NEW USE OF LUTEINISING HORMONE

Abstract

The present invention relates to luteinising hormone (LH) or to a molecule having LH-like activity or to a composition comprising it for use in inducing the activation, and/or the maturation of primordial and/or primary follicles in an individual.

Claims

1. A method of inducing activation, and/or maturation or development of primordial and/or primary follicles in an individual with luteinising hormone (LH) or with a molecule having LH-like activity or with a composition comprising it, said method comprising administering to said individual said LH or with molecule having LH-like activity or said composition comprising it, wherein the molecule having LH-like activity is selected from the group consisting of chorionic gonadotropin (CG) or an LH-agonist.

2. The method according to claim 1, wherein the maturation or the development of primordial follicles is associated with an increase in primary follicles.

3. The method according to claim 1, wherein the activation and/or maturation or the development of primordial and/or primary follicles is associated with an increase in the ultrasound count of antral follicles (AFC) and an increase in the serum values of anti-Müllerian hormone (AMH).

4. The method according to claim 3, wherein the individual has an ovarian reserve within the norm, defined as a serum concentration of AMH greater than 2 ng/ml.

5. The method according to claim 3, wherein the individual has an ovarian reserve within the norm, defined as AFC greater than 10.

6. The method according to claim 3, wherein the individual has a reduced ovarian reserve, defined as a serum concentration of AMH lower than 2 ng/ml.

7. The method according to claim 3, wherein the individual has a reduced ovarian reserve, defined as AFC lower than 10.

8. The method according to claim 1, wherein LH is human LH (hLH).

9. The method according to claim 1, wherein CG is human CG (hCG).

10. The method according to claim 1, wherein said LH is taken in an amount comprised between 150 and 450 International Units (IU) per day.

11. The method according to claim 1, wherein LH or the molecule having LH-like activity is taken for a period of time of at least 30 days.

12. The method according to claim 1, wherein LH or the molecule having LH-like activity is taken for a period of time of at least 60 days.

13. The method according to claim 1, wherein LH or the molecule having LH-like activity is taken in association or in combination with an infertility treatment in an individual.

14. The method according to claim 1, wherein LH or the molecule having LH-like activity is taken before the infertility treatment.

15. The method according to claim 1, wherein LH or the molecule having LH-like activity is taken neither in association nor in combination with FSH.

16. The method according to claim 3, wherein the individual has a serum concentration of AMH greater than 2.5 ng/ml.

17. The method according to claim 3, wherein the individual has an AFC greater than 12.

18. The method according to claim 3, wherein the individual has a serum concentration of AMH lower than 1.5 ng/ml.

19. The method according to claim 3, wherein the individual has an AFC lower than 8.

20. The method according to claim 1, wherein said LH is taken in an amount comprised between 170 and 350 IU per day.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0032] FIG. 1 shows a diagram of human gonadotropin-independent and gonadotropin-dependent folliculogenesis;

[0033] FIG. 2 shows the number of (A) primordial and (B) primary follicles (mean per histological section±SEM) in the original ovarian cortex or after two days of culture without (control) or with LH. Within every graph * denotes statistical significance versus the original biopsies whilst ** denotes significance versus the original biopsies and the controls (two-way ANOVA; P<0.05; n=6 cultures, 11-18 sections examined per sample); FIG. 3 shows a representation of the proportion of follicles in the different stages of maturation in ovarian cortex samples of the original biopsies, after two days of culture without LH or with LH. The distribution of the follicles is shown as a percentage of the total viable follicles. * denotes statistical significance in relation to the original biopsies whilst ** shows significance versus both the original biopsies and the controls (p<0.05);

[0034] FIG. 4 shows the increases in the fraction of oocytes that show cytoplasmic translocation of FOXO3a;

[0035] FIG. 5 shows the relative expression of CCN genes in primary cultures of ovarian tissue incubated for 48 hours with LH. Significant differences between the samples treated with LH and the controls are marked with * (P<0.005);

[0036] FIG. 6 shows the image of an immunoblot relating to a membrane representative of the expression of genes of the CCN family following stimulation with LH;

[0037] FIG. 7 shows the correlation between the basal LH and the percentage increase in AMH.

