WO2022090782A1 - Methods for improving sperm functionality and applications thereof - Google Patents
Methods for improving sperm functionality and applications thereof Download PDFInfo
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- WO2022090782A1 WO2022090782A1 PCT/IB2020/060230 IB2020060230W WO2022090782A1 WO 2022090782 A1 WO2022090782 A1 WO 2022090782A1 IB 2020060230 W IB2020060230 W IB 2020060230W WO 2022090782 A1 WO2022090782 A1 WO 2022090782A1
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- sperm
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- pollen
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- semen
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/0231—Chemically defined matrices, e.g. alginate gels, for immobilising, holding or storing cells, tissue or organs for preservation purposes; Chemically altering or fixing cells, tissue or organs, e.g. by cross-linking, for preservation purposes
Definitions
- the present invention is in the technical field of a sperm shipment medium/kit that can preserve sperm quality, motility and membrane integrity, more particularly, invention describes a synthesized biocompatible polymer/s for transport of biological materials.
- Semen samples are mostly transported either on dry ice, -80°C temperature, or in liquid nitrogen containing cryogenic storage tanks (-196°C).
- cryopreservation One of the most conventional method for shipment and storage of semen samples is cryopreservation. However it causes a significant loss of sperm motility, viability and DNA damage (50 to 80% loss of sperm). The fertilizing ability of the human spermatozoa decreases after cryopreservation.
- Semen collection in the laboratory or hospital environment has its own advantages and disadvantages. Although it provides labs with a quick transit of the sample and more authenticated proof, many men do find it extremely difficult produce a semen sample in unfamiliar surroundings.
- Sperm cell post-cryopres- ervation motility rates and vitality may be decreased by 25% to 75%, mainly due to the stress to which these cells are subjected during the cryopreservation process, derived chiefly from cell dehydration, high solute concentrations, recrystallization and changes in plasma membrane integrity, thus leading to sperm cell functional and structural changes affects the functionality of sperms by inducing structural damage and functional changes to sperm, such as decreased vitality, motility, speed, and reduced fertilization potential, partly due to plasma membrane integrity disorders and other still unclear factors.
- thermoresponive gel A number of new in vitro and in vivo applications of thermoresponive gel have been reported. For example, these have been used for wound dressing, microcapsules for pancreatic islets, a drug delivery system, and three- dimensional culture matrices for various cells. Extracellular matrices, including collagen and Matrigel, have been used as scaffolds for clonal expansion of cells in three-dimensional culture.
- Pollen storage is useful for breeding programmes, genetic conservation, artificial pollination and self-incompatibility.
- Germinability of a stored pollen is determined by vegetative cell membrane and this gets impacted in conventional storage methods resulting in misrepresentation of the genetic composition of original lot.
- Tricellular pollens have short lifespans and do not usually tolerate desiccation to moisture contents low enough to permit exposure to sub-zero temperatures. In cryopreservation, pollen loses its viability during cooling and rewarming process resulting in poor quality seeds and eventual yield.
- SeraGel® is a copolymer composed of thermoreversible sol-gel polymer compositions is characterized by its dynamic viscoelastic properties. Mode of Action: Pollen sample will be mixed with SeraGel® will become gel when activated and incubated at 37 degrees. Upon receiving the sample the end user will recondition the gel and centrifuge the contents to pellet the pollen sample and use it for further analysis. Water soluble compositions increased the solubility thus transition happens homogeneously without affecting the characteristic of polymer as well the biomaterial that is shipped. Our objective was to design and standardize a remote shipping medium that allows farms or research and development centres to collect pollen sample and ship it overnight to an end customer at room temperature without cold chain logistics while preserving the pollen quality parameters intact.
- this innovation can be used as a storage and transportation medium.
- the primary objective of the present invention is to provide a sperm shipment medium that can preserve sperm quality, motility and membrane integrity.
- the present invention describes a synthesized biocompatible polymer, SeraGel® that has no biological contaminants.
- SeraGel® is a proprietary mark of Seragen.
- the present invention describes SeraGel®, which is a copolymer composed of thermoresponsive polymer blocks [poly(Nisopropylacrylamide-co-n-butyl methacrylate) poly(NIPAAm-co-BMA)] and hydrophilic polymer blocks (polyethylene glycol [PEG]) and is characterized by its temperature-dependent dynamic viscoelastic properties.
- the thermoresponsive blocks are hydrophilic at temperatures below the sol-gel transition temperature and are hydrophobic at temperatures above the sol-gel transition temperature. The hydrophobic interaction results in formation of a homogenous three-dimensional polymer network in water.
- the sol-gel transition temperature can be controlled by altering the chemical composition of NIPAAm-co-BMA and PEG.
- Sperm viability can be prolonged if they are allowed to be immotile.
- a sperm is highly dynamic and keeps moving and spends its stored energy during the movement while additional energy can not be synthesised. Hence they lose their viability and quality due to lack of energy.
- immobilisation can be reversed with mechanical methods without chemical based or enzyme based methods, then this could solve the issue of maintaining sperm viability and can be transported at ambient temperatures.
- Improving the functionality of sperm by providing a sperm shipment medium that can preserve sperm quality, motility and membrane integrity.
- a synthesized biocompatible polymer that has no biological contaminants is synthesized.
- Such methods may be used in artificial insemination to reduce the number of sperm needed for insemination and to improve conception rates.
- compositions and uses of such sperm shipment medium can be used to ship any biomaterial such as, pollen grains, embryos, eggs (oocytes), cells, biopsies, tissues and similar items where temperature controlled shipping is very important.
- the possible uses of this invention include but not limited to, Individual or combination of biopolymers can be used for biomaterial transportation medium/kit.
- kits for transportation of biological samples using biopolymers especially thermoreversible polymers.
- the kits may include several transport materials, components, binding agents, containers for the transport as part of the embodiments of the invention.
- the present invention deals with methods for improving the functionality of sperm, by providing a sperm shipment medium proposed that can preserve sperm quality, motility and membrane integrity, more particularly, invention describes a synthesized biocompatible polymer that has no biological contaminants. Such methods may be used in artificial insemination to reduce the number of sperm needed for insemination and to improve conception rates. Invention also provided the compositions and uses of such sperm shipment medium.
- FIG 1 illustrates the structure of polymer [poly(Nisopropylacrylamide-co-n-butyl methacrylate) poly(NIPAAm-co-BMA)] and hydrophilic polymer blocks (polyethylene glycol 1(A) Following pictures represents chemical formula of PNIPAAm and 1(B) Representation of volume phase transition between coil (left) and globular (right) hydrogel conformations, according to the aspects of present invention.
- FIG 2 illustrates the chemical formula of PNIPAAm and 2(B) Representation of volume phase transition between coil (left) and globular (right) hydrogel conformations.
- Following pictures shows representation of 2(A) the swollen PNIPAAm hydrosol in aqueous solution below T c (32 °C) and 2(B) the shrunken dehydrated PNIPAAm hydrogel above T c (32 °C).
- FIG 3 illustrates the Flowchart describing steps for improving the functionality of sperm (including use of biopolymer), according to the aspects of present invention.
- FIG 4 illustrates the Flowchart describing steps of producing a sperm shipment medium/ Methods of invention, according to the aspects of present invention.
- FIG 5 illustrates the Flowchart describing steps/ methods to employ the use of biocompatible polymers during artificial insemination, according to the aspects of present invention.
- FIG 6 illustrates the pollen tube formation post preservation of pollen in the present invention.
