WO2022264163A1 - Recovery buffer for semen sample - Google Patents
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- WO2022264163A1 WO2022264163A1 PCT/IN2022/050521 IN2022050521W WO2022264163A1 WO 2022264163 A1 WO2022264163 A1 WO 2022264163A1 IN 2022050521 W IN2022050521 W IN 2022050521W WO 2022264163 A1 WO2022264163 A1 WO 2022264163A1
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- grams
- buffer
- sodium
- semen
- recovery
- Prior art date
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- 238000011084 recovery Methods 0.000 title claims abstract description 78
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 44
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- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims abstract description 43
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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
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
Definitions
- This invention generally relates to a recovery buffer for ejaculate semen samples that facilitates recovery/restoration/revival of the sperm motility in the samples that have been collected and transported at atmospheric temperature to the laboratory, thereby helping in the accurate prediction of the motility of the samples at the ejaculation time in semen analysis.
- a recovery buffer for ejaculate semen samples that facilitates recovery/restoration/revival of the sperm motility in the samples that have been collected and transported at atmospheric temperature to the laboratory, thereby helping in the accurate prediction of the motility of the samples at the ejaculation time in semen analysis.
- sperm motility may deteriorate leading to inaccuracies in semen analysis. This is prevented by addition of recovery buffer to the collected semen samples.
- the recovery buffer comprises sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dihydrate, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, Penicillin- G, phenol red, bovine serum albumin, EDTA (ethylenediaminetetraacetic acid), HEPES (N- (2-Hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid)) and theophylline. Further, the present disclosure describes a process for preparation of the recovery buffer for ejaculate semen sample.
- the present disclosure describes a process of recovery/restoration/revival of sperm motility in collected and transported or stored semen samples by the addition of recovery buffer.
- the recovery buffer not only helps a clinician to understand possible etiology for infertility but also to decide the suitable assisted reproductive technology (ART) to treat the patient, such as In-vitro fertilization (IVF) or Intracytoplasmic sperm injection (ICSI).
- IVF In-vitro fertilization
- ICSI Intracytoplasmic sperm injection
- Semen analysis is the cornerstone and the first step in the assessment of the male partner in a sub fertile couple.
- the seminal fluid comprises a mixture of secretions from the testes, epididymis, prostate, and seminal vesicles and the contribution from each of these glands varies by the interval of abstinence and the method used to obtain the semen samples.
- semen samples collected by masturbation in the clinic may be of a lower quality than those collected at home.
- sperm motility is extremely crucial as motility is dependent on the ATP activity of the sperm which deteriorates over time.
- sperm motility is to be measured within an hour of ejaculation by the patient. This motility assessment is performed in most laboratories by visual assessment under the microscope and quantified as percent total motility (progressive motility with non-progressive motility) and spermatozoa with no motility (immotile). Consequent to the fact that motility deteriorated over time rapidly, the semen collection and delivery should reach the lab within one hour.
- COVID-19 pandemic over the last year or more has further worsened the situation where men who are desirous to have children, have been denied access due to lockdown and male fertility is declining. Further , there is evidence now that COVID-19 may affect reproductive capacity in men, specifically impair semen parameters. As many aspects of clinical medicine and treatment moved online, male fertility assessment could not move online or remote testing, as there were technology challenges of motility impairment of semen samples if not reported within one hour.
- At-home analysis of male fertility is a cost effective, private and rapid solution to male fertility based inquiries, making it beneficial to men who are hesitant to seek medical evaluation.
- at-home analysis methods include kits/devices employing antibody reaction for color change, microscopic kits, centrifuge followed by smartphone application, microfluidic chip etc.
- Most at-home systems will provide rudimentary analysis of a sample but not a complete formal semen analysis, giving the person an idea of whether or not to pursue further testing.
- sperm analysis kits in which users collect a sample at home and then send it to the provider for analysis are also available.
- a Home Collection Kit contains a shipping container that is able to maintain semen quality enough for a proper evaluation within 26 hours and usually has an addition of a cryoprotectant which decreases motility and not an ideal method for an accurate formal semen analysis. It also includes a preservative tube, a pipette, a biohazard bag, ice packs and cooling gels to keep the sample cold, and labels to ship it back.
- At-home sperm analysis is a valuable tool for determining fertility potential, especially for couples struggling with infertility, as well as vasectomy patients.