DEFINITIONS

[0038] In the context of the present invention, the term “International Unit” (IU) as used here means a unit of measurement of the amount of a substance, based on its effect or on its biological activity.

[0039] In the context of the present invention, the term “ultrasound count of antral follicles” or “AFC” means the count of antral follicles in an individual obtained by means of a transvaginal pelvic ultrasound scan.

[0040] In the context of the present invention, the term “ovarian reserve” means the number and/or quality of oocytes, reflecting the ability to reproduce. Ovarian reserve can be assessed by any of several means. They include: female age; number of antral follicles on ultrasound; anti-Mullerian hormone levels; follicle stimulating hormone and oestradiol levels; clomiphene citrate challenge test; response to gonadotropin stimulation, and oocyte and/or embryo assessment during an ART procedure, based on number, morphology or genetic assessment of the oocytes and/or embryos.

[0041] In the context of the present invention, “CCN” means a family of extracellular matrix-associated proteins involved in intercellular signalling. Because of their dynamic role within the ECM they are considered matricellular proteins.

DETAILED DESCRIPTION OF THE INVENTION

[0042] A first aspect of the present invention relates to luteinising hormone (LH) or a molecule having LH-like activity for use in inducing the activation and/or maturation or development of primordial and/or primary follicles in an individual.

[0043] In one embodiment, the molecule having LH-like activity is selected from the group consisting of chorionic gonadotropin (CG) or an LH-agonist. Examples of LH agonists are drug-like low-molecular-weight ligands that interact allosterically within the seven transmembrane domains of the LH/CG receptors as thienopyrimidines and other pharmacological chaperones (pharmacoperones)

[0044] In fact, the Applicant has demonstrated that the administration of LH or of a molecule having LH-like activity is capable of stimulating the process leading to the activation of primordial follicles, or their maturation into primary follicles. The maturation of primordial follicles is in fact associated with an increase in primary follicles and a decrease in primordial follicles. In one embodiment, the activation and/or maturation or development of primordial and/or primary follicles is associated with an increase in the ovarian reserve of the individual. The increase in the ovarian reserve is preferably defined by means of an ultrasound count of antral follicles (AFC) and/or by means of a serum assay of anti-Müllerian hormone (AMH). The Applicant has demonstrated that the administration of LH or of a molecule having LH-like activity is capable of increasing the ovarian reserve in an individual, with a consequent increase in the AFC values and in the serum levels of AMH.

[0045] Furthermore, contrary to the prior art, the applicant has demonstrated that LH or a molecule having LH-like activity, is capable of stimulating the first phase of folliculogenesis, known as gonadotropin-independence.

[0046] Table 1 shows the differences between the present invention and the use of LH for ovarian stimulation, i.e., for stimulation of the gonadotropin-dependent phase.

TABLE-US-00001 TABLE 1 Inducing the activation, and/or the maturation of primordial Ovarian stimulation to and/or primary follicles promote folliculogenesis (Present invention) (Prior art) time Before ovarian stimulation During ovarian stimulation duration at least 30 days 14 days dose 150-450 U 75-150 IU Concomitant Not allowed Mandatory treatment with FSH

[0047] Preferably, the individual is a human subject of the female sex.

[0048] In one embodiment of the invention, the individual has an ovarian reserve within the norm. Preferably, the individual has an AFC value greater than 10, more preferably greater than 12 and/or the individual has a serum concentration of AMH greater than 2 ng/ml, more preferably greater than 2.5 ng/ml.

[0049] In one embodiment of the invention, the individual has a reduced ovarian reserve. Preferably, the individual has an AFC value lower than 10, more preferably lower than 8 and/or the individual has a serum concentration of AMH lower than 2 ng/ml, more preferably lower than 1.5 ng/ml.

[0050] In one embodiment, said LH is human LH (hLH) or said CG is human chorionic gonadotropin (hCG); preferably, said LH is recombinant human LH (rhLH) or extracted from urine or said CG is recombinant human chorionic gonadotropin (rhCG).

[0051] Said L H or said CG is preferably used as a recombinant protein or purified/isolated.

[0052] In this context, reference is preferably made to the use of the entire LH or CG protein, or to homologues, analogues, variants, derivatives or fragments of the LH or CG protein on condition that the activity of LH or CG is maintained.

