Title of Invention

COMPOSITION FOR ASSESSMENT OF HUMAN SPERM FUNCTION AND METHOD THEREOF

Abstract

The present invention relates to a composition for assessment of Human Sperm Funtion using nuclear chromatin Decondensation (NCD) test, said composition comprising sodium chloride of concentration ranging between 0.05 to 0.4% w/v, Sodium dodecyle Sulfate (SDS) of concentration ranging between 0.5 to 2.0 % w/v, and Dithiotheritol (DTT) of concentration ranging between 0.03 to 0.06 % w/v; a process for preparing the same and method thereof.

Full Text

Field of the present invention The present invention relates composition for assessment of Human Sperm Function using nuclear chromatin Decondensation (NCD) test. Background and prior art of present invention After entry of the spermatozoon into the egg during fertilization the highly condensed sperm nucleus swells and the chromatin decondenses prior to the transformation into the male pronucleus (Austin - 1951; Lopata et al. - 1978). Many studies have been carried out in an attempt to reveal the mechanism underlying the unclear swelling and the chromatin decondensation. These studies have shown that nuclear swelling and chromatin decondensation can be brought about in vitro by treatment of the mammalian spermatozoa with solutions containing thiol reagents alone (Marushige and Marushige - 1975, 1978; Witkin et al. - 1975; Wagner and Yun - 1979), thiol reagents plus SDS (Bedford et al. -1973; Mahi and Yanagimachi - 1975), thiol readgents plus Sarkosyl (Evenson et al. -1978) and thiol reagent plus a protease (Gall and Ohsumi - 1976). All of the above studies clearly demonstrated the absolute requirement of a thiol reagent for the decondensation of mammalian spermatozoa. This requirement is in accordance with the observations that the mammalian sperm heads are rich in disulfide bonds which largely form during the maturation of the spermatozoa (Calvin and Bedford -1971; Saowaros and Panyim -1979). In human the sperm head swells and the chromatin decondenses upon treatment with 20 mM dithiothreitol (DTT) (Witkin et al. - 1975) or 50 mM |3-mercaptoethanol (Wagner and Yun - 1979) or 20 mM DTT plus 1.5% Sarkosyl (Evenson et al. - 1978). Obviously, these conditions are much more severe than those expected in the ovum. To seek more physiological condition for the de condensation of the human sperm chromatin, we found that as low as 0.3 mM DTT alone could induce the decondensation within 30 min. In addition, we demonstrate the requirement of an endogenous protease in the decondensation. Further, we introduce the use of actinomycin-D binding for measuring the extent of the decondensation. [A.Incharoensakdi et al., Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok/Thailand; andrologia, 13 (1): 64-73(1981)). During spermatogenesis, histones are replaced by small proteins of highly basic character—known as protamines—that are rich in cysteine residues. During spermatogenesis and epididymal maturation, disulphide brides form between these residues, providing the chromatin with the added stability that is necessary to ensure the transport and integrity of the male genetic material. This state of chromatin condensation may be altered by various factors, such as a shortage of zinc from the prostate, or alterations in protamines, which affects the fertilizing capacity of the spermatozoon (Kvist, 1980; Kvist and Eliasson, 1980; Balhorn et al., 1998; Kvist et al., 1988; Bjorndahl and Kvist, 1996; Kramer and Krawetz (1997). The assessment of chromatin status is very important when evaluating the ability of spermatozoa to fertilize. Many techniques have been described for the evaluation of chromatin status, such as optical microscopy (Krzaaowska, 1982; Huret, 1984; Rosenborg et al., 1990), electron microscopy (Jamil, 1984; Lipitz et al., 1992) and flow cytometry (Eng. et al., 1992; Zucker et al, 1992; Molina et al., 1995; Samocha-Bone et al., 1998; Evenson et al., 1999; spoon et al., 1999). Sperm chromatin defects have been correlated with the reduced ability of spermatozoa to fertilize both in the context of assist reproduction techniques (Bianchi et al., 1996; Hoshi et al., 1996; Sakkas et al., 1996;' Hammadeh et al.,1998; Lopes et al., 1998; Filatov et al., 1999; Gopalkrishnam et al., 1999) and in the general population (Evenson et al., 1999; Hacker-Klom et al., 1999; Spano et al., 2000). Moreover, patients with fertility problems have often been characterized by an increased frequency of spermatozoa with abnormal chromatin Spano