- FIG 7 Illustrates the biomaterial transportation KIT components
- a dosage refers to one or more than one dosage.
- polypeptide peptide
- protein protein
- Polypeptide peptide
- peptide and “protein” can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins.
- subject generally refer to a human or mammals.
- sample refers to a polynucleotides, antibodies fragments, polypeptides, peptides, genomic DNA, RNA, or cDNA, polypeptides, a cell, a tissue, and derivatives thereof may comprise a bodily fluid or a soluble cell preparation, or culture media, a chromosome, an organelle, or membrane isolated or extracted from a cell.
- Body fluid refers to, but is not limited to, plasma, serum, urine, peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, or umbilical cord blood.
- CSF cerebrospinal fluid
- SeraGel® is a Thermoreversible sol-gel polymer compositions and for example, a copolymer composed of thermoresponsive polymer blocks [poly(Nisopropylacrylamide-co-n-bu- tyl methacrylate) poly(NIPAAm-co-BMA)] and hydrophilic polymer blocks (polyethylene glycol [PEG]) and is characterized by its temperature-dependent dynamic vise oelastic properties.
- thermoresponsive blocks are hydrophilic at temperatures below the sol-gel transition temperature and are hydrophobic at temperatures above the sol-gel transition temperature.
- the hydrophobic interaction results in formation of a homogenous three-dimensional polymer network in water.
- the sol-gel transition temperature can be controlled by altering the chemical composition of NIPAAm-co-BMA and PEG.
- Cells or tissues can be embedded in liquid gel solution at lower temperatures and stored, shipped or cultured three-dimension- ally in a hydrogel state at 37°C.
- a number of new in vitro and in vivo applications of ther- moresponive gel have been reported. For example, these have been used for wound dressing, microcapsules for pancreatic islets, a drug delivery system, and three-dimensional culture matrices for various cells.
- Extracellular matrices including collagen and Matrigel, have been used as scaffolds for clonal expansion of cells in three-dimensional culture. Materials from biological sources, however, cannot be absolutely free from contamination with unknown substances, including pathogens. SeraGel® is a purely synthesized biocompatible Thermoreversible sol-gel polymer compositions that has no biological contaminants.
- Sperm sample will be mixed with pre-cooled SeraGel® which will be liquid at 4 degrees and allowed to become gel when incubated at 37 degrees. Upon receiving the sample the end user will cool the gel and centrifuge the contents to pellet the sperm sample and use it for further analysis.
- compositions of the present invention provide a single sample collection, transport, and storage reagent that facilitate: 1) procuring high quality sample at the convenient of a patients residence or the current location 2) Safe handling and transport of specimens, and 3) Stabilization and preservation of sample quality and prevents degradation for prolonged periods (upto 2 weeks) at ambient temperatures.
- the results of one such study are presented in the following example: METHODOLOGY
- hydrogel-forming polymer constituting the hydrogel according to the present invention refers to a polymer has a property such that it can form a hydrogel which has a crosslinking or network structure by retaining water (in the inside thereof) on the basis of such a structure.
- the "hydrogel” refers to a gel, which comprises, at least a crosslinked or network structure comprising a polymer, and water (as a dispersion liquid) supported or retained by such a structure.
- the "dispersion liquid” retained in the crosslinked or network structure is not particularly limited, as long as it is a liquid comprising water as a main or major component. More specifically, the dispersion liquid may for example be either of water per se, an aqueous solution and/or water-containing liquid.
- the water-containing liquid may preferably contain 80 parts or more, more preferably 90 parts or more of water, based on the total 100 parts of the water-containing liquid. (Sol-gel transition temperature)
- the terms "sol state”, “gel state” and “sol-gel transition temperature” are defined in the following manner. With respect to these definitions, a paper (Polymer Journal, 18(5), 411-416 (1986)) may be referred to.
- FIG 1 illustrates the structure of polymer [poly(Nisopropylacrylamide-co-n-butyl methacrylate) poly(NIPAAm-co-BMA)] and hydrophilic polymer blocks (polyethylene glycol)
- FIG 2 illustrates the chemical formula of PNIPAAm and 2(B) Representation of volume phase transition between coil (left) and globular (right) hydrogel conformations.
- FIG 2 illustrates the chemical formula of PNIPAAm and 2(B) Representation of volume phase transition between coil (left) and globular (right) hydrogel conformations.
- Following pictures shows representation of 2(A) the swollen PNIPAAm hydrosol in aqueous solution below Tc (32 °C) and 2(B) the shrunken dehydrated PNIPAAm hydrogel above T c (32 °C).
- commercially available thermoreversible polymer(S) have been used as one of the key ingredient in the semen storage and shipment medium.
- Thermoreversible polymer (Henceforth will be referred as “Polymer” suspended in phosphate buffered saline with more than one of the following opponents as additives.
- Thermoreversible polymer (l%-50%) (For eg: Poloxamers, Poly caprolactane, NIPAAM etc)
- Vascular endothelial growth factors -VEGF vascular endothelial growth factors -VEGF(0.0001ug/ml-10ug/ml)
- Nerve Growth Factor-NGF (0.0001ug/ml-10ug/ml)
- Vitamin C (0.01mg/ml-100mg/ml)
- Vitamin E (HU-200IU/ml)
- Zinc Sulfate (O.OOlmg/ml-lOmg/ml)
- thermoreversible sol-gel composition having unique properties for use in the methods of this invention for storage and transportation of semen and sperm samples including but not limited.
- a general method of preparing the compositions of this invention can be described as the following: combining an amount of the thermoreversible polymer or a combination of two polymers with similar characteristics and an amount of a suitable aqueous solvent, wherein the amount of polymer(S) is sufficient to form a solution having more than or equal to 40% % w/w of polymer(s); stirring the mixture at a sufficiently medium speed at about or below 10 °C at for a first period of time; and rocking the mixture for a second period of time there by forming a solution.
- the aqueous solvent may be cooled /or stirred when combining with the polymer(s).
- the polymer may be added in small potions.
- One polymer(s) may be added before the other polymer is added.
- a cold aqueous solvent may be poured to the polymer(s).
- the stirring and/or rocking may or may not be continuous although continuous stirring at medium speeds is preferred for steady dissolution of the polymer(s). Further, a period of stirring may alternate with a period of rocking.
- FIG 3 illustrates the Flowchart describing steps for improving the functionality of sperm (including use of biopolymer), according to the aspects of present invention.
- FIG 4 illustrates the Flowchart describing steps of producing a sperm shipment medium/ Methods of invention, according to the aspects of present invention.
- thermally stable sol-gel refers to a composition, which undergoes a phase transition from a liquid phase to gel phase or vice versa when the temperature is raised above or reduced below a critical value, which is referred to as "transition temperature.”
- liquid phase or “liquid state” refers to a liquid or flowable form with a viscosity of less than 2000 Pascal-seconds.
- gel phase or “gel state” refers to a gel or relatively solid form with a viscosity of greater than 10,000 Pascal-seconds.
- complex viscosities of compositions are reported in Pascal-seconds and all viscosities reported in this application are reported as complex viscosities.
- thermoreversible sol-gel composition changes from a liquid state to a gel state when the temperature is raised to or above the critical value, or transition temperature, and undergoes a phase transition from the gel state to the liquid state when the temperature is lowered to or below the critical value, or transition temperature.
- phase transition from a liquid to a gel and vice versa occurs in less than 10 minutes, more preferably in less than 5 minutes and even more preferably in less than 1 minute.