[0053] In one embodiment, said LH or CG is a biologically active homologue. In one embodiment of the invention, the protein variants of LH or CG to which reference is made have modifications in the N-terminal and/or C-terminal regions, for example adapted to increase the activity of LH or CG. Said modifications are preferably selected from among deletions, additions, alterations of amino acids and combinations thereof. Alternatively, said LH or CG can be modified, preferably in its primary structure, by acetylation, carboxylation, glycosylation, phosphorylation and combinations thereof.

[0054] In a further embodiment, said LH or CG is conjugated/bound to a molecule, a metal, or a marker, for example proteins, for the preparation of fusion proteins. In a further embodiment of the invention, said LH or CG is modified by means of molecular biology techniques to improve its resistance to proteolytic degradation and/or to optimise its solubility or to improve its pharmacokinetic characteristics. In a further embodiment, said LH or CG, preferably the protein form, is conjugated to at least one molecule capable of improving its stability and/or its half-life and/or its solubility in water and/or its immunological characteristics. Said molecule, by way of example, is polyethylene glycol (PEG).

[0055] In a further embodiment of the invention, said LH or CG protein is synthesised by means of conventional protein synthesis techniques known to the skilled person. For example, the protein can be synthesized by chemical synthesis using solid-phase peptide synthesis. Alternatively, LH or CG can be produced with recombinant DNA techniques known to the person skilled in the art.

[0056] In a further embodiment of the invention, the LH or CG protein, after the synthesis thereof or production thereof with recombinant DNA techniques, is isolated or purified with methods known to the person skilled in the art. For example, LH or CG can be purified with chromatographic methods (gel-filtration, ion exchange and immunoaffinity), by means of high-performance liquid chromatography (HPLC, RP-HPLC, ion exchange HPLC, size-exclusion HPLC) or by precipitation (immunoprecipitation).

[0057] In one embodiment, said LH or the molecule having LH-like activity is used, for the medical purposes stated above, in an amount comprised between 150 and 450 International Units (IU) per day, more preferably between 170 and 350 IU per day, even more preferably comprised between 180 and 250 IU per day.

[0058] In one embodiment, LH or the molecule having LH-like activity is taken at least once a day, preferably at least twice a day. For the medical purposes described above, LH or the molecule having LH-like activity is taken for a period of time of at least 30 days, preferably for a period comprised between 40 and 120 days, more preferably comprised between 50 and 100 days. In a preferred embodiment, LH or the molecule having LH-like activity is taken per at least 60 days. Preferably, LH or the molecule having LH-like activity is administered parenterally, preferably subcutaneously or intramuscularly.

[0059] In one embodiment, LH or the molecule having LH-like activity is taken in association or in combination with an infertility treatment, preferably in association or in combination with ovarian stimulation.

[0060] LH or the molecule having LH-like activity is preferably taken before the infertility treatment, preferably before ovarian stimulation.

[0061] In one preferred embodiment, LH or the molecule having LH-like activity is taken neither in association nor in combination with FSH.

[0062] A second aspect of the present invention relates to a composition comprising LH and/or a molecule having LH-like activity for the medical uses described above. In one embodiment, the composition comprises salts, buffers, excipients, carriers, preservatives and/or combinations thereof accepted for the preparation of pharmaceutical products.

[0063] In one embodiment, the composition is formulated for parenteral administration, preferably for subcutaneous or intramuscular administration. The composition is preferably formulated in liquid form, preferably in the form of a sterile solution, emulsion or suspension, or else it is in powder form, preferably lyophilised, so as to be reconstituted to obtain a liquid formulation. In a preferred embodiment, the composition is formulated as a powder, preferably lyophilised, so as to be reconstituted to obtain a liquid formulation.

[0064] In one embodiment of the invention, said composition is formulated for enteral administration, preferably for oral administration. In particular, the composition is formulated in solid form, preferably in the form of lozenges, capsules, tablets, granular powder, hard-shelled capsules, orally dissolving granules, sachets or pills.

