between day 3 and day 10 after commencing FSH treatment.

A seventh aspect of the invention provides the use of hLH or an analogue thereof, wherein the administration of said LH is initiated before 10 days after the start of FSH treatment, preferably between 3 days and 10 days after the start of FSH treatment, in combination with COH for human patients using FSH or an analogue thereof for aiding implantation and/or reducing miscarriage rates. Another aspect of the invention provides the use of LH or an analogue thereof in the manufacture of a medicament for promoting implantation and/or reducing miscarriage rates of an embryo in a human patient, said medicament being for administration prior to ovulation or ovulation triggering.

It is a further aspect of the present invention to provide the use of LH or an analogue thereof, administered prior to ovulation or ovulation triggering, in promoting implantation and/or decreasing miscarriage rates of an embryo in a human patient.

Another aspect of the present invention is to provide a pharmaceutical composition for aiding implantation and/or reducing miscarriage rates of embryos, optionally preferably in combination with COH, said pharmaceutical composition comprising 150-1000IU LH or an analogue thereof per dose.

It is a further aspect of the present invention to provide hCG or an analogue thereof or LH or an analogue thereof for use in conjunction with COH in human patients using FSH or an analogue thereof for aiding implantation and/or reducing miscarriage rates, wherein administration of the hCG or an analogue thereof or LH or an analogue thereof is commenced before day 10 after commencing FSH treatment.

In another embodiment, the invention provides hCG or an analogue thereof or LH or an analogue thereof for promoting implantation of an embryo and/or reducing miscarriage rates in a human patient, wherein the hCG or LH or analogue thereof is administered prior to ovulation or ovulation triggering.

It is a further aspect of the present invention to provide a method of promoting implantation and/or reducing miscarriage rates in a patient, which method comprises administering to the patient hCG or an analogue thereof or LH or an analogue thereof, in combination with COH employing FSH or an analogue thereof, wherein said administration is commenced before day 10 after commencing FSH treatment.

The invention also provides a method of promoting implantation and/or reducing miscarriage rates in a patient, the method comprising administering to the patient hCG or an analogue thereof or LH or an analogue thereof, wherein the hCG or LH or an analogue thereof is administered prior to ovulation or ovulation triggering.

In another aspect the invention provides a kit for use in COH, the kit comprising 12 or more, preferably 14 or more doses of FSH per day, preferably about 75-200IU FSH per day, more preferably about 150IU FSH per day, and 4-8, 5-8 or 6-8 doses of hCG per day, for example 4, 5, 6, 7 or 8 doses of hCG per day, preferably about 25-1000IU hCG per day, more preferably about 50-100IU hCG per day.

A further aspect of the invention provides a kit for assisting implantation of an embryo and/or reducing miscarriage rates, the kit comprising 4-8, 5-8 or 6-8 doses of hCG per day, for example 4, 5, 6, 7 or 8 doses of hCG per day, about 25-1000IU hCG per day, preferably about 50-100IU hCG per day. These kits may or may not be used in conjunction with COH.

The kits and pharmaceutical compositions of the invention are preferably designed for use in the methods and uses of the invention.

Detailed Description

Human chorionic gonadotropin (hCG) is a heterodimeric glycated peptide hormone produced by the placenta during pregnancy. It appears in the serum shortly after fertilization and functions to maintain the corpus luteum after LH secretion is diminished to support the continuous secretion of estrogen and progesterone and to prevent menses. It cannot be considered as important in the natural ovulatory cycle because it is only present at high levels in pregnant womenThe application is as follows. It is well known that receptors for LH/hCG are present in the gonads, uterus, fallopian tubes, placenta, and endometrial and myometrial cells⁽⁹⁾. The half-life of hCG is longest in gonadotropins⁽¹⁰⁾。

The present inventors have unexpectedly found that administration of low doses of hCG or an analogue during the stimulatory phase of the ART cycle has a beneficial effect on pregnancy rates, for example by aiding or improving implantation and/or reducing miscarriage rates. The term "low dose" includes doses in a particular patient that are less than those typically used to prime follicular maturation prior to ovulation, i.e. the final follicular maturation ("follicular (or ovulation) priming dose"). The follicle/ovulation-initiating dose of hCG (typically in the range of 5,000 to 10,000IU hCG) varies from patient to patient.

