Use of a cyclic tripeptide for improving cellular energy metabolism

Abstract

The present invention relates to the use of a cyclic peptide comprising the tripeptide reproducing a binding site of fertilin beta to the oocyte integrin, for improving cellular energy metabolism. More particularly, the invention concerns the use of a cyclic peptide comprising the tripeptide FEEc for stimulating the energy metabolism of gametes or embryonic cells in the context of medically assisted procreation (MAP) protocols, in particular promoting the in vitro maturation of the oocyte, the fertilization rate and the birth rate.

Claims

1. A method of maturating immature mammalian oocytes in vitro comprising: contacting the immature mammalian oocytes in vitro with at least one cyclic peptide comprising a cyclic tripeptide F-E-E of SEQ ID NO: 1 with a cyclization linkage between the two end cysteines, thereby maturating the mammalian oocytes.

2. The method of claim 1, wherein the immature mammalian oocytes are human oocytes.

3. The method of claim 1, wherein the immature mammalian oocytes are incubated with 1 to 100 μM of cyclic peptide for a period of between 1 minute and 4 days.

4. The method of claim 1, wherein contacting the immature mammalian oocytes with the cyclic peptide decreases the risks of miscarriage.

5. The method of claim 1, wherein contacting the immature mammalian oocytes with the cyclic peptide decreases the risks of aneuploidy in the oocyte.

6. The method of claim 5, wherein the aneuploidy is trisomy.

7. The method of claim 1, wherein the immature mammalian oocytes are human oocytes from a woman 37 years old or older.

8. The method of claim 1, wherein the immature mammalian oocytes are human oocytes from a woman under the age of 30.

9. The method of claim 1, wherein contacting the immature mammalian oocytes with the at least one cyclic peptide improves ploidy of oocytes.

Description

FIGURE LEGENDS

(1) FIG. 1: Variation in the mitochondrial membrane potential in the presence of the FEEc (on the right) versus “scramble” control peptide (on the left). The increase in the membrane potential in the exposed spermatozoa in the majority of the patients should be noted.

(2) FIG. 2: Counting, after UV excitation, of the human spermatozoa fused with human oocytes with the pellucida removed, incubated in the absence (A) or in the presence of FEEc at 100 μM (B). The increase in the number of spermatozoa fused and the faster decondensation of their head should be noted.

(3) FIG. 3: Proportion of mature oocytes at D1 of in vitro maturation (IVM). Diagrammatic representation of the proportions of human oocytes in metaphase II (MII) from the GV stage after IVM in the control medium or the medium supplemented with FEEc at 100 μM. *p=0.02. **p=0.003. D1=24 h of IVM.

(4) FIG. 4: Proportion of atretic oocytes at D1 of in vitro maturation (IVM). Diagrammatic representation of the proportions of atretic human oocytes. Results observed at D1 starting from the GV stage after in vitro maturation (IVM) in the control medium or the medium supplemented with FEEc at 100 μM. D1=24 h of IVM.

(5) FIG. 5: Labelling of the meiotic spindle obtained on a human oocyte in MII after IVM in the standard medium (24 h). Labelling of the spindle with an anti-α-tubulin antibody and of the chromosomes with DAPI. Image obtained on a confocal microscope. A: whole oocyte. B: magnification of the metaphase plate.

(6) FIG. 6: Comparison of the fertilization rate at D1 between the young and old mice, in the presence or in the absence of fertilin, which corresponds to the QDEc peptide.

(7) Young: 7-week-old B6CBAF1 mice; old: 7-month-old B6CBAF1 mice.

(8) FIG. 7: Comparison of the average percentages of cleaved embryos at D2 and at D4 between the young and old mice, in the presence or absence of the QDEc peptide.

(9) Young: 7-week-old B6CBAF1 mice (n=108 oocytes, 56 controls, 52 QDEc); old: 7-month-old B6CBAF1 mice (n=128 oocytes, 65 controls, 63 QDEc); *p=0.02, **p=0.008, ***p=0.01.

(10) FIG. 8: Comparison of the average percentages of atresia (ATR) at D2 between the young and old mice, in the presence or absence of the QDEc peptide.

