Use of N-acetyl-5-methoxytryptamine or analogues thereof, for promoting the mechanism of implantation of the embryo and related compositions and culture media

Abstract

The present invention refers to the use of N-acetyl-5-methoxytryptamine (melatonin) and/or an analogue thereof, for use in the medical or veterinary field in the assisted reproduction for promoting the mechanism of implantation of the embryo, and in particular for the prevention of implantation failure into the uterus, by topical administration of an effective amount in a mammalian subject female in need of such treatment, and related compositions, culture media and medical devices.

Claims

1. A method for treating implantation failure in a uterus of a mammalian female subject undergoing assisted reproduction comprising topically administering an effective amount of an active ingredient selected from N-acetyl-5-methoxy tryptamine, an analogue thereof, and a combination of N-acetyl-5-methoxy tryptamine and an analogue thereof, to a mammalian female subject in need thereof, wherein the active ingredient is formulated as an endometrial irrigation or uterine washing or endometrial washing in a medium for cell culture at a final concentration ranging from 410.sup.9 g/ml to 2510.sup.9 g/ml.

2. The method of claim 1, wherein the analogue is selected from agomelatine, 6-hydroxymelatonin, serotonin, 5 hydroxytryptophan, and their derivatives.

3. The method of claim 1, wherein the mammalian female subject is a woman suffering from infertility or polyabortion.

4. The method of claim 1, wherein the step of topically administering is via endometrial irrigation or uterine washing or endometrial washing.

5. The method of claim 1, wherein the step of topically administering is carried out in a single administration at the time of oocyte retrieval.

6. The method of claim 1, wherein the N-acetyl-5-methoxy tryptamine or analogue thereof is present in a concentration ranging from 1010.sup.9 g/ml to 2510.sup.9 g/ml.

7. The method of claim 1, further comprising contemporaneously systemically administering of N-acetyl-5-methoxy tryptamine, hCG, or progesterone, or a combination thereof, from the day of oocyte retrieval.

8. The method of claim 1, wherein the assisted reproduction is a technique selected from the group consisting of planned copulation; intrauterine insemination (IUI); in vitro insemination and embryo transfer (FIVET); in vitro fertilization (IVF); intracytoplasmic sperm injection (ICSI); intracytoplasmic morphologically selected sperm injection (IMSI) techniques; and Tesa-Tese (Testicular Sperm Aspiration-Extraction).

Description

(1) The present invention will be now described in an illustrative manner, but not limitative, according to two preferred embodiments, with particular reference to the annexed figures, wherein:

(2) FIG. 1 illustrates the endometrium before treatment through the composition according to the present invention, therefore in the absence of pinopodes;

(3) FIG. 2 illustrates the endometrium 1/2 days after treatment through the composition according to the present invention: the formation of pinopodes starts to come into sight;

(4) FIG. 3 illustrates the endometrium 3/4 days after treatment through the composition according to the present invention: maximum growth of pinopodes;

(5) FIG. 4 illustrates the endometrium 5/6 days after treatment through the composition according to the present invention: it starts cleavage of pinopodes, which appear to be irregular (cell apoptosis).

(6) FIG. 5 shows the results of the FACS analysis (fluorescence intensity measurement) of the antioxidant activity of melatonin, agomelatine and 6-hydroxymelatonin (170 nM corresponding to 4010.sup.9 g/ml) carried out at H.sub.2O.sub.2 concentrations of 20 M and 200 M;

(7) FIG. 6 shows the results of apoptosis assay (% cell death) carried out at different concentrations (80 nM, 170 nM, 340 nM; corresponding respectively to 1810.sup.9 g/ml, 4010.sup.9 g/ml, 8010.sup.9 g/ml) of melatonin, agomelatine and 6-hydroxymelatonin.

(8) Merely for example, but not limitative of the present invention, hereinafter are reported the comparative studies carried out (in vitro and in vivo) by the authors of the present invention to evaluate the percentage increase of human embryonic implantation in MAR techniques through the use of melatonin in different solutions.

EXAMPLE 1

In Vivo Studies

(9) Materials and Methods

(10) Solutions of Melatonin Used for Endometrial Washings

(11) The melatonin (or N-acetyl-5-methoxy tryptamine; CAS Number 73-31-4) used has been obtained at Farmalabor as product in form of powder with title 99%.