et al., 1984; Engh et al., 1992; Foresta et al., 1992; Kosower et al., 1992; Liu and Baker, 1992; Hughes et al., 1996). In this study, different groups of patients were examined, both fertilize and infertile, in order to analyze the standard parameters of semen analysis and those that depend on the state of sperm chromatin. Thus, it was possible to observe differences between patient groups, using the technique of flow cytometry after DNA staining with the fluorochrome, propidium iodide (PI). [J.Molina et al., titled: Chromatin status in human ejaculated spermatozoa from infertile patients and relationship to seminal parameters; Human Reproduction Vol.16, No.3, pp.534-539, 2001] The nucleus of the mammalian spermatid becomes highly condensed during the latter stages of spermatogenesis. This condensation is accompanied by biochemical changes involving the replacement of the histones by the more basic, arginie- and cysteine- rich protamines. The condensed sperm nucleus appears to be chemically more resistant than nuclei of other cells. This resistance is probably due, at least partially, to the disulphide bridges existing between the adjacent protamine molecules within the sperm chromatin. After a spermatozoon has penetrated the ovum, the sperm nucleus decondenses. Swelling of the sperm nucleus was also achieved in vitro by treating spermatozoa with disulphide bond reducing agents in the presence of the detergent sodium dodecyl sulphate (SDS). However, a fraction of human sperm nuclei was found to swell in the presence of SDS alone, although high individual variations were observed. In addition, a correlation was found between the SOS-dependent swelling and the degree of condensation determined by electron microscopy. Since the degree of chromatin condensation might be related to the fertilizing potential of spermatozoa, it is of interest to study the SDS-dependent swelling which might contribute to our knowledge of the structure of the human sperm nucleus. To that aim we used turbidimetry which is less time consuming and more accurate than counting the swollen sperm nuclei. Furthermore, we showed that in the presence of SOS, turbidimetry can also be used for determination of the sperm count. [S.Jagar, et al., titled: In Vitro Swelling of the Human Sperm Nucleus in the Presence of Sodium Dodecyl Sulphate; Archives of Andrology 10:201-208 (1983)]. Sperm chromatin abnormalities in some or all of the sperm in a human ejaculate can be caused by inherent errors in sperm cell differentiation or by environmentally induced direct and/or indirect damage. To understand further how such alterations can cause early embryonic failure, it is first important to consider the components of mature sperm chromatin and the interactions between those components. Sperm chromatin is unique, characterized by a predominance of protein-complexed DNA rather than the histone-complexed DNA of somatic cells. In addition to providing the nucleus with a rigid structure, designed in part for penetration into the oocyte, this near-inert complex also confers protection to the male genetic package. Human chromatin condensation. In condensation, in contrast with that of other mammalian species, being in the posterior pole and proceeds applicably. Chromatin condensation results from protein alterations beginning with a number of post posttranslational modifications of histones, such as H4 acetylation. A testis-specific high mobility group family of proteins is expressed exclusively during nuclear elongation, and this DNA binding protein may alter chromatin structure through modulation of topoisomerase I activity. Topoisomerascs are nuclear enzymes that remove torsional stress in DNA by making endogenous nicks that arc then repaired during functionally significant structural rearrangements of chromatin. Elongating spermatids have the highest level of nicks (as might be expected, because that is the spermatid stage with the greatest amount of protein transition activity). Nick/ligation of DNA plays an important role in the reorganization of sperm nuclear chromatin, and aberrations in function can result in un-repaired DNA strand breaks, a topic of importance to loss of male genome function. Nucleosomal histones arc then replaced by transition proteins and subsequently by protamines. In the human, there are two transition proteins, TP1 and TP2, and two major protamines, PI and P2. PI, P2 and transition protein TP2 transcripts, a multigene locus on chromosome 16 expressed co-ordinately, first appear