As can be seen from the above discussion of standard COH treatment regimens, administration of such "high" or ovulation triggering doses of hCG is commonly referred to in the art as the ovulation triggering step, the oocyte maturation step or the ovulation stimulation step. However, such an ovulation stimulation or priming step or oocyte maturation step with the administration of a high dose of hCG can only be performed if there is sufficient follicular development during the stimulatory phase of the COH protocol, i.e. during the phase in which FSH or an analogue thereof is administered to induce folliculogenesis. The main difference between the method and use of the invention compared to prior art treatment regimens is that hCG is administered during the stimulatory phase, i.e. before sufficient follicular development has occurred and before ovulation has occurred or is triggered, and at a dose below that which induces oocyte maturation and ovulation stimulation, i.e. below the ovulation triggering dose. This treatment of the invention has significant advantages in implantation and abortion. Therefore, according to the invention, a low dose of hCG is administered during the stimulation phase, in combination with a COH regimen, before there is sufficient follicular development and before finally a high ovulation triggering hCG dose is administered to trigger oocyte maturation and ejection.

In these aspects of the invention, if the patient is not undergoing COH, a low dose of hCG may also be administered during the folliculogenesis stage and before sufficient follicular development has been achieved. These doses of hCG are lower than the ovulation triggering dose. As mentioned above, these patients may have a natural ovulation cycle, in which case the final ovulation triggering dose of hCG need not be administered. However, some patients who are not COH and who can be treated with a low dose of hCG according to the method of the invention may not have a natural ovulatory cycle (e.g. those who are ovulatory inducing) in which case an ovulation triggering dose of hCG is administered once sufficient follicular development has been judged to have been achieved.

As noted above, the present invention also provides for the administration of LH instead of hCG in the methods and uses of the present invention. Administration of LH is also performed during the cycle and follicular development phase as described herein for hCG.

In the case where hCG is used in each of the cases described herein, the dose used should be in the range 25-4000IU hCG per day, preferably 25-1000IU hCG per day, more preferably 30-1000 or 30-500IU hCG per day, most preferably 50-100IU or 75-125IU or 75-100IU hCG per day or 75 or 100 to 500 or 75 or 100 to 1000IU hCG per day. These doses are all lower than the ovulation triggering dose, which as mentioned above is referred to herein as "low dose" hCG. If an hCG analogue is used, equivalent doses corresponding to these hCG doses should be administered.

As noted above, the invention also provides pharmaceutical compositions or kits which may contain hCG (or LH as described herein above and below) at the dosages described above for use in the methods and uses of the invention.

In these aspects of the invention, if hCG (or an analogue thereof) is used in combination with COH using FSH or an analogue thereof, hCG (or an analogue thereof) should be administered starting before the 10 th day after commencing FSH treatment, more preferably before the 9 th day after commencing FSH treatment. The hCG is preferably administered at least 3 days after the start of FSH treatment, for example between day 3 and day 10 after the start of FSH treatment, more preferably at least 5 or 6 days after the start of FSH treatment. Preferably, the hCG should be administered only on day 7 or 8 or about day 7 or 8 after commencing FSH treatment. Therefore, hCG is administered during the follicular phase, preferably at a time point in the cycle at or around the mid-follicular phase, i.e. at least 5, 6, 7 or 8 days after commencing FSH treatment.

According to the invention, the administration of hCG may be a single bolus, in which case it should preferably be administered at day 7 or 8 or at about day 7 or 8 after commencing FSH treatment, and the dose should preferably contain 100-. Administration in single bolus is convenient for both the medical practitioner and the patient.

In another aspect, according to the invention, hCG can be administered daily until follicle maturation is triggered or ovulation is induced/triggered with a conventional pill of hCG. For daily administration, the dose should be in the range 25-4000IU hCG/day, preferably in the range 25-1000IU hCG/day, more preferably in the range 30-1000 or 30-500IU hCG/day, most preferably in the range 50-100IU or 75-125IU hCG/day or in the range 75-100 or 75 or 100 to 500 or 75 or 100 to 1000IU hCG/day. It has been found that a daily regimen starting on day 7 after commencing FSH treatment and using a dose of 50-100IU hCG per day is particularly effective. The number of administrations of hCG may also be reduced, for example every 2, 3, or 4 days, preferably every 2 days, until ovulation is triggered. In these regimens, those listed above may also be used, although the preferred dosage of the invention is 50-200 IUhCG.