- aqueous solvent refers to water or a water based solution, e.g. an aqueous salt solution, such as a saline solution, phosphate buffered saline, and other aqueous solutions suitable for dissolving the poloxamers described herein.
- An aqueous salt solution may contain one or more biocompatible salts selected from sodium chloride (NaCl), potassium chloride (KC1), sodium sulfate (Na2S04 ), sodium bisulfate (NaHS04), sodium phosphate (Na HP04 ), monosodium phosphate (NaH2P04), disodium phosphate (Na2HP04), potassium phosphate(K3PO4), monopotassium phosphate (KH2P04), dipotassium phosphate (K2HPO4), various soluble calcium and magnesium salts, such as calcium chloride (CaC12), magnesium chloride (MgC12) and other salts formed by a combination of a cation selected from the group consisting of sodium, potassium, calcium, magnesium, ammonium, and tetraalkylammonium, with an anion selected from the group consisting of chloride, bromide, tartrate, mesylate, acetate, maleate, and oxalate and other biocompatible
- PBS phosphate buffered saline
- PBS can have additional ions such as Calcium (Ca2+) or Magnesium (Mg 2+).
- the present invention is further elaborated with the help of the following examples. However, these examples should not be construed to limit the scope of the present invention.
- EXAMPLE 1 METHODS TO EMPLOY THE USE OF BIOCOMPATIBLE POLY- MERS DURING ARTIFICIAL INSEMINATION
- FIG 5 illustrates the Flowchart describing steps/ methods to employ the use of biocompatible polymers during artificial insemination, according to the aspects of present invention.
- the samples in Group 1 were immersed in liquid nitrogen throughout the experiment in cryogenic storage tanks,
- the semen samples in Group 2 were suspended in commercially available semen storage media in Group 3 were placed in a Styrofoam box kept at 4 degree for 7 days
- samples belongs to Group 4 kept in the Styrofoam box containing the samples was shipped by on-road transportation to assess the effects of shipping;
- motility indicates the functional competence of sperm cells and vitality is a way to identify living and dead sperm (Cavalcante et al., 2006); the former is one of the most severely affected variables (Watson, 1995). Verza Jr et al. (2009) studied the resistance of human sperm to cryoinjury after repeated cycles of freezing and thawing, using the quick method with liquid nitrogen vapor; the authors described a sharp drop in sperm motility after each thawing cycle. Results
- the new method will allow the laboratory also to report the exact motility without worrying about temperature changes.
- kits for practicing the as- says/methods/transportation described herein using biopolymers.
- the kits may include several transport materials, components, binding agents, containers for the transport of biological samples as part of the embodiments of the invention.
- compositions and constructs disclosed herein may be present in compositions including one or more physiologically acceptable carriers or diluents, such as water or saline.
- Such compositions may additionally contain other components, such as preservatives, stabilizers, buffers and the like. Carriers, diluents and other components suitable for use in the present compositions.
Abstract
The present invention deals with methods for improving the functionality of sperm, by providing a sperm shipment medium that can preserve sperm quality, motility and membrane integrity. Invention particularly describes a room temperature stable biomaterial shipping and storage medium specifically semen/sperm samples but not limited to where synthesised biocompatible thermoreversible polymer is a key ingredient that has no biological contaminants. Such methods may be used in semen analysis, artificial insemination to reduce the number of sperm needed for insemination and to improve conception rates. The invention provides compositions, kits and integrated systems for transportation of biological samples like semen, pollen from any plant source, tissues, embryo, oocyte but not limited to. In addition, the kits may include several transport materials, components, binding agents, containers for the transport as part of the embodiments of the invention.
Description
“METHODS FOR IMPROVING SPERM FUNCTIONALITY AND APPLICATIONS THEREOF”
TECHNICAL FIELD OF THE INVENTION
The present invention is in the technical field of a sperm shipment medium/kit that can preserve sperm quality, motility and membrane integrity, more particularly, invention describes a synthesized biocompatible polymer/s for transport of biological materials.
BACKGROUND OF THE INVENTION
Over recent decades a global decline in human sperm quality has been observed due to environmental, occupational and lifestyle factors. The diagnostic industry is undergoing a surge in infertility market.
For the male infertility, development of reliable method for shipment of semen samples at ambient temperature from remote areas to specialized labs is highly desirable. Semen samples are mostly transported either on dry ice, -80°C temperature, or in liquid nitrogen containing cryogenic storage tanks (-196°C).
One of the most conventional method for shipment and storage of semen samples is cryopreservation. However it causes a significant loss of sperm motility, viability and DNA damage (50 to 80% loss of sperm). The fertilizing ability of the human spermatozoa decreases after cryopreservation.
In addition, these methods may cause temperature fluctuations during sample transportation (Til et al 2017). Time and temperature play an important role in shipping of the sperms as they lose viability very quickly. Mitochondrial membrane integrity is another important factor in sperm viability analysis (Til et al 2016). Mitochondria provide the necessary energy to the sperm in the form of adenosine triphosphate (ATP). Mitochondrial DNA also transcribes several proteins for oxidative phosphorylation. Sperm characteristics and male fertility may be affected by changes in mitochondrial membrane potential or mutations in mtDNA(Camara et al 2008).
Newer tests methods have been established to investigate sperm physiology and functions by monitoring characteristics such as motility, capacitation, the acrosome reaction, reactive oxygen species, sperm DNA damage, chromatin structure, zona pellucida binding, and sperm-oocyte fusion.
The major problem with these assays is that they are complex to perform, difficult to standardize and are only available in a few academic centres.
Moreover, since the quality of fresh human semen deteriorates rapidly, patients requiring analysis are subjected to the inconvenience of physically delivering a freshly produced sample to the diagnostic laboratory.
Semen collection in the laboratory or hospital environment has its own advantages and disadvantages. Although it provides labs with a quick transit of the sample and more authenticated proof, many men do find it extremely difficult produce a semen sample in unfamiliar surroundings.
When such men are coerced into providing it in the laboratory only, there is a high probability that they will either fail to do so or will give an incomplete sample. Studies have confirmed that a man who is more sexually aroused can give better quality semen. Thus it is very important that these men are identified and asked to collect semen from their home.
However, a major hindrance to this type is the duration it takes to report the sample to the laboratory after collection. Usually labs ask patients to deliver it in 30 - 45 minutes. But it is quite difficult for patients who stay quite far away from the hospital.
Samples that are brought delayed will lead to temperature changes and attenuation of motility along with other parameters.
This can lead to drastic variations in the reporting of the semen analysis.
Current availability: For the male infertility, development of reliable method for shipment of semen samples at ambient temperature from remote areas to specialized labs is highly desirable. Semen samples are mostly transported either on dry ice, -80°C temperature, or in liquid nitrogen containing cryogenic storage tanks (-196°C). One of the most conventional method for shipment and storage of semen samples is cryopreservation however it causes a significant loss of sperm motility, viability and DNA damage (50 to 80% loss of sperm). The fertilizing ability of the human spermatozoa decreases after cryopreservation. Sperm cell post-cryopres- ervation motility rates and vitality may be decreased by 25% to 75%, mainly due to the stress to which these cells are subjected during the cryopreservation process, derived chiefly from cell dehydration, high solute concentrations, recrystallization and changes in plasma membrane integrity, thus leading to sperm cell functional and structural changes affects the functionality of sperms by inducing structural damage and functional changes to sperm, such as decreased vitality, motility, speed, and reduced fertilization potential, partly due to plasma membrane integrity disorders and other still unclear factors.