in association with round spermatids. There are two forms of P2: protamine 2a and 2b. Protamine 2b is identical to 2a, except that 2b lacks the first three amino-terminal amino acids . protamine-complexed DNA forms a side-by-side array of chromatin, resulting in chromatin approximately six-fold more compacted than that of somatic metaphase chromasomes. Essentially; the literature to date has implied that the protamines lie in the minor grooves of the DNA helix. However, using optically detected magnetic resonance, Prieto and colleagues have shown that the binding site of a synthetic protamine subdomain is in the major helix groove. From these data the authors have suggested that sperm protamine is in the major instead of the minor groove. The resolution of this issue will be important as models of sperm chromatin that arc compatible or incompatible with clinical fertility are developed, The mature human sperm nucleus contains a highly organized structure consisting of 27-kb loops in topologically constrained domains that are attached at there are attached at their bases to the sperm nuclear matrix. The centrosomes of the 23 human chromosomes appear to be orientated within an inner nuclear localized region, whereas the telomere regions of each chromosome are positioned along the nuclear periphery. Among the more commonly studied mammalian sperm, particle-induced X-ray emission (PIXE) studies reveal that, in bull, stallion, hamster and mouse sperm nuclei, the relative proportions of PI and P2 vary, whereas the total protamine mass to DNA mass is similar; in contrast, human sperm contains significantly less protamine. This agrees with other studies showing that about 15% of human sperm DNA remains complexed with histones. Of the approximate 15% remaining histones, a subset of both somatic and testis-specific histones constitute this complement of human sperm histones, with marked intra-and inter-individual variation in ejaculates. The trends of histone or protamine association are generally consistent for samples from the same person, but the length of the histone-associated regions can vary in different men, suggesting a fine control over the retention of histones. Sperm chromatin structure assay (SCSA) of human sperim produces person-specific dot plot patters, which are certainly related to the individual pattern of the DNA-histone-protamine complex. On the other hand, the persistence of excess histones in spermatozoa can also be considered as an abnormal characteristic, leading to poor packaging of chromatin and instability of the genome. Retention of histone and a lack of processing of P2 leads to increased DNA stainability of human sperm. This staining in independent of metachromatic staining of DNA from altered chromatin structure and DNA strand breaks. The retention of 15% histones probably contributes to this lesser stability of chromatin in human sperm. These histones are not localized in one region of the nucleus; rather, evidence indicates that they are strategically placed within the chromatin and perhaps mark sets of genes that will be preferentially activated during early development. The recent work of Gardiner-Garden and colleagues suggests that the association of sperm DNA with histones or protamines sometimes hanges within as little as 400 bp of DNA, further suggesting that there is fine control over histone retention. For example, the genes encoding 8- and y-globin, which are active in the embryonic yolk sac, contain regions that are histone associated in the sperm. No histone-associated regions are presented in the sites tested within he P and 8 globin genes, which are silent in the yolk sac. In addition to the organization between DNA and proteins, the nucleus as a whole is organized into a series of chromatin loop domains by the nuclear matrix; these loops may represent specific genes. Thus, it appears likely that the role of sperm nuclear packing is not just the containment of the male complement of DNA, but that the nature of the chromatin package is important both for its safe delivery and for early embryo function. Previous general opinion and some miscalculations have led to the view that human sperm chromatin completely fills the nuclear space defined by the nuclear envelope, and that the chromatin package is inert. However, recent atomic force microscope (AFM) measurements by Lee and co-workers showed that the hydrated human sperm nucleus has only halt of its volume taken up by the DNA and protein, leaving sufficient space for potential