As is clear from the above discussion, the hCG (or LH) used in the present invention is administered before ovulation occurs or before ovulation is triggered, for example, with an ovulation triggering dose of hCG, and continues until either an ovulation is initiated or an ovulation occurs. In all versions of the invention, hCG (or LH) administration may be continued after ovulation if deemed beneficial to the patient.

The timing of ovulation induction by administration of a "follicle/ovulation triggering dose" of hCG is well known to those skilled in the ART of ART protocols and can be determined. In general, ovulation is triggered when follicle development is deemed sufficient for the selected regimen. The level of follicular development is generally determined by measuring the size of the follicles (e.g. using ultrasound) and the serum estradiol (E) of the patient₂) The level. If the protocol is COH, then since the aim of these methods is multiple follicular development, resulting in an increasing number of mature follicles/oocytes, which are generally fertilized in vitro and then implanted in the patient, the timing of ovulation induction may be slightly different from that if the protocol is ovulation induction, in which the aim is to produce 1 or at most 2 mature follicles which are ovulated and fertilized in vivo. For example, in the example relating to the COH protocol, ovulation can be triggered with 5,000 to 10,000, e.g. 10,000IU hCG, when at least 2 follicles greater than or equal to 18mm in diameter are detected and serum estradiol levels reach 300 pg/ml. Another option is that in the case of the COH protocol, when the largest follicle has reached an average diameter of at least 18mm, at least 2 other follicles have an average diameter of at least 16mm (i.e. at least 3 follicles of at least 16mm, one of which is at least 18mm), and estradiol (E) is present for the number of follicles present₂) Ovulation can be triggered when the level is within an acceptable range (about 150 pg/ml/mature follicle). For ovulation induction, an ovulation-initiating dose of hCG can be administered when at least 1 follicle is 17mm (if more than 3 follicles are 15mm, administration is not necessary).

hCG has a relatively long half-life in vivo. Therefore, care must be taken when using multiple doses that the cumulative dose does not reach undesirably high levels. Preferably, the serum hCG level is not substantially above 50IU/L, more preferably not above 25IU/L, most preferably not above about 10IU/L prior to administration of the ovulation-inducing pill. If the hCG level is substantially higher than this, the result may be premature luteinisation. Mannarts et al reported the pharmacokinetics of a single bolus intravenous hCG injection following intramuscular and subcutaneous injections⁽¹¹⁾。

Where FSH (or an analog) is used in conjunction with the COH technique or regimen of the invention, suitable dosages and administration regimens will be apparent to those skilled in the art, and any suitable dosage and administration regimen may be used. For example, FSH may be administered daily at a dose of either about 75 to 250 or 75 to 200 IU/day, preferably either about 150 to 200 IU/day, most preferably either about 150 IU/day. Some patients respond poorly to FSH, preferably with a daily dose of up to 600 IU/day. One typical scenario is such that: the patients were evaluated for follicular development by ultrasound starting at 150IU FSH/day, 3 or 4 days later. If follicular development is sufficient, a dose of 150IU FSH/day may be maintained. If follicular development is insufficient, the dose may be increased to 225, 300, 375, 450, 525 or 600IU FSH/day. Preferably, FSH administration is continued until an ovulation triggering dose of hCG is administered. Ideally the cumulative dose of FSH should not exceed 6000 IU/cycle.

The terms "increased rate", "promoting", "assisting", "increased rate", and the like as used herein with respect to the effects of implantation and pregnancy include any measurable improvement or increase in the incidence of implantation or pregnancy in the individual or group of patients treated by the methods of the invention, for example, when compared to the level or incidence of implantation or pregnancy in one or more untreated patients, or when compared to the level or incidence of implantation or pregnancy observed in the same patient at an earlier time point (e.g., compared to a "baseline" level). For example, in embodiments in which hCG or LH is used in combination with COH, a relevant comparison is made between patients treated by the methods of these embodiments and the group of patients treated with conventional COH or the same patient treated with conventional COH. The improvement or increase is preferably statistically significant, and the P value is preferably less than 0.05. Methods for determining statistically significant results are well known and well documented in the art, and any suitable method may be used.