In addition to these factors, storage conditions also play an important role, since these samples are often transported and exposed to temperature variations, quick exposure to ambient temperature during tank changes, and longer exposures to higher temperatures when they are shipped over long distances in dry ice (-80°C) results in huge variations in the rates of recovery of sperm cell vitality and motility. This ultra low temperature storage remains a costly process that entails complicated logistical and technical implications.
Therefore, there is an urgent need in the art to overcome such a scenario and to effectively use home collection as well, there is an urgent need of a new technique that can provide patients with the comfort of collecting semen at their home and not worry about rushing to the laboratory within the stipulated time.
In addition, there have been attempts in recent studies towards addressing these issues, cells or tissues can be embedded in liquid gel solution at lower temperatures and stored, shipped or cultured three-dimensionally in a hydrogel state at 37°C. A number of new in vitro and in vivo applications of thermoresponive gel have been reported. For example, these have been used for wound dressing, microcapsules for pancreatic islets, a drug delivery system, and three-
dimensional culture matrices for various cells. Extracellular matrices, including collagen and Matrigel, have been used as scaffolds for clonal expansion of cells in three-dimensional culture.
However, materials from biological sources, cannot be absolutely free from contamination with unknown substances, including pathogens.
In summary, there is a need in the art to develop a sperm shipment medium that can preserve sperm quality, motility and membrane integrity. Moreover, such biocompatible polymer should be devoid of biological contaminants. In addition, there is a need for home collection of semen and not worry about rushing to the laboratory within the stipulated time.
Similar challenges have been faced with pollen preservation and shipment and there is a need in the art to develop a pollen shipment and storage medium that can preserve the pollen viability and germination potential. Pollen storage is useful for breeding programmes, genetic conservation, artificial pollination and self-incompatibility.
Longevity of pollen varies greatly with plant species and storage conditions. Many techniques are followed these days to maintain the viability of pollen under storage conditions. Mostly binucleate pollen can be stored for long periods of time without loss of viability as compared to tri-nucleate pollen.
Pollen stored at low temperature presented germination capacity better than high temperature. There are many factors which influence the pollen viability, however challenges continue in terms of conventional storage mechanisms.
The challenges that affect pollen storage are as follows.
Germinability of a stored pollen is determined by vegetative cell membrane and this gets impacted in conventional storage methods resulting in misrepresentation of the genetic composition of original lot.
Tricellular pollens have short lifespans and do not usually tolerate desiccation to moisture contents low enough to permit exposure to sub-zero temperatures.
In cryopreservation, pollen loses its viability during cooling and rewarming process resulting in poor quality seeds and eventual yield.
One size doesn’t fits all. Life span of pollens in storage are genetically controlled and varies with species, even under optimum storage conditions. Not all species of pollen can be stored at cryogenic temperatures because of differences in chemical composition and storage physiology.
Only LN2 tolerant pollens can be stored using Liquid Nitrogen and LN2 sensitive pollens require alternate methods
SeraGel®, the present invention is a copolymer composed of thermoreversible sol-gel polymer compositions is characterized by its dynamic viscoelastic properties. Mode of Action: Pollen sample will be mixed with SeraGel® will become gel when activated and incubated at 37 degrees. Upon receiving the sample the end user will recondition the gel and centrifuge the contents to pellet the pollen sample and use it for further analysis. Water soluble compositions increased the solubility thus transition happens homogeneously without affecting the characteristic of polymer as well the biomaterial that is shipped. Our objective was to design and standardize a remote shipping medium that allows farms or research and development centres to collect pollen sample and ship it overnight to an end customer at room temperature without cold chain logistics while preserving the pollen quality parameters intact.
Also for any biomaterial (including but not limited to semen, pollen, tissues, biopsies, oocytes, embryos, cells etc) transportation where maintaining the viability is an issue for effective end use or further analysis of any kind, this innovation can be used as a storage and transportation medium.
SUMMARY OF THE INVENTION
The primary objective of the present invention is to provide a sperm shipment medium that can preserve sperm quality, motility and membrane integrity.
In one of the embodiment, the present invention describes a synthesized biocompatible polymer, SeraGel® that has no biological contaminants. SeraGel® is a proprietary mark of Seragen.
In one of the embodiment, the present invention describes SeraGel®, which is a copolymer composed of thermoresponsive polymer blocks [poly(Nisopropylacrylamide-co-n-butyl methacrylate) poly(NIPAAm-co-BMA)] and hydrophilic polymer blocks (polyethylene glycol [PEG]) and is characterized by its temperature-dependent dynamic viscoelastic properties. The thermoresponsive blocks are hydrophilic at temperatures below the sol-gel transition temperature and are hydrophobic at temperatures above the sol-gel transition temperature. The hydrophobic interaction results in formation of a homogenous three-dimensional polymer network in water. The sol-gel transition temperature can be controlled by altering the chemical composition of NIPAAm-co-BMA and PEG.
Sperm viability can be prolonged if they are allowed to be immotile. A sperm is highly dynamic and keeps moving and spends its stored energy during the movement while additional energy can not be synthesised. Hence they lose their viability and quality due to lack of energy. Hence if there is a way to arrest their motility and at the same time immobilisation can be reversed with mechanical methods without chemical based or enzyme based methods, then this could solve the issue of maintaining sperm viability and can be transported at ambient temperatures.
As will be appreciated by a person skilled in the art the present invention provides a variety of following advantages,
Improving the functionality of sperm, by providing a sperm shipment medium that can preserve sperm quality, motility and membrane integrity.
A synthesized biocompatible polymer that has no biological contaminants.
Such methods may be used in artificial insemination to reduce the number of sperm needed for insemination and to improve conception rates.
Invention also provided the compositions and uses of such sperm shipment medium.
This invention can be used to ship any biomaterial such as, pollen grains, embryos, eggs (oocytes), cells, biopsies, tissues and similar items where temperature controlled shipping is very important.
The possible uses of this invention include but not limited to, Individual or combination of biopolymers can be used for biomaterial transportation medium/kit.
The invention provides compositions, kits and integrated systems for transportation of biological samples using biopolymers especially thermoreversible polymers. In addition, the kits may include several transport materials, components, binding agents, containers for the transport as part of the embodiments of the invention.
In summary, the present invention deals with methods for improving the functionality of sperm, by providing a sperm shipment medium proposed that can preserve sperm quality, motility and membrane integrity, more particularly, invention describes a synthesized biocompatible polymer that has no biological contaminants. Such methods may be used in artificial insemination to reduce the number of sperm needed for insemination and to improve conception rates. Invention also provided the compositions and uses of such sperm shipment medium.
Several aspects of the invention are described below with reference to examples for illustration. However, one skilled in the relevant art will recognize that the invention can be practiced without one or more of the specific details or with other methods, components, materials and so forth. In other instances, well-known structures, materials, or operations are not shown in detail to avoid obscuring the features of the invention. Furthermore, the features/aspects described can be practiced in various combinations, though only some of the combinations are described herein for conciseness.
BRIEF DESCRIPTION OF THE DRAWINGS
Example embodiments of the present invention will be described with reference to the accompanying drawings briefly described below.
FIG 1 illustrates the structure of polymer [poly(Nisopropylacrylamide-co-n-butyl methacrylate) poly(NIPAAm-co-BMA)] and hydrophilic polymer blocks (polyethylene glycol 1(A) Following pictures represents chemical formula of PNIPAAm and 1(B) Representation of volume phase transition between coil (left) and globular (right) hydrogel conformations, according to the aspects of present invention.
