GLYCOLIC ACID ENHANCES SPERM MOBILITY

Abstract

The present invention relates to glycolic acid or a pharmaceutically acceptable salt or ester thereof for use in the treatment or prevention of male infertility. The present invention also relates to an ex vivo method for increasing the mobility of spermatozoa comprising contacting glycolic acid or a pharmaceutically acceptable salt or ester thereof with spermatozoa.

Claims

1. A method of treatment or of preventing male infertility comprising: contacting glycolic acid or a pharmaceutically acceptable salt or ester thereof with spermatozoa in-vivo for the treatment or prevention of the male infertility.

2. The method of claim 1, wherein the male infertility is associated with asthenozoospermia.

3. The method of claim 1, wherein the glycolic acid or a pharmaceutically acceptable salt or ester thereof is to be applied inside the vagina and/or the cervix prior to sexual intercourse.

4. The method of claim 1, wherein the male infertility is human male infertility.

5. The method of claim 1, wherein the male infertility is male infertility of a production animal, mammalian domestic animal, zoo mammal or an endangered mammal.

6. An ex vivo method for increasing the mobility of spermatozoa comprising contacting glycolic acid or a pharmaceutically acceptable salt or ester thereof with spermatozoa.

7. The method of claim 6, wherein the spermatozoa are defrosted spermatozoa.

8. The method of claim 6, wherein the spermatozoa are human spermatozoa.

9. A composition comprising glycolic acid or a pharmaceutically acceptable salt or ester thereof, and a further compound increasing male fertility.

10. The composition of claim 9, wherein the further compound is at least one of: (i) an antioxidant, (ii) a vitamin, (iii) one or more of L-arginine, L-carnitine and L-creatine.

11. The composition of claim 9, wherein the composition is a sexual lubricating composition which promotes male fertility.

12. The composition of claim 11, wherein the sexual lubricating composition is a sexual lubricating cream or a sexual lubricating oil.

13. A vaginal ring comprising: a ring structure; a source of glycolic acid or a pharmaceutically acceptable salt or ester adapted to release the glycolic acid or a pharmaceutically acceptable salt or ester thereof when placed in a vagina.

14. The thereof composition of claim 9 wherein the glycolic acid or a pharmaceutically acceptable salt or ester thereof is at least at a concentration of 5 mM.

15. The composition of claim 9 wherein the glycolic acid or a pharmaceutically acceptable salt or ester thereof is at least in amounts of about 380.25 pg/1200 million spermatozoa.

16. The method of claim 1 wherein the glycolic acid or a pharmaceutically acceptable salt or ester thereof is at least at a concentration of 5 mM.

17. The method of claim 1 wherein the glycolic acid or a pharmaceutically acceptable salt or ester thereof is at least in amounts of about 380.25 pg/1200 million spermatozoa.

18. The method of claim 6 wherein the glycolic acid or a pharmaceutically acceptable salt or ester thereof is at least at a concentration of 5 mM.

19. The method of claim 6 wherein wherein the glycolic acid or a pharmaceutically acceptable salt or ester thereof is at least in amounts of about 380.25 pg/1200 million spermatozoa.

20. The ring of claim 13 wherein the glycolic acid or a pharmaceutically acceptable salt or ester thereof is at least at a concentration of 5 mM.

21. The ring of claim 13 wherein the glycolic acid or a pharmaceutically acceptable salt or ester thereof is at least in amounts of about 380.25 pg/1200 million spermatozoa.

Description

[0067] The figures show:

[0068] FIG. 1: Mouse sperm motility after 30 min. 10 mM glycolic acid.

[0069] FIG. 2: Mouse sperm motility after 60 min. 10 mM glycolic acid

[0070] FIG. 3: Mouse sperm motility after 90 min. 10 mM glycolic acid.

[0071] FIG. 4: Mouse sperm progressivity after 30 minutes. 10 mM glycolic acid.

[0072] FIG. 5: Sperm motility in bulls. 10 mM glycolic acid.

[0073] FIG. 6: Sperm progressivity in bulls. 10 mM glycolic acid.

[0074] FIG. 7: Comparison of sperm progressive motility in bulls after 90 min. between treatment with 10, 20 and 30 mM glycolic acid.

[0075] FIG. 8: Glycolic acid enhances sperm motility in human and bull sperm.

[0076] The examples illustrate the invention.

EXAMPLE 1

Material and Methods

[0077] Mouse:

[0078] Extract epididymis and open them in CPA medium (18% Raffinose and 3% Low fat milk powder). Let spermatozoids swim out for 5 min at room temperature.

[0079] For each condition, take 4 μl of mouse sperm in CPA and place in 196 μl of HTF medium from the incubator (at 37° C.). Let it incubate 5 min. in the 37° C. incubator, add substances to the HTF medium (see formulation below) and incubate for different times.