(11) Young: 7-week-old B6CBAF1 mice (n=108 oocytes, 56 controls, 52 QDEc); old: 7-month-old B6CBAF1 mice (n=128 oocytes, 65 controls, 63 QDEc).

(12) FIG. 9: Diagrammatic representation of the methodology of the clinical study carried out in example 5.

(13) FIG. 10: Preliminary results of the criteria for main and secondary judgement in the context of the clinical study. Pregnancy rate by fresh or frozen embryo by transfer (control group, n=17 transfers/FEEc group, n=13 transfers). “Top embryo” percentage (control group, n=75 cleaved embryos/FEEc group, n=72). Fertilization rate (control group, n=259 MII/FEEc group, n=246 MII)

(14) FIG. 11: Pregnancy rates obtained in the context of the clinical study.

(15) FIG. 12: Improvement in the rate of maturation of oocytes blocked in GV after incubation in the presence of the FEEc. This study was carried out by taking into consideration one oocyte per woman and by incubating said oocyte in the presence of the FEEc or of the control peptide.

(16) FIG. 13: Effects of FEEc on the maturation of human oocytes blocked in GV per age range.

(17) FIG. 14: Stimulation of the pre-implantation embryonic development in young mice with QDEc (*P<0.00532; **P<0.00374; ***P<0.00913; ****P<0.068; ( ) number of embryos).

MATERIALS AND METHODS

(18) The experimental examples present below were obtained using the following materials and methods:

(19) Observation of the Sperm Movement Parameters

(20) The sperms studied were each divided into 2 aliquots, one of which was incubated with the FEEc and other of which was incubated with a “scramble” peptide containing the same amino acids but in a random order. The inventors incubated, for 3 h at 37° C., spermatozoa from human beings in the presence of 100 μM of the FEEc peptide or of the scramble peptide and then observed the sperm movement parameters according to an automated analysis (Computed Assisted Sperm Analysis, CASA).

(21) The sperm parameters tested are the following: smoothed VAP, VSL, VCL and ALH. They correspond respectively to the average path velocity, the straight line velocity, the curvilinear velocity and the lateral head displacement. The study showed a significant increase in the percentage of hyperactivated spermatozoa (according to the criteria of Mortimer et al.). This can explain the increase in fertilization rates observed in the presence of the peptide.

(22) Measurement of the Mitochondrial Membrane Potential

(23) The inventors incubated, for 3 h at 37° C., spermatozoa from human beings in the presence either of the FEEc peptide, or of the “scramble” peptide which comprises the same amino acids in a random order and thus constitutes the control group. After washing, the spermatozoa are labelled using a lipophilic fluorescent dye, DIOC6.

(24) The mitochondrial membrane potential (proton gradient at the level of the mitochondrial inner membrane) was then measured by flow cytometry. It is found to be increased in the spermatozoa after exposure to FEEc.

(25) Measurement of the Fertilization Index

(26) Human oocytes with the pellucida removed were incubated with human spermatozoa in the absence or presence of FEEc at 100 μM. The fused spermatozoa were counted after UV excitation. The spermatozoa were considered to be fused when the nucleus was labelled with Hoechst 33342. Furthermore, the heads of the spermatozoa having penetrated the oocyte in the presence of the FEEc peptide are not only greater in number but also have a blurred appearance attesting to the decondensation of their sperm head. This decondensation is one of the first steps of oocyte activation after penetration of the spermatozoan. It can therefore be concluded from this that not only does the FEEc improve the fertilizing ability of the spermatozoan, but it also activates the fertilized oocyte.

(27) Collection of Human Oocytes with a View to In Vitro Maturation (IVM)

(28) Immature human oocytes given to research were collected from the in vitro fertilization (IVF) laboratory of the Medically Assisted Procreation (MAP) Center of the Cochin hospital (Paris, France). Two hours after the oocyte puncture, the oocytes intended to be microinjected had their corona removed with hyaluronidase (Origio, Limonest, France). After observation under an inverted microscope (Hoffman), the immature oocytes at the germinal vesicle (GV) stage were retained for the remainder of the experiments.