[0065] A third aspect of the present invention relates to a method for inducing the activation and/or the maturation or development of primordial and/or primary follicles in an individual. Said method comprises at least a step of administering an effective amount of LH or of a molecule having LH-like activity or of a composition comprising it to an individual who has a need for it.

[0066] In one embodiment, the activation and/or maturation of primordial and/or primary follicles is associated with an increase in the ovarian reserve of the individual. The increase in the ovarian reserve is preferably defined by means of an ultrasound count of antral follicles (AFC) and/or by means of a serum assay of anti-Müllerian hormone (AMH).

[0067] Preferably, the individual is a human subject of the female sex.

[0068] In one embodiment of the invention, the individual has an ovarian reserve within the norm. The individual preferably has an AFC value greater than 10, more preferably greater than 12 and/or the individual has a serum concentration of AMH greater than 2 ng/ml, more preferably greater than 2.5 ng/ml.

[0069] In one embodiment of the invention, the individual has a reduced ovarian reserve. The individual preferably has an AFC value lower than 10, more preferably lower than 8 and/or the individual shows a serum concentration of AMH lower than 2 ng/ml, more preferably lower than 1.5 ng/ml.

[0070] In one embodiment, the method for inducing the activation and/or the maturation of primordial and/or primary follicles in an individual is associated or combined with a method for treating infertility in an individual.

[0071] In one embodiment, LH or the molecule having LH-like activity is taken in association or in combination with an infertility treatment in an individual, preferably in association or in combination with ovarian stimulation.

[0072] LH or the molecule having LH-like activity is preferably taken before the infertility treatment, preferably before ovarian stimulation.

[0073] In one preferred embodiment, LH or the molecule having LH-like activity is taken on its own and not in association with FSH.

Example

[0074] In Vitro Study

[0075] In mammals, the ovaries contain a limited and finite number of oocytes organised in primordial follicles. The majority of primordial follicles are maintained quiescent as a reserve for the duration of reproductive life. Only a few of them are activated and develop into more advanced follicular stages. Although the molecular mechanisms that regulate the maintenance of dormancy and the activation of primordial follicles have not been completely understood, various studies have demonstrated that they depend on coordinated actions of inhibiting/activating molecules produced by the oocyte and the communication of the oocyte itself with somatic and intraoocyte cells.

[0076] For these reasons, the aims of this study are to investigate a) whether luteinising hormone (LH) can promote the cytoplasmic-nuclear translocation of FOXO3a and b) whether LH can be capable of activating members of the CCN family in in-vitro cultures of ovarian tissue.

[0077] Materials and Methods

[0078] Ovarian Tissue Donors

[0079] 2×2×2 mm.sup.3 pieces of ovarian cortical tissue were obtained from biopsies taken from patients undergoing diagnostic laparoscopy and salpingochromoscopy as a workup in the study of couple infertility. The patients enrolled in this study historically had a regular menstrual cycle and stated that they had no pathologies at the time of sampling. All patients were asked for and granted written consent to the use of the samples and of the clinical data for research purposes.

[0080] Tissue Preparation

[0081] The ovarian tissue was obtained under conditions of sterility and transferred to a Petri dish containing 1× PBS, where the medulla tissue was separated from the cortex by using a scalpel. The ovarian cortical tissue was further divided into smaller pieces with a size of 1 mm.sup.3. A single piece deriving from the initial biopsy was directly fixed in 4% paraformaldehyde at 4° C. overnight for the immunohistochemical analysis. A single piece deriving from the initial biopsy was instead treated for total RNA extraction with the method described below. The remaining pieces were deposited in a 12-well multiwell plate (one piece per well) and covered with a pre-balanced culture medium (500 μl/well; 1 hour at 37° C.). The culture medium was composed of 25 mM HEPES, 1 mM penicillin/streptomycin, 1 mM L-glutamine, 1 mM amphotericin B, and 10% foetal bovine serum (FBS) dissolved in the basic medium McCoy 5A. All the reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). The samples were cultured at 37° C. under conditions of a humidified atmosphere containing 5% CO2 for 24 hours before the treatments.

[0082] Treatments

[0083] After 24 hours of primary culture, the samples were treated with 10 μM of LH (dissolved in the culture medium) for an additional 48 hours (one change of medium after the first 24 hours). The control samples were cultured only in the medium without LH. The samples thus treated were processed for total RNA extraction or for the immunohistochemical analysis as described below.