cell functions to occur. Fertilization requires fusion of both sperm and egg nucleus. At that time, the condensed sperm head undergoes decondensation, a process which is physiologically relevant prior to nuclear fusion. Nuclear material or chromatin in sperms get very much condensed in order to get accommodated inside the limited space available in the sperm head. The process of chromatin condensation need not be always optimal. Since millions of sperms are being produced, few may demonstrate subnormal or above-normal condensation in their nuclear chromatin packaging. Nuclear chromatin decondensation test is a laboratory protocol to determine the status of sperm nuclear chromatin that can be assessed in vitro. In normal semen sample, the test shows > 70% swollen sperm heads. If the percentage is more, it does not matter. But if it is less than 70% it is a matter of concern and needs to be re-looked and re-evaluated again. In such cases, fertilization may not occur and if there is fertilization there may be a possibility of early abortion. Objects of the present invention The main object of the present invention is to develop a composition for assessment of Human Sperm Function using nuclear chromatin Decondensation (NCD) test. Another object of the present invention is to develop a process for preparing the composition for assessment of Human Sperm Function using nuclear chromatin Decondensation (NCD) test. Yet another object of the present invention is to develop a modified Nuclear Chromatin Decondensation (NCD) test method for assessment of Human Sperm Function, said method comprising steps of: Statement of the present invention The present invention relates to a composition for assessment of Human Sperm Function using nuclear chromatin Decondensation (NCD) test, said composition comprising sodium chloride of concentration ranging between 0.05 to 0.4% w/v, Sodium dodecyle Sulfate (SDS) of concentration ranging between 0.5 to 2.0 % w/v, and Dithiotheritol (DTT) of concentration ranging between 0.03 to 0.06 % w/v. Detailed description of the present invention Accordingly, the present invention relates to a composition for assessment of Human Sperm Function using nuclear chromatin Decondensation (NCD) test, said composition comprising sodium chloride of concentration ranging between 0.05 to 0.4% w/v, Sodium dodecyle Sulfate (SDS) of concentration ranging between 0.5 to 2.0 % w/v, and Dithiotheritol (DTT) of concentration ranging between 0.03 to 0.06 % w/v. In an embodiment of the present invention, the concentration of sodium chloride is about 0.2% w/v. In another embodiment of the present invention, concentration of Sodium Dodecyle Sulfate (SDS) is about 1.0% w/v. In yet another embodiment of the present invention, concentration of Dithiotheritol (DTT) is ranging between 0.045 % w/v. In still another embodiment of the present invention, the invention relates to a process of preparing a composition for assessment of Human Sperm Function using nuclear chromatin Decondensation (NCD) test, said process comprising steps of mixing sodium chloride of concentration ranging between 0.05 to 0.4% w/v, Sodium dodecyle Sulfate (SDS) of concentration ranging between 0.5 to 2.0 % w/v, and Dithiotheritol (DTT) of concentration ranging between 0.03 to 0.06 % w/v and obtaining the composition. In still another embodiment of the present invention, the concentration of sodium chloride is about 0.2% w/v. In still another embodiment of the present invention, concentration of Sodium Dodecyle Sulfate (SDS) is about 1.0% w/v. In still another embodiment of the present invention, concentration of Dithiotheritol (DTT) is ranging between 0.045 % w/v. In still another embodiment of the present invention, the invention relates to a modified Nuclear Chromatin Decondensation (NCD) test method for assessment of Human Sperm Function, said method comprising steps of: mixing pre-warmed composition comprising sodium chloride of concentration ranging between 0.05 to 0.4% w/v, Sodium dodecyle Sulfate (SDS) of concentration ranging between 0.5 to 2.0 % w/v, and Dithiotheritol (DTT) of concentration ranging between 0.03 to 0.06 % w/v with semen, incubating the mixture for 5 to 10 minutes, stopping the incubation with stop solution, and assessing Nuclear Chromatin Decondensation. In still another embodiment of the present invention, stop solution is selected from a group comprising dye Rose Bengal and Cyansine of concentration ranging between 1-10% w/v in 0.5 to 7% v/v aldehyde, selected from a