(12) Two clinical trials have been carried out providing the administration of melatonin (concentration 1010.sup.9 g/ml) through endometrial washing in: 1) culture medium Sydney IVF Blastocyst medium (Clinical trial 1) supplied by Cook Ireland Ltd (Catalogue Number G20722 and G20929). Said culture medium is normally used to improve cleavage, differentiation and expansion in vitro of blastocyst. The medium contains D-glucose, all the 20 essential L-amino acids, L-taurine, Gentamicin, human serum albumin, calcium lactate, calcium pantothenate; sodium chloride, sodium bicarbonate and sodium pyruvate; potassium chloride, potassium phosphate; magnesium chloride, magnesium sulfate; purified water. Further characteristics of the culture medium: pH: 7.5-7.8 (use of bicarbonate buffer) Osmolarity: 280-290 mOsm/kg Endotoxins: <0.4 EU/ml MEA: 80% 2) physiological solution (Clinical Trial 2)
Statistical Analysis:

(13) It has been carried out a statistical analysis (IMPRUN.TXT) of linear regression with more variables.

(14) Statistical Methods:

(15) The role of the number of implanted embryos, of their quality and of the washing process with the solution containing melatonin in the increasing of the chance of pregnancy has been computed by means of non-conditional logistic regression, by adjustment according to the mother's age.

(16) The ratio between the chance of pregnancy associated with each procedure (number of embryos, their quality and application of the washing process with saline solution or Blastocyst medium containing melatonin) and the chance of pregnancy with reference respectively to the lower category of number of embryos (one), to the quality less than optimal of implanted embryos, or to the absence of the washing process, has been defined as Odds Ratio (OR), and it has been computed through non-conditional logistic regression, by adjustment according to the mother's age.

(17) In the logistic regression, OR corresponds to the antilogarithm on a natural basis of the regression coefficient associated with each covariate in the regression model.

(18) The value thus calculated expresses therefore the advantage obtained through each procedure, irrespective of the other conditions.

(19) The two-tailed confidence intervals at 95% (IF95%) of the OR have been calculated through the formula of Wald (e(z.sub./2*se.sub.)).

(20) Sterility Controls (Culture Medium)

(21) To carry out the sterility controls of the culture medium supplemented with melatonin it has been used the BACT/ALERT 3D-60 device (BIOMERIEUX). The bottles of Bact/ALERT culture are applied to systems of microbial detection in qualitative procedures for recovering and detecting optional anaerobic and aerobic (bacteria) in the blood and other fluids normally sterile as the Blastocyst medium used in the present experimentation.

(22) The Bact/ALERT system of microbial detection is used to determine whether microorganisms are present in the samples of blood or of other fluids normally sterile as the blastocyst medium used in our invention, with suspected bacteremia. The Bact/ALERT system and the culture bottles offer a system of microbial detection and a culture medium with environmental and nutritional conditions suitable for microorganisms commonly present in blood infections and other fluids normally sterile.

(23) The inoculated bottles (from a minimum of 5 ml to a maximum of 10 ml of sample at issue) are incubated in the device, where they are subjected to continuous monitoring in order to detect a possible growth of microorganisms in the Bact/ALERT bottles.

(24) The Bact/ALERT system of microbial detection uses a colorimetric sensor and reflected light for monitoring the presence and the production of carbon dioxide (CO.sub.2) dissolved in the culture medium.

(25) The microorganisms possibly present in the sample metabolize the substrates in the culture medium producing carbon dioxide. The production of CO.sub.2 determined by the growth of microorganisms induces the gas permeable green-blue sensor present on the bottom of each culture bottle to take a yellow colour.

(26) The lighter colour indicates an increase of the units of monitoring reflectance of the system.

(27) The reflectance of the bottle is monitored and traced by the device every 10 minutes.

(28) The culture bottles are established as being positive or negative by the managing software of Bact/ALERT systems of microbial detection after 6 days of incubation.

(29) No intervention is necessary up to the moment in which the Bact/ALERT device alerts that a culture bottle is positive or negative.

(30) Before carrying out any endometrial irrigation culture tests have been carried out for testing sterility in 2-4-6 days and once the negative culture was found it was used into the uterus.

(31) Criteria of Inclusion

(32) Restricted criteria of inclusion have been used for selecting the patient population involved in the studies: 3) age 20 and <44 years 4) body mass index (BMI) 20 and 28 kg/m.sup.2 5) Basal FSH19 UI/l 6) Male and female factors
Patients

(33) The patients involved in all the two clinical studies, after collecting informed consent, have been subjected to assisted reproduction.