[0084] RT-qPCR

[0085] The single piece of ovary deriving from the original biopsy and the samples treated with 10 μM of LH (and the corresponding controls) were directly lysed in 1 ml of Tri-Reagent® (Sigma Aldrich) and immediately processed for total RNA extraction according to the protocol described by the manufacturer. The extracted RNA was placed in 20 μl RNase-free water and digested with DNase I (Promega, Madison, WI, USA). The purified RNA was quantified using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientic, Waltham, MA, USA). The messenger RNA corresponding to every sample was reverse transcribed into cDNA using M-MuLV reverse transcriptase (NEB, Ipswich, MA, USA).

[0086] The cDNAs were used as a template for RT-qPCR reactions.

[0087] For every gene considered, the samples were evaluated in triplicate and expressed as a mean in order to allow an analysis of gene expression. The results were normalised using the expression of the β-actin gene expressed constitutively. The negative controls of the reactions amplified distilled water in place of the cDNA. The genes evaluated were: CCN2, CCN3 and CCN5. The relative expression of every gene was calculated by applying the 2.sup.−ΔΔCt method described by Livak and Schmittgen, 2001.

[0088] Slide Preparation

[0089] The samples deriving from the original biopsy, the samples treated with LH (and the corresponding control samples) were fixed in 4% paraformaldehyde at 4° C. overnight. The samples were subsequently dehydrated with an ascending alcohol series and embedded, after clearing in xylene, in blocks of paraffin. Serial slices of ovary were obtained by cutting with a microtome (7 μm) and used for immunohistochemical analysis or morphological staining (haematoxylin and eosin) to enable a count of the follicles.

[0090] Immunohistochemistry

[0091] The paraffin was removed from the slides with xylene and they were rehydrated with a descending alcohol series. The slides were unmasked to allow the exposure of nuclear antigens. The slides were treated with the primary anti-FOXO3a or anti-p-FOXO3a antibody (mouse anti-human; 1:100) and subsequently incubated with a secondary antibody conjugated to TRITC or FITC (goat anti-mouse; 1:10000). All of the antibodies used were purchased from Santa Cruz, CA, USA. The samples were counterstained with DAPI to enable identification of the nuclei. A double-blind count was made of the positive follicles by two different operators who randomly selected at least 5 fields per slide.

[0092] SDS-PAGE

[0093] The proteins contained in the individual samples were extracted with RIPA buffer and quantified with the Bradford assay. A same amount of proteins (50 μg) was mixed with 2× loading buffer (4% SDS; 20% glycerol; 0.004% bromophenol blue; 0.125 M Tris-HCl at pH 6.8; 10% 2-mercaptoethanol), 1× protease inhibitor cocktail and 5 mM phenylmethylsulfonyl fluoride (PMSF, Sigma Aldrich, St. Louis, MO, USA) and finally boiled for 10 minutes to linearize the proteins. The protein extracts were resolved by means of a 12% polyacrylamide/bis-polyacrylamide gel under denaturing conditions. The ColorBurst Electrophoresis Marker (Sigma Aldrich, St. Louis, MO, USA) was used as a molecular weight marker. The samples were loaded in triplicate in three identical gels. The proteins were run for 90 minutes at a constant voltage (120 V).