(29) In Vitro Maturation (IVM) of Immature Human Oocytes

(30) The immature human oocytes at the GV stage were randomized, either in the control culture medium (Global, JCD, La Mulatière, France) (n=203) or in the same medium supplemented with 100 μM of FEEc (n=193). The immature human oocytes were categorized in two groups: those belonging to women under the age of 37 and those belonging to women aged 37 and over. On the day of the follicular puncture, the two groups of oocytes at the GV stage were incubated in 20 μl drops covered with oil, and maintained at 37° C. under 5% CO.sub.2 so as to be observed under an inverted microscope (Hoffman) at D1 (24 h of incubation) and D2 (48 h of incubation). The oocytes were categorized as metaphase II (1.sup.st polar globule in the perivitelline space), germinal vesicle (GV), metaphase I (rupture of the germinal vesicle without expulsion of the polar globule) or atretic.

(31) Collection of Murine Oocytes with a View to In Vitro Maturation (IVM)

(32) B6CBAF1 females (between 5 and 8 weeks old) supplied by the Charles River laboratory (L'Arbresle, France) were stimulated by injection of PMSG (Pregnant Mare Serum Gonadotrophin) at 10 IU (Sigma-Aldrich, Saint-Quentin Fallavier, France) without triggering ovulation. The immature oocytes were collected from the ovaries 48 h after the latter injection, then had their cumulus removed by means of hyaluronidase and were washed three times in M2 culture medium. Only the oocytes categorized as GV were retained for the remainder of the experiments.

(33) In Vitro Maturation (IVM) of the Immature Mouse Oocytes

(34) The mouse oocytes were incubated in a manner randomized between, on the one hand, the standard medium and, on the other hand, the medium supplemented with 100 μM QDEc. The culture dishes were prepared the day before and incubated at 37° C. under 5% CO.sub.2. The oocytes were observed at D0 (8 h post-sacrifice) and D1 (24 h).

(35) Immunofluorescence

(36) The human oocytes resulting from the IVM were fixed in 2% paraformaldehyde (PFA) for 1 h at ambient temperature and then washed in PBS containing 0.5% of BSA. The permeabilization was carried out by incubation of the oocytes in a solution containing 0.5% of BSA, 0.1% of Triton X-100, 0.05% of Tween-20 and 5% of normal goat serum. The oocytes were then washed in PBS-05% BSA before being incubated overnight in a dilution to 1/200 of anti-human α-tubulin antibody (Sigma-Aldrich) in PBS containing 0.5% of BSA. The oocytes were then incubated for 1 h in the presence of the Alexa Fluor-conjugated IgG secondary antibody (Life Technologies, Alfortville, France). After a washing step, the oocytes were incubated for 10 min in DAPI (diluted to 1/1000) before being mounted on a slide and observed by confocal microscopy in the dark. For the spindle analysis, the oocytes with distinct and well-organized microtubules fibers associated with perfect alignment of the chromosomes at the level of the metaphase plate are identified as normal.

(37) Stimulation and Mating of the Mice

(38) 7-week-old “young” and 7-month-old “old” B6CBAF1 females were mated with C57N males after superovulation, the latter consisting in injecting PMSG at 10 IU (Sigma-Aldrich) followed by triggering of ovulation by administration of hCG (Human Chorionic Gonadotrophin) at 10 IU (Sigma-Aldrich), 46-48 h afterwards.

(39) The day after the mating, the mice exhibiting a vaginal plug were sacrificed. The oocytes were collected from the oviducts 15-16 h after the injection of hCG and the fertilization rate was evaluated by the presence of the second polar globule in the perivitelline space.

(40) Incubation of the Fertilized Mouse Oocytes

(41) The mouse oocytes fertilized after mating and collected were randomized into 4 groups (young exposed to the cyclic QDE peptide (QDEc), young controls, old exposed to QDEc, old controls) and placed in drops of culture medium (KSOM) of 20 μl for the controls, and supplemented with 100 μM of QDEc for the exposed ones. The exposure lasted from D1 to D4 after the mating in vivo (D0). The QDEc is equivalent to the human FEEc. The culture dishes are incubated at 37° C. under 5% of CO.sub.2 and they are covered with mineral oil.

(42) The oocytes were observed every day with a binocular magnifying lens in order to assess the signs of embryonic development. Normal development kinetics correspond, at a minimum, on D2, to an embryo cleaved into 2 cells and, on D5, to an embryo at the morula or blastocyst stage.