(34) In this first two-arms randomized multi-centre clinical study (control and study group) involved about 430 patients/arm.

(35) The patients undergoing assisted fertilization have been randomized the day of oocyte retrieval in three groups:

(36) Group A: 430 patients to be subjected to endometrial irrigation with 1.5 ml of physiological solution (controls).

(37) Group B: 436 patients to be subjected to endometrial irrigation with a solution of 1.5 ml of culture medium supplemented with final concentration of melatonin (1010.sup.9 g/ml).

(38) Group C: patients not subjected to endometrial irrigation.

(39) The study has been carried out involving patients in several private centers specialized in MAR, such as the Center of BRA, the Promea Center of Torino and Cagliari, the Genera Center of Perugia and the polyclinic Public Center of Bari. To these centres it has been sent the culture medium supplemented with melatonin after having been subjected to sterility tests to carry out the following protocol of treatment.

(40) After in vivo enrolling for assisted fertilization by endometrial irrigation with known concentration (final concentration of 1010.sup.9 g/ml) of melatonin at the time of oocyte retrieval, patients were subjected to related ultrasound-guided visualization of the diameter of liquid stratum created on the bottom of the uterus.

(41) The second clinical study with a single prospective centre involved 64 patients in the group of control and 92 patients in the group of study.

(42) In said study melatonin has been administered in sterile physiological solution, through endometrial washing inside the uterine cavity of patients, following to oocyte retrieval.

(43) Endometrial Washing

(44) Briefly, the treatment consists in endometrial irrigation inside the uterine cavity without any cell in co-culture, using the physiological solution or the culture medium admixed with melatonin with final concentration of 1010.sup.9 g/ml (preceding studies had been effected with one third and the half of the final concentration then maintained without giving the same resultsnot shown data).

(45) After oocyte retrieval, (the time correspondent to the ovulation after the administration of hCG in the protocols of MAR) after having controlled the vaginal haemostasis, it has been jointed a sterile syringe from 2.5 ml to a single-lumen intrauterine catheter with apical opening and the culture medium (Blastocyst medium) or the physiological solution has been suctioned, modified through addition of melatonin up to a volume of 1.5 ml.

(46) In the case of the physiological solution, the catheter can be pre-filled with physiological solution supplemented with melatonin.

(47) The catheter has been introduced in the internal uterine orifice with the same steps through which the operator carries out the conventional embryo-transfer (ET).

(48) Then, it has been slowly injected the saline solution supplemented with melatonin or the modified medium and, at the end of irrigation, the thickness of the endocavitary liquid stratum has been measured by transabdominal echography (washing and echography must be simultaneous, the liquid stratum arrives at about 10 mm and then quickly disappears).

(49) Results

(50) Clinical Study 1

(51) The multi-centre in vivo study involved 863 patients subjected to assisted fertilization and to endometrial irrigation (echographic measurements of the liquid stratum) with melatonin and 3-4 days after that, they have been subjected to embryonic transfer. For the latter, the CI (confidence interval or trust interval) has been calculated through statistical analysis (IMPRUN. TXT) of linear regression with more variables of 95%.

(52) The univariate analysis shows that the implantation of 3 embryos implies a significant increase, equal to about 3 times (OR=2.8, IF 95% 1.7-4.4) the chance of pregnancy.

(53) A number of two embryos does not appear instead to be sufficient to increase the chance of pregnancy (OR=1.0, IF 95% 1.7-4.4).

(54) On the other hand, also the optimal quality of the implanted embryos seems to be an important factor in increasing the chance of pregnancy (OR=2.9, IF 95% 1.8-4.7). In the univariate analysis, the same rate of relevance appears to be associated with the use of the irrigation procedure (OR=2.2, IF 95% 1.6-2.9).

(55) The multivariate analysis allows to control the independent effect of each variable, therefore totally irrespective of the effect of the other applied procedures and of the mother's age. In this case, the use of the washing procedure doubles the chance of success of pregnancy (OR=2.2, IF 95% 1.6-3.0).

(56) A similar improvement appears to be associated with the implantation of three embryos, rather than the implantation of a single embryo (OR=2.1, IF 95% 1.2-3.7), and to the use of embryos of optimal quality (OR=1.9, IF 95% 1.1-3.3).