[0094] Immunoblot

[0095] The proteins separated on the basis of molecular weight were transferred using the Trans-Blot SD semi-dry transfer cell (Bio-Rad, Hercules, CA, USA) on three membranes (one per gel) made of polyvinylidene fluoride (PVDF, Thermo scientific, Rockford, IL, USA). The membranes were blocked overnight in a solution of Tris-buffered saline (TBS, pH 7.4) and 5% partially skimmed milk (skim milk powder, Sigma Aldrich, St. Louis, MO, USA). The membranes were subsequently incubated for 1 hour individually with anti-CCN2, —CCN3 or —CCN5 antibody (Santa Cruz Biotechnology, Dallas, TX, USA) diluted 1:200. After three washes with 0.05% Tween 20 (Sigma Aldrich, St. Louis, MO, USA) in TBS, the membranes were further incubated with the rabbit anti-goat secondary antibody, horseradish peroxidase (HRP) conjugated (Bethyl, Montgomery, TX, USA) 1:10000. Finally, the membranes were covered with ECL Western Blotting Substrate (Thermo Scientific, Rockford, IL, USA) and the bands were detected by chemiluminescence using ChemiDoc XRS+ (Bio-Rad, Hercules, CA, USA). The signals were acquired and semi-quantified by means of a system for the analysis of digital images (VersaDoc Imaging System and QuantityOne software, Bio-Rad Laboratories Inc.). After the acquisition of the images, the membranes were washed and rehybridised with the anti-human β tubulin primary antibody (Abcam, Cambridge, UK) obtained in rabbits diluted 1:500 and processed as described previously. After development, the intensity of the β tubulin bands was used as an internal control on the initially loaded quantity of proteins.

[0096] Statistical Analysis

[0097] A statistical analysis was conducted on the relative expressions obtained with RT-qPCR by combining the Kruskal-Wallis test followed by Dunn-Bonferroni's test (P<0.005). A statistical analysis of the bands after immunoblotting was conducted using GraphPad Prism software and applying a two-way ANOVA test, considering a probability P<0.05 as significant. For the count of ovarian follicles, the number of follicles was compared within the treated groups using an ANOVA test followed by a post hoc test. Differences with a probability P<0.05 were considered significant.

[0098] Results

[0099] As shown in FIG. 2, the number of primary follicles was increased by the treatment with LH (with a consequent decrease in the number of primordial follicles). A well-documented phenomenon of spontaneous activation of primordial follicles following the culturing of ovarian tissue is also present in the experiments. In fact, the controls (ovarian tissue in culture without LH) show a significant increase in the number of primary follicles compared to those present in the original biopsy. LH revealed to be capable of increasing the number of the primary follicles versus both the controls and the original biopsies. FIG. 3 shows the percentage of follicles in the various stages of maturation in relation to the total present in the initial biopsy. The changes in the percentages of the various subpopulations were significant both in the control samples and in the samples treated with LH. LH showed to be effective in increasing not only the number of follicles which, on passing through the phenotype transition, reach the primary follicle stage, but also the number of the follicles that progress beyond this phase to reach the pre-antral phenotype. FIG. 4 summarises the percentage of oocytes showing to be positive at the cytoplasmic level compared to the percentage of oocytes wherein FOXO3a was localised in the nucleus. LH induced the activation of primordial follicles by promoting the translocation of FOXO3a from the nucleus to the cytoplasm. LH was capable of inducing the relative expression of all three genes and of the proteins belonging to the CCN family after two days of treatment, confirming that the phosphatidylinositol-3-kinase pathway was activated (FIGS. 5 and 6).

[0100] In Vivo Study

[0101] A prospective pilot trial was conducted on 30 patients affected by idiopathic infertility. The aim of the study was to investigate the effects of a medium/long-term treatment with exogenous LH on the ovarian reserve, namely the number of antral follicles (AFC) and the circulating AMH. The hypothesis behind this study was that LH may activate or promote the growth of primordial and/or primary follicles hence leading to an increase in preantral and antral follicles which may be recognized in vivo by ultrasound (the antral follicle count-AFC) or by measuring AMH (the hormone produced by the antral follicles).

[0102] The age of the enrolled patients ranged from 18 to 40 years. Exclusion criteria were: presence of known infertility factors (endocrine-ovulatory factor, tubal factor, endometriosis), presence of ovarian cysts, presence of endocrine/metabolic disorders, and male factor. The demographic characteristics of the patients are shown in table 2. All of the patients had regular menstrual cycles (every 28-35 days).