(43) Randomized Prospective Study in Human IVF

(44) A clinical trial was begun in the IVF laboratory of the MAP Center of Cochin, on Sep. 8, 2014, on 66 couples, the average age being 34.3±4.2 years old for the women and 37.0±5.2 for their partners. It is a randomized, single-center prospective study of in vitro fertilization (IVF) carried out in the presence or absence of FEEc. The oocytes, recovered from their cumulus, were divided up into two groups alternately into one and then into the other according to their order of recovery. When all the oocytes were recovered, a technician who had not participated in the recovery of the cumuli determined by randomization which of the two groups was inseminated in the presence of FEEc and which served as control. One part of the oocytes is incubated in the standard culture medium (Global, JCD), the other part in this same medium supplemented with 100 μM FEEc.

(45) The methodology of this study, represented diagrammatically in FIG. 9, is presented below. Human oocytes originating from women 18 to 43 years old were recovered by ovarian puncture after hormonal stimulation by gynecologists. They were divided up in a randomized manner into 2 groups: the oocytes incubated in the presence of a standard culture medium supplemented with 100 μM FEEc or of a standard culture medium (Global, JCD), then placed in an incubator at 37° C. under an atmosphere of 5% of CO.sub.2. The partner's spermatozoa were recovered in the laboratory and the most mobile selected according to a standard preparation of the MAP centers. The IVF consists in bringing the spermatozoa into contact with the oocytes in the insemination medium at a concentration of 10.sup.5 spermatozoa selected/ml in drops of 20 microliters under oil. The insemination is carried out in an incubator at 37° C. under 5% of CO.sub.2 for 18 hours. 18 h after the insemination (D1), the corona is removed from the oocytes. The fertilized oocytes are washed and transferred into another drop of medium and placed in culture for a further 24 h. At the time of the transfer in utero, they are washed three times, then placed in a transfer medium and placed in the uterine cavity. The embryos are transferred according to their apparent quality without paying any attention to the group of origin. One or more embryos are transferred according to the age, the indication of the IVF, the attempt rank and the quality of the embryos obtained, and with the agreement of the couples. Some embryos may be placed in prolonged culture over the course of 5 days (at the blastocyst stage) either immediately, or after transfer of embryos at D2.

(46) The main evaluation criterion is the clinical pregnancy rate by transfer of fresh or frozen embryos and the miscarriage rate taking into account the 3 groups: homogeneous transfers (controls and treated) and mixed transfers (mixture of the two).

(47) The secondary criteria are: The fertilization rate, i.e. the ratio of the number of zygotes having two pronuclei in the cytoplasm, 18 hours after insemination, relative to the number of ooctyes in metaphase 2 in the cohort. The percentage of embryos of good quality, i.e. of embryos of which the cleavage sequence corresponds to the ideal sequence, that is to say: 4 to 5 cells at D2 and 8 to 9 cells at D3 and of which the blastomer fragmentation is of type A (when the volume occupied by the fragments is less than 10% of the embryonic volume) or B (when the volume occupied by the fragments is between 10% and 30% of the total volume of the embryo). Thus, these “Top” embryos correspond to the ratio of the number of embryos of each type relative to the total number of embryos for each group.

(48) This protocol was approved by the West VI Ethics Committee on Dec. 13, 2012. The study obtained authorization from the Agence de la Biomédecine [French Biomedicine Agency] on Jul. 8, 2013. The IVF is carried out with strict adherence to good clinical practice. Each couple agreeing to participate in the study signed a free and informed consent document.

(49) Statistical Analysis

(50) The quantitative variables were studied by means of their numbers, mean and standard deviation. The data were compared between the exposed and non-exposed group using an appropriate test (Student's test or Wilcoxon test) for the quantitative variables. The comparisons of percentages were carried out using a Chi-squared (χ.sup.2) test or Fisher's exact test. The differences between the compared data were considered to be statistically significant when the p value (significance threshold) was less than 0.05.