The terms "reduce", "reducing" and the like as used herein with respect to the effects of miscarriage, refer to any measurable reduction or decrease in the incidence of miscarriage in the individual or group of patients treated with the methods of the invention, e.g., when compared to the incidence of miscarriage in one or more untreated patients, or when compared to the incidence of miscarriage observed in the same patient at an earlier time point (e.g., compared to a "baseline" level). For example, in embodiments in which hCG or LH is used in combination with COH, a relevant comparison is made between patients treated by the methods of these embodiments and the group of patients treated with conventional COH or the same patient treated with conventional COH. The reduction is preferably statistically significant, and the P value is preferably less than 0.05. Most preferably, the use and method of the present invention is capable of preventing miscarriage. These terms also include the prevention of miscarriage.

The hCG (or LH) use of the invention can be used on any patient whose infertility is believed to be due to preterm birth or failure to become implanted, regardless of whether the patient is receiving other exogenous gonadotropins.

Miscarriage is defined as delivery of the fetus before it has independent viability. Preterm birth refers to those abortions that occur during the first 1 month of fetal development. The methods and uses of the present invention are particularly useful for reducing the level of preterm labor.

The use of hCG (and of course LH) in accordance with the invention in conjunction with COH will generally be used in conjunction with in vitro fertilization techniques. However, the use of hCG (and LH) as described herein in conjunction with COH may also be used in conjunction with in vivo fertilization.

In addition, the hCG (and LH) use of the invention may also be used in combination with in vivo fertilization in patients who do not undergo COH, for example, in regimens that include natural ovulation and, for example, ovulation induction with an antiestrogen or aromatase inhibitor (i.e., regimens that do not include the administration of exogenous gonadotropins). When used in patients who do not receive other exogenous gonadotropins, hCG should be administered beginning before ovulation is expected in any given cycle, preferably at or about day 6, 7 or 8 or 6, 7 or 8 after menstruation. A single bolus may be administered (e.g. the doses described above in relation to the administration of a single bolus, in particular 100-500IU hCG), or daily (e.g. the doses described above in relation to the daily administration, in particular 50-100IU hCG), or every 2 days (e.g. the doses described above, in particular 50-200IU hCG), until natural ovulation or, if desired, ovulation is triggered with the ovulation-triggering dose of hCG described above.

In embodiments of the invention where hCG (or LH) is used in patients who do not undergo COH, these patients may optionally be induced to ovulate with exogenous gonadotropins (e.g. exogenous FSH). Suitable hCG (or LH) doses for use are as described above and below. Ovulation is triggered at the appropriate time point with a high dose of hCG, as described above.

Thus, as indicated above, it will be appreciated that the use of hCG (or LH) according to the invention may be advantageous in conjunction with a COH regime employing FSH, and that hCG (or LH) may also be used to improve the embryo implantation rate and/or reduce miscarriage rates in human patients who are not undergoing a COH regime, but who wish to improve the chances of successful conception. These patients (or their spouses) often have some fertility problems, i.e. infertility or low fertility. In addition, patients may not have significant fertility problems such as ovulation and fertilization, but may be infertile due to susceptibility to premature delivery and/or failure to seed. Elderly women, such as women over the age of 35, who are also well suited for treatment by this method, typically suffer from problems of implantation and high miscarriage rates. These patients may ovulate spontaneously or may be subjected to an ovulation induction protocol other than the COH protocol, for example, using aromatase inhibitors or the like which stimulate endogenous FSH secretion, or the ovulation induction protocol discussed above in which exogenous FSH is administered. Such ovulation induction protocols are standard in the art and are fully described.