- Men who are reluctant to seek conventional clinical testing due to high cost, long wait time, inconvenience, or social stigma might be more willing to use home-based sperm analysis kits, which overcome those problems. With these kits, men are able to rapidly evaluate their fertility potential with ease at a low-cost from the comfort and privacy of their own homes, unlike the traditionally used methods (these only provide a rough estimate of semen quality and are used as a guide to fertility).
- an embodiment herein provides a recovery buffer for ejaculate semen sample which facilitates recovery of sperm motility in collected, stored or transported semen sample at atmospheric temperature, wherein the recovery buffer comprises sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dihydrate, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, Penicillin-G, streptomycin, phenol red, bovine serum albumin, EDTA (ethylenediaminetetraacetic acid), HEPES (N-(2-
- the recovery buffer at a volume of 1000 ml comprises about 4-7 grams of sodium chloride, about 0.2-0.4 grams of potassium chloride, about 0.03- 0.08 grams of magnesium sulphate heptahydrate, about 0.03-0.06 grams of potassium dihydrogen phosphate, about 0.2-0.4 grams of calcium chloride dihydrate, about 1.5-2.5 grams of sodium bicarbonate, about 0.4-0.6 grams of glucose, about 0.025-0.04 grams of sodium pyruvate, about 3.00- 4.00 ml of sodium lactate, about 0.065-0.080 grams of Penicillin-G, about 0.04-0.06 grams of streptomycin, about 0.001-0.003 grams of phenol red, about 3.5-5.5 grams of bovine serum albumin, about 0.04-0.06 grams of EDTA (ethylenediaminetetraacetic acid), about 0.15-0.40 grams of HEPES (N-(2 -Hydroxy ethyl) piperazine-N'-(2-aminol) piperazine-N'
- the present disclosure relates to a process for preparation of a recovery buffer for ejaculate semen sample comprising the steps of: a) taking Milli-Q water;b) adding buffer components gradually into the water with gentle stirring;c) adding bovine serum albumin (BSA) slowly and taking care to avoid frothing, to result in the buffer; d) checking if the pH of the buffer is 7.2-7.8 and osmolality of the buffer is 270-290 mOsmols/Kg water; e) making the volume upto a desired level with Milli-Q water; f) sterilizing the buffer by passing through sterile filters, disposable filter units; and g) storing the buffer in sterile storage bottles at 4°C until use.
- BSA bovine serum albumin
- aprocess for preparing 250 ml of the recovery buffer comprises the steps of: a) taking 150 ml of Milli-Q water in a 500 ml glass beaker with a stirrer-bar, placed onto a magnetic stirrer; b) adding buffer components gradually into the water with gentle stirring, wherein the buffer components are sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dehydrate, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, Penicillin-G, streptomycin, phenol red, EDTA (ethylenediaminetetraacetic acid), HEPES (N-(2-Hydroxyethyl) piperazine-N'-(2- ethanesulfonic acid)), c) adding bovine serum albumin (BSA) slowly and taking care to avoid frothing, to result in the buffer; then add theophylline gradually into the water with gentle stirring
- BSA bovine serum albumin
- the present disclosure relates to a kit to facilitate recovery of sperm motility in collected, stored or transported semen sample, wherein the kit comprises a recovery buffer vial; a pasteur pipette to measure out 2ml of the recovery buffer and dispense the same into the sample; and an instructions manual, wherein the recovery buffer comprises sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dihydrate, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, Penicillin-G, streptomycin, phenol red, bovine serum albumin, EDTA (ethylenediaminetetraacetic acid), HEPES (N-(2-Hydroxyethyl) piperazine-N'-(2- ethanesulfonic acid)), and theophylline.