Example 1: Improvement in the Spermatozoan Movement Parameters and in the Percentage of Hyperactivated Spermatozoa in Men

(51) Preliminary experiments showed that, in an 18-hour survival test of sperms incubated in the presence of the FEE peptide, the survival is significantly improved in the group treated with the FEE at the concentration of 100 μM compared with the control.

Example 1a: Automated Analysis of the Sperm Movement Parameters after Incubation in the Presence of FEEc and of a Scramble Peptide for Control

(52) The results presented in table 1 below show an increase in the smoothed VAP (p=0.008), in the VSL (p=0.048), in the VCL (p<0.0001) and in the ALH (p=0.002), resulting in a 29% increase in hyperactivated spermatozoa (p=0.009) compared with the control group. This improvement in the percentage of hyperactivated spermatozoa explains the improvement in their fusiogenic capacity and the increase in the fertilization rates recorded in mice on intact oocyte cumulus complexes.

(53) TABLE-US-00001 TABLE 1 Automated analysis of sperm movement parameters compared between the control group and the group after incubation in the presence of FEEc. Parameters Control FEE P VAP (μm/s) 81.0 ± 16.8 86.3 ± 13.7 0.008 7% VSL (μm/s) 67.3 ± 16.7 71.8 ± 15.3 0.048 7% VCL (μm/s) 137.5 ± 26.7  148.0 ± 26.6  <0.0001 8% ALH (μm) 6.1 ± 1.3 6.6 ± 1.3 0.002 8% BCF (Hz) 32.7 ± 3.4  33.0 ± 3.9  NS — STR (%) 85.8 ± 11.4 84.9 ± 10.0 NS — LIN (%) 51.1 ± 12.6 50.9 ± 12.2 NS — Hyperactivation 12.3 ± 9.7  15.9 ± 11.6 0.009 29% 

Example 1b: Measurement of the Mitochondrial Membrane Potential

(54) The mitochondrial membrane potential is found to be increased by 21% in the presence of the FEEc peptide compared with the “scramble” peptide (p<0.001) (FIG. 1).

(55) The FEEc therefore improves the sperm movement parameters by increasing the sperm mitochondrial membrane potential.

Example 1c: Study of the Fertilization Index

(56) The results presented in FIG. 2 show that not only a larger number of spermatozoa fused with the oocytes with the pellucida removed, in the presence of the FEEc peptide, but said spermatozoa are also decondensed, contrary to those of the control group.

(57) A mean of 19.0±4.6 spermatozoa is counted in the cytoplasm on the control oocytes, whereas an increase with 36.9±11.7 spermatozoa fused by oocytes is reported after incubation with the FEEc at 100 μM (p<0.001). This phenomenon suggests an increase in the fertilization capacity of the spermatozoa and an oocyte activation mediated by the FEEc peptide.

(58) The sperm movement parameters of 37 patients were analyzed in the presence or absence of FEEc (incubation for 3 hours). There is a significant increase in the percentage of hyperactivated spermatozoa according to the Mortimer criteria, which explains the increase in their fertilization capacity (see table 1 above).

Example 2: Improvement in the Percentages of In Vitro Maturation of Immature Human Oocytes

(59) A significant increase in oocyte maturation with FEEc was demonstrated at D1, for all of the human oocytes tested. The results obtained are the following: 42.3% ( 69/163) of oocytes in metaphase II (MII) with FEEc versus 30.0% ( 52/173) in the control group, p=0.02 (FIG. 3). The increase in maturation is even more marked for the oocytes from women aged 37 and over as soon as D1. The results obtained are the following: 47.9% ( 23/48) of oocytes in MII with FEEc versus 20.4% ( 11/54) in the control group (p=0.003).

(60) Although small for the oocytes from women less than 37 years old, the improvement in oocyte IVM in human beings is very significant for oocytes of older women since the same maturation rate as for the oocytes of the young women was obtained (47.9% of the oocytes in metaphase II in the presence of FEEc versus 20.4% in the control group, p=0.003).

(61) The results obtained with the 336 human oocytes at the GV stage, incubated in randomized manner in the presence or in the absence of the FEEc peptide, show that the presence of the FEEc improves the maturation rate of human oocytes.