In applications where a COH regimen is not employed, the timing of administration of hCG (or LH) will generally be calculated from the day of onset of menstruation, although in an ovulation induction regimen using FSH, the timing of administration may be calculated from the day of onset of FSH treatment. Appropriate times after menstruation or after commencing FSH treatment are discussed above. As mentioned above, the administration of hCG (or LH) is commenced prior to ovulation at the follicular stage (which may be either natural ovulation or ovulation triggering with an ovulation triggering dose of hCG), which is preferably preceded by the mid-ovulation phase of the cycle, e.g. 5 to 8 days after menstruation, e.g. day 6, 7 or 8 after menstruation, or at least 5 or 6 days after commencing FSH treatment, e.g. 5, 6, 7 or 8 days after commencing FSH treatment. It follows that although the treatment regimen for these patients differs from those patients undergoing a COH regimen using FSH, the preferred timing of hCG (or LH) administration is similar, i.e. at or around the mid-ovulatory phase of the cycle.

When LH is used in combination with a method which does not involve COH, the preferred timing of administration of LH may be with reference to hCG. Preferably, LH is administered daily or half daily at a daily dose of 125-7000IU LH or 150-1000IU LH, more preferably 150-700 IU LH or 350-700IU LH. The other optional dosage is 10-200IU LH, 10-150IU LH or 20-100IU LH/day. A daily dosage of 150IU LH is particularly effective for the methods of the invention and is the preferred dosage to be used. In accordance with the present invention, administration of LH is particularly effective in women at least 35 years of age.

The LH, FSH and hCG for use in the present invention may be formulated for administration by any convenient route, typically in combination with a pharmaceutically acceptable carrier, diluent or excipient. Suitable formulations and routes of administration of LH, FSH and hCG are well known in the art and have been described, and any suitable route and formulation may be used. Thus, the pharmaceutical compositions of the present invention will generally contain a pharmaceutically acceptable carrier, diluent or excipient, and an appropriate active ingredient.

As noted above, patients who may benefit from the methods and uses described herein are any patients who are certain forms of infertility or have low fertility, or who wish to reduce miscarriage rates and/or problems associated with inappropriate implantation, such as those at high risk of inappropriate implantation and/or miscarriage, e.g., patients at least 35 years old or who have had implantation and/or miscarriage problems in the past. Suitable patients may have a natural drain cycle or may undergo ovulation induction or COH protocols.

According to the invention, administration of hCG (or LH) is useful for those patients being treated with a GnRH agonist or antagonist. According to the invention, hCG or LH administration is particularly useful in combination with IVF or IVF/ICSI. The method can improve implantation rate and pregnancy rate of the first 3 months. The use of hCG or LH may even help implantation in some patients who have previously been diagnosed as being unable to undergo IVF regimens due to non-ovulation problems.

The hCG or LH use of the invention is particularly useful for treating patients with low endogenous LH levels, such as patients with hypogonadotropic hypogonadism.

The hCG or LH use of the invention may also be used in patients who were previously unable to or do not remain pregnant using FSH alone, for example in standard COH or ovulation induction protocols.

Other suitable patients are, for example, those with polycystic ovarian disease (PCOD), patients with luteal phase and immune factor deficiencies, and patients aged 35 years and older ("advanced age" patients). The age of the patient is preferably not more than 45 years, more preferably not more than 42 years.

The hCG used may be obtained from any source, provided that it is not contaminated with any substances that would significantly affect its action, in particular other gonadotropins. Although the use of recombinant hCG (rhCC) is preferred because of its high purity, urinary hCG can also be used. Such conditions are equally applicable to the resources of hLH used in the present invention.

Analogs of hCG include all molecules that have the same physiological, biochemical or biological effects as hCG, and/or bind to the same receptor of hCG. Luteinizing Hormone (LH) is known to have some of the same physiological effects as hCG.