FIG 2 illustrates the chemical formula of PNIPAAm and 2(B) Representation of volume phase transition between coil (left) and globular (right) hydrogel conformations. Following pictures shows representation of 2(A) the swollen PNIPAAm hydrosol in aqueous solution below Tc (32 °C) and 2(B) the shrunken dehydrated PNIPAAm hydrogel above Tc (32 °C). Adapted with permission from Reference [641. Copyright € 2015 Springer Science & Business Media Singapore, according to the aspects of present invention.
FIG 3 illustrates the Flowchart describing steps for improving the functionality of sperm (including use of biopolymer), according to the aspects of present invention.
FIG 4 illustrates the Flowchart describing steps of producing a sperm shipment medium/ Methods of invention, according to the aspects of present invention.
FIG 5 illustrates the Flowchart describing steps/ methods to employ the use of biocompatible polymers during artificial insemination, according to the aspects of present invention.
FIG 6: illustrates the pollen tube formation post preservation of pollen in the present invention.
FIG 7 Illustrates the biomaterial transportation KIT components
In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.
DETAILED DESCRIPTION OF THE INVENTION
It is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or
illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.
In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.
The use of “including”, “comprising” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item. Further, the use of terms “first”, “second”, and “third”, and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.
As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise. By way of example, “a dosage” refers to one or more than one dosage.
The terms “comprising”, “comprises” and “comprised of’ as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps.
All documents cited in the present specification are hereby incorporated by reference in their totality. In particular, the teachings of all documents herein specifically referred to are incorporated by reference.
Example embodiments of the present invention are described with reference to the accompanying figures.
In the drawings, like reference numbers generally indicate identical, functionally similar,
and/or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.
Definitions
The following terms are used as defined below throughout this application unless otherwise indicated.
The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. "Polypeptide," "peptide" and "protein” can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins.
The terms "subject", "patient" or "individual" generally refer to a human or mammals.
[058] "Sample" refers to a polynucleotides, antibodies fragments, polypeptides, peptides, genomic DNA, RNA, or cDNA, polypeptides, a cell, a tissue, and derivatives thereof may comprise a bodily fluid or a soluble cell preparation, or culture media, a chromosome, an organelle, or membrane isolated or extracted from a cell.
“Body fluid” refers to, but is not limited to, plasma, serum, urine, peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, or umbilical cord blood.
EMBODIMENTS OF THE INVENTION
Inventors objective was to design and standardize a remote sperm shipping medium that allows patients/donors/diagnostic centre or IVF clinics to collect semen sample and ship it overnight to a sperm bank or diagnostic centre or an IVF clinic at room temperature without cold chain logistics while preserving the semen quality parameters intact.
SeraGel® is a Thermoreversible sol-gel polymer compositions and for example, a copolymer composed of thermoresponsive polymer blocks [poly(Nisopropylacrylamide-co-n-bu- tyl methacrylate) poly(NIPAAm-co-BMA)] and hydrophilic polymer blocks (polyethylene glycol [PEG]) and is characterized by its temperature-dependent dynamic vise oelastic properties. The thermoresponsive blocks are hydrophilic at temperatures below the sol-gel transition temperature and are hydrophobic at temperatures above the sol-gel transition temperature. The hydrophobic interaction results in formation of a homogenous three-dimensional polymer network in water. The sol-gel transition temperature can be controlled by altering the chemical composition of NIPAAm-co-BMA and PEG. Cells or tissues can be embedded in liquid gel solution at lower temperatures and stored, shipped or cultured three-dimension- ally in a hydrogel state at 37°C. A number of new in vitro and in vivo applications of ther- moresponive gel have been reported. For example, these have been used for wound dressing, microcapsules for pancreatic islets, a drug delivery system, and three-dimensional culture matrices for various cells. Extracellular matrices, including collagen and Matrigel, have been used as scaffolds for clonal expansion of cells in three-dimensional culture. Materials from biological sources, however, cannot be absolutely free from contamination with unknown substances, including pathogens. SeraGel® is a purely synthesized biocompatible Thermoreversible sol-gel polymer compositions that has no biological contaminants.
Mode of Action: Sperm sample will be mixed with pre-cooled SeraGel® which will be liquid at 4 degrees and allowed to become gel when incubated at 37 degrees. Upon receiving the sample the end user will cool the gel and centrifuge the contents to pellet the sperm sample and use it for further analysis.
The compositions of the present invention provide a single sample collection, transport, and storage reagent that facilitate: 1) procuring high quality sample at the convenient of a patients residence or the current location 2) Safe handling and transport of specimens, and 3) Stabilization and preservation of sample quality and prevents degradation for prolonged periods (upto 2 weeks) at ambient temperatures. The results of one such study are presented in the following example:
METHODOLOGY
Hydrogel-forming polymer The hydrogel-forming polymer constituting the hydrogel according to the present invention refers to a polymer has a property such that it can form a hydrogel which has a crosslinking or network structure by retaining water (in the inside thereof) on the basis of such a structure. Further, the "hydrogel" refers to a gel, which comprises, at least a crosslinked or network structure comprising a polymer, and water (as a dispersion liquid) supported or retained by such a structure.
The "dispersion liquid" retained in the crosslinked or network structure is not particularly limited, as long as it is a liquid comprising water as a main or major component. More specifically, the dispersion liquid may for example be either of water per se, an aqueous solution and/or water-containing liquid. The water-containing liquid may preferably contain 80 parts or more, more preferably 90 parts or more of water, based on the total 100 parts of the water-containing liquid. (Sol-gel transition temperature)
In the present invention, the terms "sol state", "gel state" and "sol-gel transition temperature" are defined in the following manner. With respect to these definitions, a paper (Polymer Journal, 18(5), 411-416 (1986)) may be referred to.
FIG 1 illustrates the structure of polymer [poly(Nisopropylacrylamide-co-n-butyl methacrylate) poly(NIPAAm-co-BMA)] and hydrophilic polymer blocks (polyethylene glycol
1(A) Following pictures represents chemical formula of PNIPAAm and 1(B) Representation of volume phase transition between coil (left) and globular (right) hydrogel conformations, according to the aspects of present invention. FIG 2 illustrates the chemical formula of PNIPAAm and 2(B) Representation of volume phase transition between coil (left) and globular (right) hydrogel conformations. Following pictures shows representation of 2(A) the swollen PNIPAAm hydrosol in aqueous solution below Tc (32 °C) and 2(B) the shrunken dehydrated PNIPAAm hydrogel above Tc (32 °C). Adapted with permission from Reference [641. Copyright € 2015 Springer Science & Business Media Singapore, according to the aspects of present invention.
For the present invention commercially available thermoreversible polymer(S) have been used as one of the key ingredient in the semen storage and shipment medium.