TABLE-US-00002 TABLE 2 Patient characteristics (n = 30) Age (M ± SD) 32 ± 5  BMI  22 ± 2.5 Duration of infertility (months) 18 ± 9  Primary infertility (%) 90% Average duration of cycle (days) 29 ± 2  AMH (ng/ml) 1.6 ± 0.6 D3FSH (IU/L) 6.5 ± 1.9 D3E2 (pg/ml) 45 ± 23 D3 LH (IU/L) 4.5 ± 2.6 AFC (n)  12 ± 4.4

[0103] The 30 patients in the study were treated with LH at a dosage of 187.5 U per day (75 U in the morning and 112.5 U in the evening) for two months. They underwent an analysis of the ovarian reserve by means of an assay of serum AMH and an ultrasound count of antral follicles (AFC) at time 0, after one month and at the end of the therapy (2 months). Of the 30 patients, 11 had undergone a cycle of in vitro Fertilization (IVF) in a time interval ranging from 10 days to 90 days before the start of the therapy with LH. These patients repeated a second cycle of IVF after the two months of therapy with LH.

[0104] Aims of the Study

[0105] Primary aim of the study: to evaluate the effect of LH administration on ovarian reserve markers, namely AFC and AMH.

[0106] Secondary aim: to evaluate the predictive criteria of ovarian response to the LH therapy. To evaluate the outcome of IVF cycles before and after the long-lasting therapy with LH.

[0107] Results of the Study

[0108] It emerged from the study that, after treatment with LH for two months, there was an average 60% increase in AFC and 52% increase in AMH (Table 3)

TABLE-US-00003 TABLE 3 Ovarian reserve markers after one and two months of therapy 1st month of 2nd month of Basal therapy therapy AMH (ng/ml) 1.6 (±0.61) 2.1 (±0.92) 2.43 (±1.2) (mean ± SD) % change in AMH Na 31% 52% AFC (mean ± SD) 12 (±4.4)  15 (±7.7)  19.2 (±8.4) % change in AFC Na 25% 60%

[0109] Of the 30 patients enrolled, 12 showed an extremely reduced ovarian reserve (defined as AMH <1 ng/ml and/or AFC<7). In these patients the response to the therapy was comparable to that found in the patients with a normal ovarian reserve.

[0110] Using multiple regression analysis, we then investigated which variables can predict a positive response to therapy with LH and it emerged that a younger age and low serum levels of FSH and LH are the criteria predictive of a good response to therapy with exogenous LH. In particular, the percentage increase in AMH was greater when the age was lower and the basal plasma levels of LH were lower (FIG. 7).

TABLE-US-00004 TABLE 4 Predictors of the Increase in AMH after therapy with LH Dependent Y Increase in AMH Analytical Enter Sample size 30 Coefficient of Determination R.sup.2 0.7226 R.sup.2-adapted 0.7021 Multiple correlation coefficient 0.8501 Residual standard deviation 15.3146

[0111] Regression Equation

TABLE-US-00005 Independent variables Coefficient Std. Error t P (Constant) 185.61452 age −2.68351 0.86582 −3.099 0.0045 LH −10.24955 2.99391 −3.423 0.0020

[0112] Analysis of Variance

TABLE-US-00006 Source DF Sum of squares Mean squares Regression 2 16497.6808 8248.8404 Residual 27 6332.5261 234.5380 F-Ratio 35.1706 Significance P < 0.001

[0113] Zero-Order Correlation Coefficients

TABLE-US-00007 Variable R age −0.776 LH −0.790

[0114] Pre-treatment with LH and IVF outcome Some of the recruited patients had undergone a cycle of ovarian stimulation in the 3 months preceding the therapy with LH, without achieving pregnancy. These patients underwent a second IVF cycle after two months of therapy with LH. The comparison between the two ovarian stimulations is shown in table 5. It emerged that the treatment with LH led to an increase in the embryos obtained. In particular, it enabled more blastocysts to be obtained and more patients had cryopreserved embryos after the ovarian stimulation treatment.

TABLE-US-00008 TABLE 5 Cycle before the Cycle after the LH treatment LH treatment N 11 11 Age 34 ± 5  34 ± 5  Basal AMH (ng/ml) 1.2 ± 0.3 1.7 ± 0.5 AFC (n) 10 ± 4  14 ± 6  Starting dose of FSH 225 IU 225 IU % patients with d2/d3 embryos 91% 64% % patients with d5 embryos  9% 36% Average number of cleavage stage 2 ± 1 4 ± 2 embryos available for transfer Number of blastocysts 1 ± 0 2 ± 1 available for transfer % of patients who have 18% 54% supernumerary embryos Clinical pregnancy  0 36%