Some of the hCG analogues include single-chain hCG in which the C-terminus of the β -subunit is fused to the N-terminus of the α -subunit (Sugahara et al, PNAS, 92, 1995, 2041-. Examples of other analogues that have been disclosed can be found in European patent EP0322226(Applied Research System), WO 92/22568 (University of Medicine & Dentistry of New Jersey), WO96/05224 (University of Washington), WO 90/09800 (University of Washington), WO 93/06844 (University of Washington), WO 98/43999 (University of Washington), WO 99/25849 (University of Washington).

hCG can be detected by any suitable technique, e.g., Vaitukaitis et al⁽¹²⁾The radioimmunoassay and ELISA assay⁽¹³⁾. The biological activity of hCG can be measured by any suitable technique, for example, the mouse Leydig cell bioassay (Leydig cell bioassay)⁽¹⁴⁾。

As mentioned above, the use of LH or an analogue thereof is beneficial during the post-stimulation phase of the ART cycle. In the COH regimen (i.e. in the case where LH is used in combination with COH), LH should be administered starting before the 10 th day after the start of FSH administration. Because LH has a short half-life, it is preferably administered daily or half daily. In the case of the combination of LH with COH in the present invention, preferred timing of administration of LH is as described above for hCG. In the most preferred COH modification, LH is administered beginning on day 6 or 7 or about day 6 or 7 after commencing FSH treatment. Preferably, LH should be administered 3 days after the start of FSH treatment. Daily doses of 125-7000IU LH or 150-1000IU LH, more preferably 150-700 or 350-700IU LH, may be administered daily until ovulation is induced. The other optional dosage is 10-200IU LH, 10-150IU LH or 20-100IU LH/day. A daily dosage of 150IU LH is particularly effective for the methods of the invention and is the preferred dosage to be used.

In the present invention, similar daily doses may be used when LH is administered to patients who are not undergoing COH.

LH analogs include all molecules that have the same physiological, biochemical, or biological effect as LH and/or bind to the same receptor of LH. It is well known that hCG has some of the same physiological effects as LH. Examples of some LH analogues that have been disclosed are disclosed in european patent EP0322226(Applied Research Systems), WO 92/22568 (university of medicine and dentistry, new jersey), WO96/05224 (university of washington), WO 90/09800 (university of washington), WO 93/06844 (university of washington), WO 98/43999 (university of washington), WO 99/25849 (university of washington), WO 00/61586(Akxo Nobel).

In embodiments of the invention employing FSH, it will be appreciated by those skilled in the art that FSH may be replaced by a biologically active analog or by a compound that stimulates secretion of endogenous FSH. The latter case includes aromatase inhibitors and antiestrogens such as tamoxifen (tamoxifen) and Chlorostilbene Citrate (CC). These compounds stimulate endogenous FSH secretion by eliminating the negative feedback exerted by estrogen on the hypothalamus (either by antagonizing estrogen receptors, which acts like CC and tamoxifen, or by greatly reducing estrogen concentration, which acts like aromatase inhibitors).

The preferred form of FSH for use in conjunction with the hCG application of the present invention is known as FSH-CTP. Such long-acting human FSH has been described in WO 93/06844 and has a wild-type FSH α -subunit and a β -subunit consisting of the wild-type FSH β -subunit fused at its carboxy terminus to the carboxy-terminal peptide (CTP) of the hCG β -subunit (residues 112-118 to 145 of the native hCG β sequence). Other classes of FSH analogues include, for example, single chain FSH analogues in which the β -subunit is fused to the CTP of hCG, which in turn is fused to the FSH α -subunit, as described in WO96/05224 (single chain FSH-CTP).

As described above with respect to LH and hCG, the FSH used in the methods of the present invention may be obtained from any source. These resources are well known to those skilled in the art of ovulation induction and COH procedures. FSH urine preparations, such as hMG with a 1: 1 ratio of FSH to LH activity, may be used. Since the purity of recombinant fsh (rFSH) is high, rFSH is preferably used.

In the ovulation induction and COH stimulation phase of IVF, FSH is replaced with human menopausal gonadotropin (hMG). hMG is a relatively coarse hormone extract extracted from the urine of postmenopausal women, and has FSH and LH activity (1: 1). Non-proprietary hMG may contain as little as 2% of the active hormone, and therefore, up to 98% of the protein content may be a urinary contaminant. When hMG is used instead of FSH in the methods of the invention, as described above, supplementation with hCG also proves advantageous, for example, in aiding implantation and/or preventing or reducing miscarriage. The hCG should be administered beginning before the 10 th day after the start of the hMG treatment, more preferably beginning before the 9 th day after the start of the hMG treatment, and most preferably beginning at or about the 7 th or 8 th day after the start of the hMG treatment. hCG is preferably administered 3 days after the start of hMG therapy. Dosage and dosing regimen reference is made to the dosage and dosing regimen of hCG in combination with FSH. Preferably the dosage is 150 IU/day, more preferably 50 or 100IU hCG/day.