Product composition/preparation: Thermoreversible polymer (Henceforth will be referred as “Polymer” suspended in phosphate buffered saline with more than one of the following opponents as additives. Thermoreversible polymer (l%-50%) (For eg: Poloxamers, Poly caprolactane, NIPAAM etc)
Glycerol (1. l%-30%)
Calcium chloride (0.1mM-20 mM)
Hepes (lmM-40 mM)
Sodium dihydrogen phosphate (O.OlmM-lOmM)
Sodium hydrogen carbonate (lmM-50mM)
Sodium chloride (lmM-1000 mM)
Glycine (lmM-30mM)
Potassium chloride (lmM-50 mM)
Magnesium Chloride (O.OlmM-lOmM)
Sodium lactate ( lmM-40mM)
Glucose (0.5mM-20mM)/Sucrose (10mM-500mM)
Gentamicin sulfate (1ug/ml-l00ug/ml)
Ultra-fdtered egg yolk (l%-50%)
Vascular endothelial growth factors -VEGF(0.0001ug/ml-10ug/ml)
Nerve Growth Factor-NGF(0.0001ug/ml-10ug/ml)
Epithelial Growth Facotr-EGF(0.0001ug/ml-10ug/ml)
Platelet rich plasma-lysate (0.1%-50%)
Vitamin C(0.01mg/ml-100mg/ml)
Vitamin E (HU-200IU/ml)
CoQlO (O.OOlug/ml-lOOug/ml)
L-camitine (O.OOlug/ml-lOOug/ml)
Selenium (0.000 lug/ml-lOug/ml)
N-acetyl -cysteine (0.00 lug/ml- 1 OOug/ml)
Zinc Sulfate (O.OOlmg/ml-lOmg/ml)
Folic acid(0.01mg/ml-100mg/ml)
Ubiquinone (0.01 mg/ml- 1 OOmg/ml)
Vitamin B12 (O.OOOlug/ml-lOug/ml)
This invention provides a thermoreversible sol-gel composition having unique properties for use in the methods of this invention for storage and transportation of semen and sperm samples including but not limited. A general method of preparing the compositions of this invention can be described as the following: combining an amount of the thermoreversible polymer or a combination of two polymers with similar characteristics and an amount of a suitable aqueous solvent, wherein the amount of polymer(S) is sufficient to form a solution having more than or equal to 40% % w/w of polymer(s); stirring the mixture at a sufficiently medium speed at about or below 10 °C at for a first period of time; and rocking the mixture for a second period of time there by forming a solution. It is to be understood that the above steps may be combined and that the sequence of the steps may be altered. For example, the aqueous solvent may be cooled /or stirred when combining with the polymer(s). The polymer may be added in small potions. One polymer(s) may be added before the other polymer is added. Alternatively, a cold aqueous solvent may be poured to the polymer(s). The stirring and/or rocking may or may not be continuous although continuous stirring at medium speeds is preferred for steady dissolution of the polymer(s). Further, a period of stirring may alternate with a period of rocking.
FIG 3 illustrates the Flowchart describing steps for improving the functionality of sperm (including use of biopolymer), according to the aspects of present invention. FIG 4 illustrates the Flowchart describing steps of producing a sperm shipment medium/ Methods of invention, according to the aspects of present invention.
Nature/characteristics of the product
The term "thermoreversible sol-gel" refers to a composition, which undergoes a phase transition from a liquid phase to gel phase or vice versa when the temperature is raised above or reduced below a critical value, which is referred to as "transition temperature." The term "liquid phase" or "liquid state" refers to a liquid or flowable form with a viscosity of less than 2000 Pascal-seconds. The term "gel phase" or "gel state" refers to a gel or relatively
solid form with a viscosity of greater than 10,000 Pascal-seconds. As is well known in the science of rheology, complex viscosities of compositions are reported in Pascal-seconds and all viscosities reported in this application are reported as complex viscosities. Such phase transition is reversible. Thus, a thermoreversible sol-gel composition changes from a liquid state to a gel state when the temperature is raised to or above the critical value, or transition temperature, and undergoes a phase transition from the gel state to the liquid state when the temperature is lowered to or below the critical value, or transition temperature. Preferably the phase transition from a liquid to a gel and vice versa occurs in less than 10 minutes, more preferably in less than 5 minutes and even more preferably in less than 1 minute.
The term "aqueous solvent" refers to water or a water based solution, e.g. an aqueous salt solution, such as a saline solution, phosphate buffered saline, and other aqueous solutions suitable for dissolving the poloxamers described herein. An aqueous salt solution may contain one or more biocompatible salts selected from sodium chloride (NaCl), potassium chloride (KC1), sodium sulfate (Na2S04 ), sodium bisulfate (NaHS04), sodium phosphate (Na HP04 ), monosodium phosphate (NaH2P04), disodium phosphate (Na2HP04), potassium phosphate(K3PO4), monopotassium phosphate (KH2P04), dipotassium phosphate (K2HPO4), various soluble calcium and magnesium salts, such as calcium chloride (CaC12), magnesium chloride (MgC12) and other salts formed by a combination of a cation selected from the group consisting of sodium, potassium, calcium, magnesium, ammonium, and tetraalkylammonium, with an anion selected from the group consisting of chloride, bromide, tartrate, mesylate, acetate, maleate, and oxalate and other biocompatible, water soluble salts including those described in P . Heinrich Stahl, Camille G. Wermuth (Eds.), Handbook of Pharmaceutical Salts Properties, Selection, and Use; 2002. The term "phosphate buffered saline" or "PBS" refers to a buffer solution which help to maintain a physiological pH and a physiological ionic strength (i.e. isotonic). The final salt concentration of PBS is approximately 137 millimolar (mM) sodium chloride (NaCl), 10 mM phosphate, 2.7 mM potassium chloride, with a pH of 7.4. It may be prepared by diluting a stock solution ten times, which stock solution is prepared by dissolving 800 g sodium chloride (KC1), 20 g potassium chloride, 144 g disodium phosphate (Na2HP04) and 24g monopotassium phosphate (KH2P04 ) in 8 liters (L) of distilled water, and topping up to 10 L. PBS can have additional ions such as Calcium (Ca2+) or Magnesium (Mg 2+).
The present invention is further elaborated with the help of the following examples. However, these examples should not be construed to limit the scope of the present invention.
EXAMPLE 1: METHODS TO EMPLOY THE USE OF BIOCOMPATIBLE POLY- MERS DURING ARTIFICIAL INSEMINATION
Sperm sample were mixed with pre-cooled SeraGel® which are liquid at 4 degrees and allowed to become gel when incubated at 37 degrees. Upon receiving the sample the end user will cool the gel and centrifuge the contents to pellet the sperm sample and use it for further analysis .
FIG 5 illustrates the Flowchart describing steps/ methods to employ the use of biocompatible polymers during artificial insemination, according to the aspects of present invention.
TABLE 1: Sperm quality of specimens shipped via SeraGel transport medium (n = 10)
Experimental Data:
A total of 12 semen samples and two transport methods SeraGel R media and Cryofreezing were tested for maintaining desired shipping temperature. Ten semen samples were assessed for pre- and post-shipment changes in sperm motility, membrane integrity, total motile spermatozoa and recovery of motile spermatozoa. Even though motility, membrane integrity and total motile spermatozoa declined in samples examined under cryogenic freezing conditions and in SeraGel loaded overnight-shipped samples, the observed motility and total motile spermatozoa were higher in SeraGel group and they were sufficient for use with assisted reproductive techniques. Further well planned studies with more sperm function analysis and embryology work is necessary to further validate this first set of encouraging results.
EXAMPLE 2: TEST THE EFFECTS ON SPERM VIABILITY OF TRANSPORT¬
ING SEMEN
This innovation aimed to test the effects on sperm viability of transporting semen samples suspended in the proposed “Semen storage and transportation medium”. The growing use of donor semen means that more samples are being shipped over long geographical distances. In sperm donation programs and assisted reproduction technology procedures, safe sample transportation across the country is of the utmost importance. Semen specimens are most often transported either on dry ice, at a temperature of about -80°C, or in cryogenic storage tanks containing liquid nitrogen (-196°C). Other factors such as temperature fluctuations during sample transportation and handling may negatively affect the quality of thawed sperm. (Carrell et al., 1996).