- the recovery buffer comprises sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dihydrate, sodium bicarbonate,
- the recovery buffer at a volume of 1000 ml comprises about 4-7 grams of sodium chloride, about 0.2-0.4 grams of potassium chloride, about 0.03- 0.08 grams of magnesium sulphate heptahydrate, about 0.03-0.06 grams of potassium dihydrogen phosphate, about 0.2-0.4 grams of calcium chloride dehydrate, about 1.5-2.5 grams of sodium bicarbonate, about 0.4-0.6 grams of glucose, about 0.025-0.04 grams of sodium pyruvate, about 3.00- 4.00 ml of sodium lactate, about 0.065-0.080 grams of Penicillin-G, about 0.04-0.06 grams of streptomycin, about 0.001-0.003 grams of phenol red, about 3.5-5.5 grams of bovine serum albumin, about 0.04-0.06 grams of EDTA (ethylenediaminetetraaceticacid), about 0.15-0.40 grams of HEPES (N-(2 -Hydroxy ethyl) piperazine-N'-(2-aminol) piperazine-N'
- the present disclosure relates to a method of recovering sperm motility in collected, stored or transported semen samples by measuring 2 ml of a recovery buffer and dispensing to a sample, wherein the recovery buffer comprises sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dihydrate, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, Penicillin- G, streptomycin, phenol red, bovine serum albumin, EDTA (ethylenediaminetetraacetic acid), HEPES (N-(2-Hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid)) and theophylline, and wherein the sample comprises semen and transport buffer.
- the recovery buffer comprises sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dihydrate, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, Penicillin- G
- the semen samples are transported in a transport buffer comprising sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dihydrate, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, Penicillin-G, streptomycin, phenol red, bovine serum albumin, EDTA (ethylenediaminetetraaceticacid), HEPES (N-(2- Hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid)), and trehalose.
- a transport buffer comprising sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dihydrate, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, Penicillin-G, streptomycin, phenol red, bovine serum albumin, EDTA (ethylenediaminetetraaceticacid), HEPES (N-(2- Hydroxyethyl) piperazine-N'
- FIG.l illustrates the average fraction sperm motility at 6th hour versus 0th hour in three sets of semen samples stored at different buffer storage conditions, i.e. room temperature, at 45°C and at 55°C.
- FIG.2 shows the effect of incubation time on mean progressive motility of semen samples in transport buffer stored at three different temperatures (room temperature, at 45°C and at 55°C) and treated with recovery buffer after 5 hours.
- FIG.3 illustrates the mean fraction sperm motility Embodiments
- the present invention relates to a recovery buffer for ejaculate semen samples that facilitates recovery/restoration/revival of the sperm motility in the samples that have been collected and transported at atmospheric temperature to the laboratory, thereby helping in the accurate prediction of the motility of the samples at the ejaculation time in semen analysis.
- a recovery buffer for ejaculate semen samples that facilitates recovery/restoration/revival of the sperm motility in the samples that have been collected and transported at atmospheric temperature to the laboratory, thereby helping in the accurate prediction of the motility of the samples at the ejaculation time in semen analysis.
- the sperm motility may deteriorate leading to inaccuracies in semen analysis. This is prevented by addition of recovery buffer to the collected semen samples.
- the recovery buffer comprises sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dihydrate, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, Penicillin- G, Streptomycin, phenol red, bovine serum albumin, EDTA (ethylenediaminetetraacetic acid), HEPES (N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)) and theophylline. Further, the present disclosure describes a process for preparation of the recovery buffer for ejaculate semen sample.
- the present disclosure describes a process of recovery/restoration of sperm motility in collected and transported/stored semen samples by the addition of recovery buffer.
- 1000 ml of the recovery buffer comprises about 4-7 grams of sodium chloride, about 0.2-0.4 grams of potassium chloride, about 0.03- 0.08 grams of magnesium sulphate heptahydrate, about 0.03-0.06 grams of potassium dihydrogen phosphate, about 0.2-0.4 grams of calcium chloride dehydrate, about 1.5-2.5 grams of sodium bicarbonate, about 0.4-0.6 grams of glucose, about 0.025-0.04 grams of sodium pyruvate, about 3.00- 4.00 ml of sodium lactate, about 0.065-0.080 grams of Penicillin-G, about 0.04-0.06 grams of streptomycin, about 0.001-0.003 grams of phenol red, about 3.5-5.5 grams of bovine serum albumin, about 0.04-0.06 grams of EDTA (ethylenediaminetetraacetic acid), about 0.15-0
- the transport buffer of the present disclosure helps to promote efficient semen testing by transporting semen efficiently for semen analysis without affecting sperm motility parameters and viability, which in turn has potential therapeutic application in assessing infertility associated with male factor (Oligospermia, asthenospermia and teratospermia).