group comprising formaldehyde and glutaraldehyde. In still another embodiment of the present invention, wherein incubating the mixture for about 7 minutes. In still another embodiment of the present invention, the composition is pre-warmed in an incubator at 45 to 50°C. In still another embodiment of the present invention, the semen is with sperm count ranging between 1 to 10 million. In still another embodiment of the present invention, the method is performed in about 10 minutes. In still another embodiment of the present invention, the invention relates to a kit for assessment of Human Sperm Function using nuclear chromatin Decondensation (NCD) test, said kit comprising: 1. Sodium Chloride of concentration ranging between 0.05 to 0.4% w/v, Sodium Dodecyle Sulfate (SDS) of concentration ranging between 0.5 to 2.0 % w/v, Dithiotheritol (DTT) of concentration ranging between 0.003 to 0.007 % w/v. dye of concentration ranging between 1-10% w/v selected from a group comprising Rose Bengal and Cyansine, and optionally along with distilled water, test tube(s), micropipette, slide(s) with cover slip(s). In still another embodiment of the present invention, the dye is prepared in 0.5 to 7% v/v aldehyde, selected from a group comprising formaldehyde and glutaraldehyde. In still another embodiment of the present invention, the sachet contains: Sodium Chloride (NaCl) Sodium dodecyle Sulfate (SDS) Dithiotheritol (DTT) When dissolved in 10 ml of water, the final concentration of the ingredients is- NaCl—0.2%, SDS— 1%, DTT- 0.045% The colored stop solution contains: 5% Rose Bengal in 2.7% Formaldehyde Cyansine as a dye can also be used in place of Rose Bengal. Similarly Glutaraldehyde can replace Formaldehyde. In still another embodiment of the present invention, the temperature range is in between 45-50°C. In still another embodiment of the present invention, workable range for incubation time duration of 7 minutes, preferably in the time duration is between 5 to 10 minutes. In still another embodiment of the present invention, range of semen volume to be taken 50 µl to 100 µl containing 5-10 millions of sperms. In still another embodiment of the present invention, the workable range for incubation duration of 7 minutes, preferably the duration should be between 5 to 10 minutes. Brief description of the accompanying drawings Figure 1 shows swelled heads of the sperms observed during the modified nuclear chromatin decondensation test of instant invention. Figure 2 shows Flowcytometric analysis indicates M3 peak of control sample gets shifted to right side of the window following nuclear chromatin decondensation (NCD). The invention is further elaborated with the help of following examples. However, these examples should not be construed to limit the scope of the invention Example 1 1. Evaluation of Nuclear Chromatin Decondensation (NCD) in proven fertile subjects (Table removed) Observation: In proven fertile subjects the values of NCD obtained with the new method was > 70 %, which is normal as per published literature. Example 2 2. Evaluation of Nuclear Chromatin Decondensation (NCD) in subjects with normal sperm count, motility, viability and morphology (Table removed) Observation: The results of the present NCD test as evaluated in the above list of subjects clearly indicate that it is closely aligned to the normal values compared to the values obtained in the old protocol. Example 3 3. Evaluation of Nuclear Chromatin Decondensation (NCD) in subjects with normal sperm count and subnormal motility, viability and morphology (Table removed) Observation: Subjects with normal sperm count but subnormal motility, viability and morphology showed subnormal NCD values using both new and old protocol. Example 4 4. Evaluation of Nuclear Chromatin Decondensation (NCD) in Oligospermic subjects with subnormal motility, viability and morphology (Table removed) (Table removed) Observation: In oligospermic subjects with subnormal motility, viability and morphology, the values of NCD obtained with both new and old protocols were found subnormal. Advantages of the present invention A. With the present methodology, the time of completion of one test protocol is significantly reduced. Time taken for one test Existing: 70 minutes; Instant invention: 10 minutes B. Assessment sperm head: Status of sperm head swelling is comparatively better. It makes the distinction of swollen and non-swollen sperm heads very clear. C. The present method is much simpler with limited steps and with only one