[0080] At each time point, fill a Leja-Slide with 25 μl of the HTF buffer containing the diluted sperm and place in the sperm counting machine (Hamilton-Thorne, USA). The machine takes 10 pictures per samples and analyses the different parameters (motility, progressivity,etc. . . . ). Sperm motility analysis was done blindly. Glycolic acid and 0-lactate were given to the technician from the facility as Substance 1 and Substance 2, only later did she get to know which belong to what compound.

[0081] Bull:

[0082] Frozen bull sperm was defrosted in a warm bath and centrifuged. The supernatant was removed and the sperms were resuspended in HTF-like medium alone or with glycolic acid. Sperms were incubated with the 10, 20 and 30 mM glycolic acid for 10 min. before the measurements began. Measurements were performed by an experienced technician from the urology department.

[0083] Human:

[0084] Human sperm was collected from donors after ejaculation and used on the same day. Frozen human sperm was defrosted in a warm bath before starting the experiments. The regional ethical committee approved sample collection and use of human sperm. Both fresh and frozen human sperm were centrifuged, the supernatant was removed and the sperms were resuspended in HTF-like medium alone or with glycolic acid. Sperms were incubated with the 30 and 60 mM glycolic acid for 10 min before the measurements began. Measurements were performed by an experienced technician from the urology department.

EXAMPLE 2

Experimental Outline

[0085] The examples tested the effect of 10 to 30 mM of glycolic acid and D-lactate on mouse sperm motility and progressivity or on bull motility. For this, epididymic mouse sperm from two strains (WT and DJ-1) or ejaculated frozen bull sperm was obtained. Sperm from each individual was divided and treated either with glycolic acid or with the vehicle for up to 90 minutes. Mouse sperm motility and progressivity was determined with the help of an automatized machine (CASA system, Hamilton-Thorne Research, http://www.hamiltonthorne.com/index.php/products/casa-products/sperm-analysis-systems-research) that uses high frequency photo images and image analysis to analyze these parameters. Frozen ejaculated bull sperm motility was assessed by an experienced technician. Mouse sperm from WT and DJ-1 mice was divided by the software into motile and non-motile sperm according to standardized parameters and results are given as percentages of the total population identified as motile sperm (FIG. 1-4). Sperm progressivity alone was analyzed only on the mouse sperm, as only the software from the CASA system is sensitive enough to make this analysis and this machine can only analyze mouse sperm. Bull sperm was divided into progressive motile, motile (the sum of progressive motile and motile on the spot) and the rest (FIGS. 5, 6 and 7).

EXAMPLE 3

Experimental Results

[0086] The results depicted in FIGS. 1 to 5 show that mouse and bull sperm motility and progressivity are significantly enhanced upon appliance of glycolic acid.

[0087] Addition of 10 mM of Glycolic acid to ejaculated sperm increases sperm motility (percentage of motile spermatozoids) and sperm progressivity (percentage of motile spermatozoids that move forward or progressive) by an average of 19% (motility) and 28% (progressivity) respectively 30 minutes after the application in mouse and bull sperm (FIGS. 1-6). Similar results were obtained after 60 and 90 minutes (FIG. 1-6). A further increase in bull sperm motility and progressivity values when compared to 10 mM glycolic acid was obtained when using higher concentrations (20 and 30 mM) of glycolic acid. Sperm motility and sperm progressivity are key indicators of sperm quality and are strongly associated with fecundation rates and male fertility. Thus, improving sperm motility and progressivity is expected to have a direct effect on the fecundation rate. This can be observed both in WT and DJ-1 mice.

[0088] This enhancement has been consistently observed over all experiments and may vary with dosage, time and between species in a range from 10 to 40% when compared to controls. Importantly, this increase in motility is almost all due to an increase in the proportion of progressive motile spermatozoids. Thus, suggesting that, in these concentrations, glycolic acid has a strong effect on the quality of the movement. Interestingly, in mouse sperm a significance difference in the sperm treated with GA has been observed but not with D-lactate when compared to the controls. The enhancement is sustained over time up to 90 min in both species.

EXAMPLE 4

Results on Sperm Motility and Mitochondrial Activity Upon Application of Glycolic Acid

[0089] FIG. 8 shows that glycolic acid enhances sperm motility in human and bull sperm. This enhancement can be observed in human sperm, both defrosted and fresh, and defrosted bull sperm. Two-way ANOVA analysis was used (much more restrictive than a t-Test). * means p<0.05, **p<0.01, ***p<0.001 and ***p<0.0001. On top of the differences for each time point between the different treatment groups, if all time-points were taken together and analyzed as a whole, the results show that GA induced a significant (p<0.05) increase in sperm motility throughout the whole experiment when compared to the control (i.e. GA treated sperm was higher in every time point when compared to controls). Interestingly, glycolic acid did not increase the mitochondrial membrane potential, and may even decrease it (see FIG. 8). This was measured using JC-1 and FACS sorting to determine the percentage of sperms that was active and the percentage that was inactive. Both sperm motility and mitochondrial measurements are normalized to a control at time-point 0.