Methods
Twenty semen samples sent for laboratory analysis were included in the study. Clarification on the study was provided at the time the patients came to schedule their semen collection appointments, and the individuals who agreed to join the study signed an informed consent form. Only the samples classified as normozoospermic according to the WHO criteria were included in the study (2010). Twenty normozoospermic semen samples were collected and divided into five groups.
The samples in Group 1 were immersed in liquid nitrogen throughout the experiment in cryogenic storage tanks,
The semen samples in Group 2 were suspended in commercially available semen storage media in Group 3 were placed in a Styrofoam box kept at 4 degree for 7 days
In Group 3, the semen samples suspended in proposed invention in Group 2 were placed in a Styrofoam box kept under room temperature for 7 days
In Group 4, the semen samples suspended in proposed invention in Group 2 were placed in a Styrofoam box kept at 37 degrees for 7 days
In Group5, samples belongs to Group 4 kept in the Styrofoam box containing the samples was shipped by on-road transportation to assess the effects of shipping;
End of 7th day, sperm parameters were analyzed for viability, vitality, and motility; spermatozoa were also tested for mitochondrial activity. The main variables assessed in semen preservation techniques are motility and vitality. Motility indicates the functional competence of sperm cells and vitality is a way to identify living and dead sperm (Cavalcante et al., 2006); the former is one of the most severely affected variables (Watson, 1995). Verza Jr et al. (2009) studied the resistance of human sperm to cryoinjury after repeated cycles of freezing and thawing, using the quick method with liquid nitrogen vapor; the authors described a sharp drop in sperm motility after each thawing cycle.
Results
Compared to fresh samples, all groups had reduced vitality and motility. Significant decreases in motility recovery rates (P=0.01) and vitality (P=0.001) were observed in group 2 when compared to the control group (Group 1 -Cry opreservation). Mitochondrial activity was signifi- cantly decreased only in Group 2 (P=0.04), as evidenced by greater numbers of sperm cells not stained by reagent 3,3 ’-diaminobenzidine. The percentage of motility, morphology and count from Group 3, 4 and 5 were comparable with Group- 1, served as control (Table 2). In terms of mitochondrial activity, no significant differences were observed between groups for cells in classes I, II and III, even when compared to fresh samples (Table 3).
Conclusions
Storage medium and transportation in the proposed invention did not affect the quality of semen samples stored in the proposed invention, but dry ice as a means to preserve the samples during transportation had detrimental effects upon the sperm parameters assessed in this study.
Major advantages of the invention over the available prior arts:
Major advantage of the current invention are,
The new method will allow the laboratory also to report the exact motility without worrying about temperature changes.
In cases of preparation for lUI-Intra Uterine Insemination, it allows for the maximum number of motile spermatozoa to be retrieved results in better pregnancy outcome.
In case of preparations for IVF, quality sperms will lead to quality embryos and successful pregnancy and live birth.
Because such logistical concerns are a major encumbrance when conducting studies in clinical andrology, there exists a need for the ambient temperature storage and transportation of unprocessed human semen. With the aid of such a medium, samples could be mailed to a centralized, accredited, diagnostic laboratory in which the facilities and expertise for assessing the functional competence of human spermatozoa are concentrated.
Such an advance would not only improve the availability of such assays, but would also help standardize laboratory assessments of semen quality in the context of multicentre clinical trials. The present paper addresses this issue and describes the basis for creating a medium in which excellent preservation of sperm function is observed at ambient temperatures over a period of 24 h.
The invention provides compositions, kits and integrated systems for practicing the as- says/methods/transportation described herein using biopolymers. In addition, the kits may include several transport materials, components, binding agents, containers for the transport of biological samples as part of the embodiments of the invention.
Those of skill in the art will appreciate that for use in the disclosed methods, the compositions and constructs disclosed herein may be present in compositions including one or more physiologically acceptable carriers or diluents, such as water or saline. Such compositions may additionally contain other components, such as preservatives, stabilizers, buffers and the like. Carriers, diluents and other components suitable for use in the present compositions.
Merely for illustration, only representative number/type of graph, chart, block, and sub-block diagrams were shown. Many environments often contain many more block and sub-block diagrams or systems and sub-systems, both in number and type, depending on the purpose for which the environment is designed.
While specific embodiments of the invention have been shown and described in detail to illustrate the inventive principles, it will be understood that the invention may be embodied otherwise without departing from such principles.
Reference throughout this specification to “one embodiment”, “an embodiment”, or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment”, “in an embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.
It should be understood that the figures and/or screen shots illustrated in the attachments highlighting the functionality and advantages of the present invention are presented for example
purposes only. The present invention is sufficiently flexible and configurable, such that it may be utilized in ways other than that shown in the accompanying figures.
It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
REFERENCES
1) Appell RA, Evans PR (1977) The effect of temperature on sperm motility and viability. Fertil Steril 28:1329-1332.
2) Appell RA, Evans PR, Blandy JP (1977) The effect of temperature on the motility and viability of sperm. Br J Urol 49:751-756.
3) Bjomdahl L, Kirkman-Brown J, Hart G, Rattle S, Barratt CL (2006) Development of a novel home sperm test. Hum Reprod 21:145-149.
4) Botchan A, Karpol S, Lehavi O, Paz G, Kleiman SE, Yogev L, Yavetz H, Hauser R (2013) Preservation of sperm of cancer patients: extent of use and pregnancy outcome in a tertiary infertility center. Asian J Androl 15:382-386.
5) Cohen J, Fehilly CB, Walters DE (1985) Prolonged storage of human spermatozoa at room temperature or in a refrigerator. Fertil Steril 44:254-262.
6) Daudin M, Rives N, Walschaerts M, Drouineaud V, Szerman E, Koscinski I, Eustache F, Saias- Magnan J, Papaxanthos- Roche A, Cabry-Goubet R, Brugnon F, Le Lannon D, Barth el emy C, Rigot JM, Fr eour T, Berthaut I, Giscard d’Estaing S, Touati F, M elin-Blocquaux MC, BlagosklonovO, Thomas C, Benhamed M, Schmitt F, Kunstmann JM, Thonneau P, Bujan L. (2015) Sperm cryopreservation in adolescents and young adults with cancer: results of the French national sperm banking network (CECOS). FertilSteril 103 :478-486.
7) Elzanaty S, Malm J (2008) Comparison of semen parameters in samples collected by masturbation at a clinic and at home. Fertil Steril 89:1718-1722.
8) Esfandiari N, Saleh RA, Blaut AP, Sharma RK, Nelson DR, Thomas AJ Jr, Falcone T, Agarwal A (2002) Effects of temperature on sperm motion characteristics and reactive oxygen species. Int J Fertil Womens Med 47:227-233.
9) Esteves SC, Sharma RK, Thomas AJ Jr, Agarwal A (1996) Suitability of the hypo-osmotic swelling test for assessing the viability of cryopreserved sperm. Fertil Steril 66:798-804.
10) Gangrade BK (2013) Cryopreservation of testicular and epididymal sperm: techniques and clinical outcomes of assisted conception. Clinics 68:131-140.