The method of the invention can also use hMG as a resource of hLH, i.e. hMG can be used as a resource of urine hLH.

The invention will now be further described by way of the following non-limiting examples.

Examples

Example 1

Stimulation protocols

Control group 1: on day 1 of menstruation, patients were given daily injections of triptorelin (decapeptyl) (0.1 mg) for desensitization. After 14 days sonography was performed, no cysts were present and stimulation was initiated with rFSH (150 to 200 IU/day). After 7 days, follicular growth was examined by sonography and E was determined₂Blood concentration. The patient is examined daily, and when at least 2 follicles with a diameter of 18mm or more are detected, serum E is added₂When the level of (D) reached 300pg/mL, ovulation was triggered with 10,000IU of hCG.

Control group 2: on day 7 of control 1, control 2 received hMG (150 IU/day) in addition to rFSH.

Experimental groups: on day 7 of the control group, the experimental group received 50-100IU hCG per day and rFSH was administered until ovulation was triggered as described above.

The oocyte is fertilized in vitro. After 4 hours, they were washed and placed in the culture medium (ISM 1). After 20 hours, the fertilization was checked and the embryos were cultured in the same medium for up to 48 hours. Then, they were transferred to a second culture broth (ISM 2). The 2 best embryos were then removed and transplanted into patients and culture continued until blastocyst formation (5-6 days).

Results

The results of the experiments are shown in tables 1, 2 and 3. Tables 1 and 2 show the results of comparison of control group 1 (administration of rFSH only) with experimental group (administration of rFSH + hCG). Table 3 shows the results of comparison between the control group 2 (administration of rFSH + hMG) and the experimental group (administration of rFSH + hCG).

There was no difference in the time used in the stimulation phase. Experimental group E receiving hCG₂The level rises. The two groups did not differ much in graft difference: receiving hCG: 92%, control group: 86%, (p ═ 0.1). Two groups obtained from supernumerary embryos formed blastocysts nearly equally (received hCG: 185/4)11-45%, control: 292/627-46.5%, p-0.622). The implantation rate in the hCG group (receiving hCG: 24.5%, control: 14.6%, p ═ 0.0134) and pregnancy rate per transfer (receiving hCG: 37.5%, control: 23.6%, p ═ 0.0246) increased, averaging 1.9 embryos per transfer.

Table 1: low dose hCG assay results Using rFSH during the stimulation phase

	Has hCG	No hCG
			Number of patients	96	127
Grade (year of old)	2.2(32.8±4.1)	1.5(32.1±4.5)(p＝0.1068)
			Transplantation	89(92％)	109(86％)
Stimulation time (sky)	11.7	12.3
			Recovered oocytes	10	9.7
Embryo (m)	6.2(62％)	5.4(56％)
			Implantation bed	43/175(24.5％)	30/206(14.6％)(p＝0.0134)
Embryo/transplantation	1.97	1.89
			Pregnancy	36/96(37.5％)	30/127(23.6％)(p＝0.0246)
Blastocyst	185/411(45％)	292/627(46.5％)(p＝0.6221)

Table 2: low dose hCG assay results Using rFSH during the stimulation phase

	Has hCG	No hCG	Total up to
				Pregnancy	109	230	339
Dislocation	2	6	8(2.4％)(p＝0.95)
				Abortion	8(7.3％)	36(15.6％)	44(13％)(p＝0.03)
Total number of miscarriages + ectopic sites	10(9，17)	42(18.3)	52(15.7)(p＝0.03)

Table 3: low dose hCG and hMG assay results using rFSH during the stimulation phase

	FSH+hCG	hMG
			Pregnancy	109	76
Dislocation	2	2
			Abortion	8	13(p＝0.049)
Total number of miscarriages + ectopic sites	10	15(p＝0.049)

Example 2

The following study compares pregnancy outcomes in patients treated with COH with FSH alone and patients treated with FSH and starting LH on day 6.