(62) No significant difference between the oocyte atresia rates among the 2 groups was detected (16.5% in the presence of the peptide versus 16.8% for the control group) (FIG. 4). In the sample of women aged 37 and over, a non-significant tendency toward a decrease in the atresia rate is noted in the presence of the FEEc peptide (16.7%, 8/48) compared with the control medium (24.1%, 13/54) p>0.05.

(63) The study was continued and supplementary results were obtained. These results confirm the results described above and demonstrate other effects of the FEEc peptide, as described below.

(64) In total 600 oocytes were matured in vitro. For the analysis, only one oocyte per woman was included in the study in order for all the events to be independent. In the presence of fertilin in the medium, the maturation rate went from 38.3% to 59.0% (P<1.6×10.sup.−4) (FIG. 12). When the analysis is carried out as a function of the age of the woman from whom the oocytes in GV originate, it is seen that the improvement in the maturation is relatively modest for women under the age of 37 (42.6% to 51.8%, P<0.2), but that it is much greater in the oocytes originating from women aged 37 and over (35.1% to 65.9%, P<3.91×10.sup.−5). Fertilin is therefore able to stimulate the in vivo maturation of the oocyte with the corona removed, and the expulsion of the first polar globule. Apparently, the greater the age-related energy deficit of the oocyte, the better the fertilin performs this stimulation. This study also made it possible to demonstrate an increase in the maturation rate more particularly of the oocytes in GV of women over the age of 37 and under the age of 30 (FIG. 13).

Example 3: Organization of the Meiotic Spindle of Human Oocytes Matured In Vitro

(65) FIG. 5 shows the incomplete and aberrant organization of the alignment of the chromosomes on the metaphase plate with non-aligned chromosomes. The image is obtained from a human oocyte in MII after 24 h of IVM in the presence of the control medium.

Example 4: Improvement in the Rate of Fertilization and Early Embryonic Development in Mice

(66) At D1, the fertilization rate remains unchanged in the young mice, whereas it goes from 39% to 51% in the old mice (p<0.03) (FIG. 6).

(67) At D2, among the young mice, 50.0% ( 26/52) of the oocytes are cleaved in the QDEc group compared with 32.4% ( 18/56) in the control group (P=0.02) (FIG. 7). Regarding the old mice, there are significantly more cleaved embryos at D2 in the presence of QDEc (58.7%, 37/63) compared with the control (35.4%, 23/65), p=0.008. At D4, the proportion of cleaved embryos from young mice that reach the morula or blastocyst stage is 34.6% ( 7/16) for the control group compared with 63.0% ( 17/26) for the medium supplemented with QDEc, p<0.03. This proportion reaches 86.3% ( 63/73) for the old mice in the presence of the peptide compared with 28.3% ( 15/53) in the control medium, p=0.001.

(68) Advantageously, the blast formation in the mice is improved in the presence of the QDEc peptide.

(69) The percentages of atresic embryos are not significantly different between the 2 groups among the young mice: 17.3% ( 9/52) in the presence of QDEc and 30.4% ( 17/56) for the control (p=0.1). Compared with the young mice, the percentage of atresia is greater in the group of old mice with rates similar between the QDEc (38.1%, 24/63) and control (49.2%, 32/65) groups (p=0.2) (FIG. 8).

(70) In order to more finely study the preimplantation embryonic development, the following protocol was carried out: mice were mated at D0. At D1, the oocytes were recovered by laceration of the tube bulbs. They were then dispensed into two groups in randomized fashion and placed in culture with or without QDEc, before fertilization.

(71) The results show an increase in the morulas at D3, in the blastocysts at D4 and among these an increase in the expanded blastocysts when the oocyte was incubated in the presence of QDEc (31.1% vs 45.9%, P<0.009) (FIG. 14). Furthermore, after transfer of these blastocysts into pseudogestational females, there is a significant increase in the number of progeny obtained by transferred embryos, especially for the embryos originating from young mice (table 2 below).

(72) TABLE-US-00002 TABLE 2 Birth rates after transfer of the control embryos and embryos treated with QDEc in mice Embryos transferred Progeny Birth Mice (n) (n) rate P Young Control 109 37 34% 0.05 Fertilin 132 61 46% Old Control 58 20 35% 0.61 Fertilin 30 12 40% Total Control 167 57 34% 0.04 Fertilin 162 73 46%

Example 5: Increase in the Clinical Pregnancy Rates by In Vitro Fertilization

(73) The results presented below were obtained in the context of the “fertilin” study.