pre- incubating solution. Preparations of different solutions are done away with. D. It is presented in the form of a laboratory test kit. No such kit is presently available. E. It is 100 % reproducible F. The values are better aligned to the normal range than those obtained with the existing protocol. Claim: 1. A composition for assessment of Human Sperm Function using nuclear chromatin Decondensation (NCD) test, said composition comprising sodium chloride of concentration ranging between 0.05 to 0.4% w/v, Sodium dodecyle Sulfate (SDS) of concentration ranging between 0.5 to 2.0 % w/v, and Dithiotheritol (DTT) of concentration ranging between 0.03 to 0.06 % w/v. 2. A composition as claimed in claim 1, wherein the concentration of sodium chloride is about 0.2% w/v. 3. A composition as claimed in claim 1, wherein concentration of Sodium Dodecyle Sulfate (SDS) is about 1.0% w/v. 4. A composition as claimed in claim 1, wherein, concentration of Dithiotheritol (DTT) is ranging between 0.045 % w/v. 5. A process of preparing a composition for assessment of Human Sperm Function using nuclear chromatin Decondensation (NCD) test, said process comprising steps of mixing sodium chloride of concentration ranging between 0.05 to 0.4% w/v, Sodium dodecyle Sulfate (SDS) of concentration ranging between 0.5 to 2.0 % w/v, and Dithiotheritol (DTT) of concentration ranging between 0.03 to 0.06 % w/v and obtaining the composition. 6. A process as claimed in claim 5, wherein the concentration of sodium chloride is about 0.2% w/v. 7. A process as claimed in claim 5, wherein concentration of Sodium Dodecyle Sulfate (SDS) is about 1.0% w/v. 8. A process as claimed in claim 5, wherein concentration of Dithiotheritol (DTT) is ranging between 0.045 % w/v. 9. A modified Nuclear Chromatin Decondensation (NCD) test method for assessment of Human Sperm Function, said method comprising steps of: a. mixing pre-warmed composition comprising sodium chloride of concentration ranging between 0.05 to 0.4% w/v, Sodium dodecyle Sulfate (SDS) of concentration ranging between 0.5 to 2.0 % w/v, and Dithiotheritol (DTT) of concentration ranging between 0.03 to 0.06 % w/v with semen, b. incubating the mixture for 5 to 10 minutes, c. stopping the incubation with stop solution, and d. assessing Nuclear Chromatin Decondensation. 10. A method as claimed in claim 9, wherein stop solution is selected from a group comprising dye Rose Bengal and Cyansine of concentration ranging between 1-10% w/v in 0.5 to 7% v/v aldehyde, selected from a group comprising formaldehyde and glutaraldehyde. 11. A method as claimed in claim 9, wherein incubating the mixture for about 7 minutes. 12. A method as claimed in claim 9, wherein the composition is pre-warmed in an incubator at 45 to 50°C. 13. A method as claimed in claim 9, wherein the semen is with sperm count ranging between 1 to 10 million. 14. A method as claimed in claim 9, wherein the method is performed in about 10 minutes. 15. A kit for assessment of Human Sperm Function using nuclear chromatin Decondensation (NCD) test, said kit comprising: a. Sodium Chloride of concentration ranging between 0.05 to 0.4% w/v, b. Sodium Dodecyle Sulfate (SDS) of concentration ranging between 0.5 to 2.0 % w/v, c. Dithiotheritol (DTT) of concentration ranging between 0.003 to 0.007 % w/v. d. dye of concentration ranging between 1-10% w/v selected from a group comprising Rose Bengal and Cyansine, and e. optionally along with distilled water, test tube(s), micropipette, slide(s) with cover slip(s). 16. A kit as claimed in claim 15, wherein the dye is prepared in 0.5 to 7% v/v aldehyde, selected from a group comprising formaldehyde and glutaraldehyde. A composition, a modified Nuclear Chromatin Decondensation (NCD) test method for assessment of Human Sperm Function, and a kit, substantially as herein described with reference to the accompanying drawings. 16

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3508-del-2005-Abstract-(22-10-2013).pdf

3508-del-2005-abstract.pdf

3508-DEL-2005-Assignment (29-05-2009)..pdf

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3508-del-2005-Claims-(22-10-2013).pdf

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3508-del-2005-description (complete).pdf

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3508-DEL-2005-Form-13 (06-07-2009).pdf

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3508-DEL-2005-Form-18 (24-07-2009).pdf

3508-del-2005-form-18.pdf

3508-del-2005-form-2.pdf

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