(57) The use of all the three procedures as above reported seems to be therefore the best choice in the procedures of assisted fertilization.

(58) In conclusion, the in vivo studies have proved that the irrigation or the washing with melatonin of endometrial cells allows to double the number of pregnancies with respect to the other two groups, which did not differentiated significantly.

(59) Clinical Study 2

(60) The single centre study involved 64 patients in the group of control and 92 patients in the group of study.

(61) The patients have been subjected to assisted reproduction and to endometrial washing (echographic measurements of the liquid stratum) with melatonin in sterile saline solution, and 3-4 days after they have been subjected to embryonic transfer.

(62) The primary endpoint of the study is the rate of clinical pregnancies (fetal heart) due to embryonic transfer (ET); the secondary endpoint is the rate of ongoing pregnancies and aborts.

(63) The rate of clinical pregnancies resulted to be equal to 37% in the group of study vs 17.2% in the group of control, whereas the rate of ongoing pregnancies resulted to be equal to 32.6% in the group of study vs 14.1% in the group of control.

(64) The results have been reported in the following Table 1.

(65) TABLE-US-00001 TABLE 1 CONTROL WASHING No (%) No (%) No ET 64 92 Age (years SD) 38.8 1.2 36 4 No embryos 2.7 1.1 2.5 1.08 transferred (Average SD) No embryos 1.5 1.sup. 1.3 0.8 Grade A (Average SD) No embryos 0.9 0.7 1.17 0.8 Grade B (Average SD) No embryos 0.2 0.5 0.1 0.3 Grade C (Average SD) Day ET 3.5 0.5 3.5 1.2 (Average SD) BHCG Positive 12 18.8% 41 44.6% (on total of ET) Clinical 11 17.2% 34 37.0% Pregnancy/ET Ongoing 9 14.1% 30 32.6% Pregnancy/ET Aborts 1 9.1% 4 11.8%

(66) The endometrial washing carried out with the various solutions containing melatonin, allows to double the rate of clinical pregnancies and ongoing pregnancies vs the group of control in the infertile female population.

(67) The data seem to confirm that the removal of endometrial exudate using the physiological solution with melatonin can create a physiological endometrial environment and improve implantation success.

(68) This allows to obtain a valid and innovative means for increasing the implantation rates in assisted reproduction technologies (ART).

EXAMPLE 2

In Vitro Study

(69) Materials and Methods

(70) The melatonin and the culture medium used are the same as those used for the in vivo study.

(71) Electron Microscopy (SEM)

(72) It has been used a field emission at high resolution electron microscope, FE HITACHI S 4000 model, operating at 15-20 KW.

(73) Preparation of Samples

(74) Soon after retrieval from the patient, small pieces of endometrial tissue of about 1 mm in size have been placed in a fixing solution composed of paraformaldehyde 1% and glutaraldehyde 0.5% in buffer cacodylate 0.1 M pH 7.2 for 3 hours.

(75) After this fixing the pieces are washed in PBS 20 min3 times (1 hour total) and then the post fixing is carried out in a solution of osmium tetroxide 2% and potassium ferrocyanide 2.5%.

(76) The preparation is kept in the dark for 3 hours to be then accurately washed rotating in PBS, with 4 replacements of 20 min each.

(77) It follows dehydration of the samples of tissue with acetone in ascending succession with 3 replacements in 1 hour for each gradation 50%-70%-80%-90%-95% and 2 replacements with pure acetone.

(78) At this stage it follows drying up to the critical point with liquid CO.sub.2 in the apposite high-pressure device which substitutes acetone 10% with liquid CO.sub.2, it is then slowly brought to the critical point temperature and CO.sub.2 evaporates. At this point the samples of tissue are perfectly anhydrous and can be arranged on the trays of the electron microscope.

(79) The samples are fixed through a special electrically conductive double sided adhesive and for their arrangement two very thin needles are used and the preparation is ready to be observed with scanning electron microscope (SEM).

(80) Biological Samples

(81) Under prior informed consent, they have been placed under culture fragments of endometrium withdrawn from patients subjected to diagnostic hysteroscopy.

(82) For each patient, two parts have been set: one part of the tissue has been placed under culture through a conventional Blastocyst Medium void of melatonin, the other part through a conventional Blastocyst medium supplemented with melatonin.