(74) In a first step, 56 couples were included. The average age is 33.9+/−4.1 years old among the women and 36.7+/−5.3 for the partners. No significant difference was noted, either between the fertilization rates or between the percentage of top-quality embryos among the 2 groups, FEEc versus control. A non-significant tendency toward increasing the pregnancy rate by transfer is noted in the presence of the peptide compared with the control and with the mixed group, 46.1% ( 6/13) compared with 29.4% ( 5/17) and 40% (⅖), respectively (FIG. 10 and table 2 below). A miscarriage was reported after transfer of one embryo from the control group. No adverse effect has been reported to date.

(75) To date, 66 couples have been included in the study. 54 transfers were carried out, 26 with control embryos, 22 with embryos of the FEE group, 6 with embryos of the two groups (mixed transfers). The rate of cumulative pregnancy was respectively 34.6%, 45.5% and 33.3% in the 3 groups. The rate of spontaneous miscarriage was respectively 33.3%, 10% and 0%. The rate of clinical pregnancy is therefore respectively 23%, 41% and 33.3%. An essential fact when the rate of clinical pregnancy is relative to the age of the patient is that, in the young patients, it is seen that the rate of evolutive clinical pregnancy (that is to say pregnancy to term) is significantly increased from 20% to 57.1% (P<0.03).

(76) TABLE-US-00003 TABLE 3 Pregnancy rate by transfer (for 66 patients) Embryo transfer Control FEEc Mixed Total Patients included 66 Deferred transfers 21 Cumulative 26 22 6 54 transferred Pregnancy rate n (%) 9 (34.6%) 10 (45.5%) 2 (33.3%) 38.9% Miscarriages n (%) 3 (33.3%)  1 (10.0%) 0 4 (19%)

(77) The results also show an increase of 21% in clinical pregnancies after transfer of an embryo from the FEEc group, 40.0% ( 8/20) compared with 33.3% ( 8/24) in the control group (p>0.05). The fertilization rates are 66.2% among the controls compared with 67.6% in the group exposed to the FEEc (p>0.05). The rate of early spontaneous miscarriage reaches 37.5% (⅜) in the control group compared with 11.1% ( 1/9) when the embryo has been exposed to the FEEc (p>0.05) (FIG. 11).

(78) For the women under the age of 37, after exposure to the FEEc, the fertilization rates are 70.9% compared with 68.3% in the control group. In the case where the oocyte was exposed to the FEEc, the pregnancy rates reach 57.1% compared with 20.0% in the control group (table 4 below).

(79) TABLE-US-00004 TABLE 4 Fertilization rate and pregnancy rate according to age of the partner (≥37 or <37 years old) Fertili- Fertili- Pregnancy Pregnancy Woman's Number of zation rate zation rate rate rate age couples Control FEEc Control FEEc (year) (n) (%) (%) (%) (%) 26-36 47 68.3% 70.9% 20.0% 57.1%* 37-43 19 60.2% 60.3% 33.0% 20.0% *P < 0.03

(80) Of the first 66 couples included in the study up to now, 51 transfers have been carried out, the others being deferred and the results show that of these 51 transfers: 21 were carried out using embryos of the control group; 18 using the group in the presence of the FEEc; 12 with embryos of the two groups.

(81) The pregnancy rate is higher and there are fewer miscarriages for the group in the presence of the FEEc in comparison with the other groups (33% of miscarriages compared with 9% for the embryos in the presence of FEEc) (tables 5 and 6 below).

(82) The pregnancy rates for the young women go from 20% to 57.1% (p<0.03) in the presence of FEEc compared with the control.