After negative quantitative serum pregnancy tests, conditioned pituitary glands were desensitized with 0.5 mg daily dose of gonadotropin releasing hormone agonist (GnRH-a) Lupron ® 7 to 8 days after ovulation estimation until serum estradiol (E)₂) Desensitization was achieved at levels < 75 pg/mL. At that time, treatment was initiated by subcutaneous injection of recombinant human FSH at an initial dose of 225IU per day. This dose is maintained for the first 5 treatment days and may be increased by 75 to 150 IU/day every 1 to 2 days if the ovarian response in the patient is perceived to be slow. The maximum allowable dose of FSH is 450 IU/day, the maximum cumulative dose cannot exceed 6,000IU per cycle. For those patients randomized to FSH and r-hLH treatment with r-hLH was started at a daily dose of 150IU on day 6 of stimulation. The dose of r-hLH was unchanged. After reaching down-regulation, Lupron (leuprolide acetate) treatment was continued at a dose of 0.25 mg daily throughout the stimulation period until hCG was administered. Estradiol levels and ultrasound measurements are evaluated throughout the stimulation cycle to determine the patient's response to treatment. Daily administration of FSH or FSH plus r-hLH is continued until follicle development is considered sufficient. The criteria for hCG administration (for ovulation triggering) are met when the mean diameter of the largest follicle is > 18mm and the mean diameter of at least 2 other follicles is > 16 mm. For the number of follicles present, E of the patient₂The levels are also within the range acceptable to the investigator (about 150 pg/ml/mature follicle). At that time, the patient received a single intramuscular injection of 10,000USP units of hCG for the final stage of follicular maturation. According to the customary practice at the study site, the oocytes are recovered vaginally, 34 to 36 hours after administration of hCG, monitored by ultrasound. Then, intracytoplasmic single sperm injection (ICSI) was performed at each site according to standard degree. A maximum of 3 embryos can be replaced 2 to 3 days after egg removal. All patients who started receiving hCG in the evening of the day of oocyte retrieval were supported for the luteal phase with natural progesterone in oil for a minimum of 7 days; next, the investigator may choose to use progesterone suppositories. The total support time for the luteal phase is determined by the investigator as the case may be.

All patients undergoing embryo transfer received hCG injection and blood samples were collected 15 to 17 days later to determine serum levels of β -hCG. If the test result is positive (biochemical pregnancy), the test is repeated after 2 to 7 days.

All patients were subjected to post-treatment visits including general physical examinations and clinical laboratory tests 15 to 17 days after administration of hCG (when the patients were subjected to a pregnancy test for a review) or within one week of menstruation. All patients who had been pregnant were subjected to ultrasonography 35 to 42 days after administration of hCG and the number of foetal sacs and foetal heart activity ("clinical pregnancy") were recorded. If there is clinical pregnancy, these women are followed to determine the pregnancy outcome.

Patients were divided into ages 35 and above ("advanced patients") and under 35 years and the data was analyzed. Clinical pregnancy data are shown in table 4. Patients 35 years old and older were treated with FSH + LH, which showed a significant improvement in pregnancy outcome (FSH + LH: 48.8%, FSH only: 21.6%).

Table 4: pregnancy data for patients treated with FSH only versus patients treated with FSH + LH (starting administration of LH on day 6)
								FSH+LH			FSH
Age of patient	＜35	≥35	Total number of	＜35	≥35	Total number of
							Number of patients	103	41	144	107	37	144
Clinical pregnancy (%)	46(44.7％)	20(48.8％)	66(45.8％)	48(44.9％)	8(21.6％)	56(38.9％)

Reference to the literature

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Claims (4)

1. Use of human luteinising hormone (hLH) in the manufacture of a medicament for use in combination with Controlled Ovarian Hyperstimulation (COH) in human patients using FSH to reduce miscarriage rates.

2. The use of claim 1, wherein the patient is at least 35 years old.

3. The use as claimed in claim 1 or claim 2, wherein the hLH is recombinant hLH.

4. The use as claimed in claim 1 or claim 2, wherein the hLH is urinary hLH.