(83) Culture Set

(84) In vitro culture of endometrium (withdrawn through hysteroscopy from patients subjected to assisted pre-fertilization controls) with melatonin for 3-6 days and correlated visualization of pinopodes through electron microscopy (SEM).

(85) Results

(86) In vitro studies have shown that the co-culture from 3 to 5 days of endometrial tissue in test-tube with permanent addition of melatonin, involves an unquestionable increase of pinopodes (under electron microscopy (SEM)), irrespective of the age of the patient and of the menstrual cycle phase in spontaneous cycles or manipulated through pharmacological stimulation of super-ovulation, with respect to the culture of control. Such pinopodes are structures which are defined as the most important implantation marker which for each woman (with a variability of 5 days, that is, a woman can menstruate after 26 days with short cycle or menstruate after 33 days) is of 48 hours. In fact, two thirds of success of becoming pregnant depends on the correct moment of implantation, the other third depends on the quality of the embryo.

(87) The examination under the electron microscope SEM has shown that the addition of melatonin leads to a complete morphological expression of pinopodes as illustrated in FIGS. 1-4.

(88) This outlines the importance of pinopodes during the implantation window and the increase of their expression induced in culture through mediums supplemented with melatonin.

EXAMPLE 3

Study on the Effects of Melatonin and Analogues on Oxidation and Apoptosis of Endometrial Cell Line HEC-1-A

(89) Tested Compounds

(90) The analogues of melatonin which has been used for comparison with melatonin (M5250), are agomelatine (A1362) and 6-hydroxy melatonin (H0627), all purchased from Sigma Aldrich.

(91) They were used in different concentrations (i.e. 40 nM, 80 nM, 170 nM and 340 nM, corresponding respectively to 9.410.sup.9 g/ml, 1810.sup.9 g/ml, 4010.sup.9 g/ml and 8010.sup.9 g/ml).

(92) Cell Line

(93) HEC-1A cells were obtained by ATCC and culture following instructions of the provider.

(94) HEC-1-A is an epithelial stabilized cell line isolated by H. Kuramoto from adenocarcinoma patient (21), which provide a valuable model to study endometrial epithelial cell in vitro (22).

(95) Antioxidant Activity Assay

(96) The HEC-1-A cell line was cultured with 170 nM (40 ng/ml) of Melatonin, 6-hydroxy-melatonin and agomelatine and two different concentrations of H.sub.2O.sub.2 (20 and 200 mcg/ml).

(97) Reactive Oxidative Substances (ROS) were measured by FACS as previously described by Italiano et al. (23) in two independent replicates.

(98) Results

(99) Melatonin and the two analogues asserted the same anti-oxidative effect, on the in-vitro cell line model, at the H.sub.2O.sub.2 concentration of 20 mcg/ml.

(100) When the H.sub.2O.sub.2 concentration is increased up to 200 mcg/ml melatonin and agomelatine continued to protect cell line from oxidation, whereas the 6-hydroxy-melatonin does not. However, increasing the 6-hydroxy-melatonin dose up to 340 nM the anti-oxidative effect of this analogue is reached (data not shown).

(101) Results are illustrated in the chart of 5.

(102) These results are in line with data published by Duan et al. (24) on the anti-oxidative effect of melatonin in a different cell model and demonstrate that melatonin and analogues assayed have a similar effect at least when 20 mcg/ml of H.sub.2O.sub.2 is used on endometrial cell line.

(103) Apoptosis Assay

(104) The apoptosis (% cell death) of the endometrial cells is a physiological phenomenon generally observed at 10-20% rate during cell culture.

(105) In order to determine if melatonin and its analogs induce cell death in this specific experimental context, we have treated the HEC-1-A cell line with 340 nM (80 ng/ml), 170 nM (40 ng/ml) and 80 nM (18.8 ng/ml) of melatonin, 6-hydroxy-melatonin and agomelatine, respectively, for 24 hours.

(106) Apoptosis levels were assayed by Propidium Iodide staining and FACS analysis. Percentage of sub-G1 events is shown for one of two experiments performed.

(107) Results

(108) In all the concentrations tested, Melatonin exerts a protective effect on apoptosis. Agomelatin has a protective effect at 170 and 340 nM, while no effect on apoptosis is shown by 6-hydroxy-melatonin up to concentration of 170 nM. Results are shown in 6.

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