(83) TABLE-US-00005 TABLE 5 Pregnancy rate by transfer (for 66 patients) Transfer of embryos Control FEEc Mixed Total Patients included 66 Deferred transfers 15 Transferred patients 21 18 12 51 Pregnancy rate n (%) 34.6%   45.5%   33.3% Miscarriages n (%) 33% 10%  0 Evolutive pregnancy 23% 41%   33% rate (%)

(84) TABLE-US-00006 TABLE 6 Pregnancy rate by transfer as a function of the age of the women Fertilization Pregnancy Couples rate rate/transfer Age F No. Control(%) FEE(%) Control(%) FEE(%) Fisher 26-36 47 68.3% 70.9% 20% (4/20) 57.1% (8/14) P = 0.03 37-43 19 60.2% 60.3% 33% (2/6) 10% (1/10) P = 0.5 Total 66 66.23% 67.6% 23% (6/26) 37.5% (9/24) P = 0.2

(85) The continuation of the fertilin study made it possible to obtain additional results. In total, 66 couples were included in the study. The results are reported in the tables below.

(86) The overall data of the attempts are reported in table 7. The overall fertilization rates were virtually unmodified. However, the percentage of attempts for which there was a paucity of fertilization (less than 20% fertilization rate) fell from 37.9% to 27.3% in the presence of FEEc, suggesting a better fertilization capacity of the gametes in the presence of fertilin. Likewise, the polyspermy is of the same order of magnitude in the fertilin group as in the control group (4.0% vs 5.2%) showing that the normal mechanism for blocking polyspermy was not modified.

(87) TABLE-US-00007 TABLE 7 Overall results of the fertilin study on 66 patients General data Total Control Fertilin No. couples 66 Age man   37 ± 5.2 Age woman 34.3 ± 4.2 No. oocytes 647  325 322 No. M2 592(91.4%) 302(92.9%)  290(90.0%)  No. 2PN 396  200 196 No. 3PN 30  17  13 Paucity of 25(37.9%) 18(27.3%) fertilization Fertilization 10 6(9.1%) 4(6.1%) failure Useful blast 133(33.6%) 70(35.0%) 63(32.1%) formation

(88) The results of all the couples having had a transfer of embryos are reported in table 8. This table excludes the couples who had an unexplained fertilization failure in the 2 groups of oocytes with or without fertilin (n=6).

(89) TABLE-US-00008 TABLE 8 Results obtained in the 66 couples of the fertilin study All patients (66 couples included) No. transfer Weight Embryos No. Fert. Blast Implant Pregnancy Spont. Evolutive at birth transferred ooc. rate (%) rate n Miscar. pregnancy (n babies) Control 151 69.5% 37.1% 36(39.5%) 16 5(33.3%) 11(30.5%) (6)2951 g FEEc 98 78.6% 37.7% 34(35.1%) 13 2(15.4%) 11(32.5%) (9)3041 g (NS)

(90) As shown in table 8, for the patients having had an embryo transfer, the fertilization rate went from 69.5% to 78.6%, which is an improvement of 13% but which does not reach significance in the present cohort. The embryonic development up to the blastocyst stage is not modified, nor is the rate of implantation of the embryos in the uterus. On the other hand, the rate of miscarriages is decreased by close to 50% in the fertilin group (15.4% vs 30.5%).

(91) The results of the couples having had an embryo transfer and in which the woman is under the age of 37 are reported in table 9.

(92) If one takes into consideration only the couples in which the woman is under the age of 37 (n=47) and which correspond to more than 70% of the patients treated, it is seen that the fertilization rate is significantly improved from 70.9% to 83.3% (P<0.05).

(93) The rate of miscarriages goes from 36.3% for the patients having received an embryo of the control group to 9.1%, that is to say four times lower, for those having received an embryo of the fertilin group. In fact, the rate of evolutive pregnancies giving birth to a child goes from 28.6% in the group of the control embryos to 41.6% in that of the embryos of the fertilin group.

(94) TABLE-US-00009 TABLE 9 Results obtained in the couples in which the woman is under the age of 37 in the context of the fertilin study Patients <37 years old (47 couples included) No. transfer Weight Embryos No. Fert. Blast Implant Pregnancy Spont. Evolutive at birth transferred ooc. rate (%) rate n Miscar. pregnancy (n babies) Control 127 70.9% 36.7% 28(37.9%) 12 4(36.3%) 8(28.6%) (7)2894 g FEEc 72 83.3% 36.7% 24(44.0%) 11 1(9.1%)  10(41.6) .sup.   (8)3271 g P < 0.05