11) Hourvitz A, Goldschlag DE, Davis OK, Gosden LV, Palermo GD, Rosenwaks Z (2008) Intra- cytoplasmic sperm injection (ICSI) using cryopreserved sperm from men with malignant neoplasm yields high pregnancy rates. Fertil Steril 90:557-563.
12) Howlader N, Noone AM, Krapcho M, Garshell J, Miller D, Altekruse SF, Kosary CL, Yu M, Ruhl J, Tatalovich Z, Mariotto A, Lewis DR, Chen HS, Feuer EJ, Cronin KA (eds). SEER Cancer Statistics Review, 1975-2012, National Cancer Institute. Bethesda, MD, http://seer.can- cer.gov/csr/1975_2012/, based on November 2014 SEER data submission, posted to the SEER web site, April 2015.
13) Krzyzosiak J, Molan P, McGowan L, Vishwanath R (2001) Effect of sperm number and oxygenation state of the storage media on in vitro fertility of bovine sperm stored at ambient temperature. Theriogenology 55:1401-1415.
14) Licht RS, Handel L, Sigman M (2008) Site of semen collection and its effect on semen analysis parameters. Fertil Steril 89:395-397.
15) Loren AW, Mangu PB, Beck LN, Brennan L, Magdalinski AJ, Partridge AH, Quinn G, Wallace WH, Oktay K (2013) Fertility preservation for patients with cancer: American Society of Clinical Oncology clinical practice guideline update. J Clin Oncol 31 :2500-2510.
16) Maxwell WM, Stojanov T (1996) Liquid storage of ram semen in the absence or presence of some antioxidants. Reprod Fertil Dev 8: 1013-1020.
17) Nangia AK, Krieg SA, Kim SS (2013) Clinical guidelines for sperm cryopreservation in cancer patients. Fertil Steril 100:1203-1209.
18) Niederberger C (2014) Re: spermbanking is of key importance inpatients with prostate cancer. J Urol 191:754.
19) Pacey AA, Eiser C (2014) The importance of fertility preservation in cancer patients. Expert Rev Anticancer Ther 14:487-489.
20) Robinson RD, Knudtson JF (2014) Fertility preservation in patients receiving chemotherapy or radiotherapy. Mol Med 111:434-438.
21) Rosen A, Jayram G, Drazer M, Eggener SE (2011) Global trends in testicular cancer incidence and mortality. EurUrol 60:374-349.
22) Royster MO, Lobdell DT, Mendola P, Perreault SD, Selevan SG, Rothmann SA, Robbins WA (2000).
23) Evaluation of a container for collection and shipment of semen with potential uses in population based, clinical, and occupational settings. J Androl 21:478-484.
24) Rybar R, Prinosilova P, Kopecka V, Hlavicova J, Veznik Z,Zajicova A, Rubes J (2012) The effect of bacterial contamination of semen on sperm chromatin integrity and standard semen
parameters in men from infertile couples. Andrologia 44:410-418. ) Said TM, Tellez S, Evenson DP, Del Valle AP (2009) Assessment of sperm quality, DNA integrity and cryopreservation protocols in men diagnosed with testicular and systemic malignancies. Andrologia 41:377-382. ) Salonia A, Capogrosso P, Castiglione F, Russo A, Gallina A, Ferrari M, Clementi MC, Castagna G, Brigand A, Cantiello F, Damiano R, Montorsi F (2013) Sperm banking is of key importance inpatients with prostate cancer. Fertil Steril 100:367-372. ) Sharma RK, Vemulapalli S, Kohn S, Agarwal A (1997) Effect of centrifuge speed, refrigeration medium, and sperm washing medium on cryopreserved sperm quality after thawing. Arch An- drol 39:33-38. ) Song GJ, Herko R, Lewis V (2007) Location of semen collection and time interval from collection to use for intrauterine insemination. Fertil Steril 88:1689-1691. ) Tomlinson M (2010) Therapeutic sperm cryopreservation. In: Clinical Andrology EAU/ESAU Course Guidelines. ) Bjomdahl L, Giwercman A, Toumaye H, Weidner W (eds). Informa Health Care, London, UK, pp 124-133. ) Toumaye H, Dohle GR, Barratt CLR (2014) Fertility preservation in men with cancer. Lancet 384: 1295-1301. Williams DH (2010) Sperm banking and the cancer patient. Ther Adv Urol 2:19-34. ) Williams DHT (2013) Fertility preservation in the male with cancer. Curr Urol Rep 14:315— 326. World Health Organization (2010) Laboratory Manual for the Examination and Processing of Human Semen, 5th edn. Switzerland Press, Geneva.
Claims
1. Shipment box
2. Sample collection container fdled/coated with Seragel
3. Glass slides
4. Pasteur pipette wherein the kit wherein the medium that can preserve biomolecule quality, motility and membrane integrity.
11) A kit storage and/or transportation of Pollen at ambient temperature, wherein the kit comprising of
1. Shipment box
2. Sample collection container fdled/coated with Seragel mixed in Pollen Germination medium
3. Glass slides
4. Pasteur pipette wherein the kit wherein the medium that can preserve biomolecule quality, motility and membrane integrity.
12) A method for shipping the pollen from any sources (and improving the functionality of pollen as the pollen is not allowed to dry completely and immobilised and not subjected to conventional methods with harsh fluctuating temperature conditions), comprising the acts of:
1. mixing pollen sample with a pre-cooled SeraGel® suspended in pollen germination medium;
2. leaving the mix (a) undisturbed at room temperature or placing on top of a warmer maintaining at 30 degree centigrade;
3. packing (b) in a cover provided with the container; and
4. delivering (c) to the end user for respective applications.
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|---|---|---|---|
| PCT/IB2020/060230 WO2022090782A1 (en) | 2020-10-30 | 2020-10-30 | Methods for improving sperm functionality and applications thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/IB2020/060230 WO2022090782A1 (en) | 2020-10-30 | 2020-10-30 | Methods for improving sperm functionality and applications thereof |
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| WO2022090782A1 true WO2022090782A1 (en) | 2022-05-05 |
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ID=81383094
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| WO (1) | WO2022090782A1 (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001067859A2 (en) * | 2000-03-14 | 2001-09-20 | Alnis Biosciences, Inc. | Cryoprotective system |
| US20030104347A1 (en) * | 2000-03-21 | 2003-06-05 | Yuichi Mori | Coating material for living organism tissue, coated product from living organism tissue and method of coating living organism material |
| US20110105835A1 (en) * | 2007-12-27 | 2011-05-05 | Michele Magistrini | Method for preserving sperm and applications thereof |
| WO2011099872A1 (en) * | 2010-02-12 | 2011-08-18 | Androgenix Ltd | Methods for improving sperm functionality |
| IN201941016363A (en) * | 2019-04-25 | 2020-10-30 |
-
2020
- 2020-10-30 WO PCT/IB2020/060230 patent/WO2022090782A1/en active Application Filing
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001067859A2 (en) * | 2000-03-14 | 2001-09-20 | Alnis Biosciences, Inc. | Cryoprotective system |
| US20030104347A1 (en) * | 2000-03-21 | 2003-06-05 | Yuichi Mori | Coating material for living organism tissue, coated product from living organism tissue and method of coating living organism material |
| US20110105835A1 (en) * | 2007-12-27 | 2011-05-05 | Michele Magistrini | Method for preserving sperm and applications thereof |
| WO2011099872A1 (en) * | 2010-02-12 | 2011-08-18 | Androgenix Ltd | Methods for improving sperm functionality |
| IN201941016363A (en) * | 2019-04-25 | 2020-10-30 |
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