- Semen analysis is the cornerstone for the assessment of the male partner in a sub fertile couple. Semen analysis is the first step in identifying male factor infertility. Compared to many other tests used in the assessment of the infertile couple, semen analysis has been standardized throughout the world. Standardized methods of semen analysis are available, however, semen transportation medium for efficient semen quality and sperm viability preservation is not available. Hence, the present disclosure provides a buffer for semen sample that allows accurate assessment of sperm quality. Measures of semen quality are used as surrogate measures of male fertility in clinical andrology, reproductive toxicology, epidemiology, and risk assessment. This could also be very useful to study the impact of COVID-19 on reproductive health by remotely collecting samples.
- the present disclosure describes a process of recovery/restoration/revival of sperm motility in collected and transported/stored semen samples by the addition of recovery buffer.
- a person who wishes to get his semen sample tested in a laboratory, without physically visiting a laboratory may employ a transport buffer to facilitate preservation of the sample collected at his residence, and may then transport the sample to the laboratory via courier or by other means of transport.
- the transport buffer comprises sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dihydrate, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, Penicillin-G, streptomycin, phenol red, bovine serum albumin, EDTA
- HEPES N-(2-Hydroxyethyl)piperazine-N'-(2- ethanesulfonic acid)
- trehalose ethylenediaminetetraacetic acid
- HEPES N-(2-Hydroxyethyl)piperazine-N'-(2- ethanesulfonic acid)
- trehalose ethylenediaminetetraacetic acid
- HEPES N-(2-Hydroxyethyl)piperazine-N'-(2- ethanesulfonic acid)
- Another embodiment of the present disclosure relates to a kit comprising a recovery buffer vial; a pasteur pipette to measure out 2ml of the recovery buffer and dispense the same into a sample; and an instructions manual, wherein the sample comprises semen mixed with 2ml of a transport buffer.
- the process of recovery/restoration/revival of sperm motility in collected and transported/stored semen samples involves the application of the kit, wherein the recovery buffer is measured out and dispensed to the collected and transported/stored semen samples.
- the recovery buffer comprises sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dihydrate, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, Penicillin-G, Streptomycin, phenol red, bovine serum albumin, EDTA (ethylenediaminetetraacetic acid), HEPES (N-(2-Hydroxyethyl)piperazine-N'-(2- ethanesulfonic acid)), and theophylline.
- the recovery buffer for semen samples is prepared by the following procedure:
- Buffer components and other reagents are acquired from reputed vendors: Sigma Chemical Co.., St Louise, MO, USA; In-Vitrogen, USA or any other local vendors.
- the standard buffer composition of recovery buffer is comprehensively described in Table 1 above. 250 -500 ml (desired volume) of the buffer is prepared on the day before the experiment. 150 ml of Milli-Q water is taken in a 500 ml glass beaker with stirrer-bar, placed onto a magnetic stirrer. To this, the required buffer components are slowly added one by one (refer to Table 1) into water with gentle stirring. It is to be noted that 60% syrup of sodium lactate has a large osmotic effect and must be measured very accurately. CaChis dissolved in 5 mL of EbO separately and added to the solution. . Thereafter, BSA is slowly added at the end and care is taken to avoid frothing.
- pH and osmolality of the buffer pH and osmolality of the buffer (pH: 7.2-7.8; osmolality: 270-290 mOsmols/Kg water) is checked.
- the volume is made up to 250 ml with Milli-Q water in a volumetric flask.
- the buffer is sterilized by passing through 0.2 pm sterile filters, disposable filter units and stored in sterile storage bottles.
- the buffer is stored at 4°C until use.
- Semen samples were collected after informed consent. The samples were divided into four parts. Three parts were transported to the laboratory in 2ml of transport buffer stored at RT, 45°C and 55°C (comprising trehalose as one of the constituents) and another part as it is (raw sample).
- the transport buffer comprises sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dihydrate, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, Penicillin- G, streptomycin, phenol red, bovine serum albumin, EDTA (ethylenediaminetetraacetic acid), HEPES (N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)), and trehalose.
- the samples were labelled/categorized as four groups: raw sample or sample without the transport buffer, sample treated with RT transport buffer and incubated at room temperature , - sample treated with 45°C -transport media and incubated at room temperature sample treated with 55°C transport media and incubated at room temperature
- Preliminary testing of all parameters of semen testing was carried out, such as Volume, Ph, Pus cells, Vitality, RBC, debris, Concentration, Motility (in accordance with WHO 5th Manual and draft 6th manual, Morphology) with staining.
- Motility was assessed subsequently every hour, as it is only parameter that deteriorates for both Control samples (i.e. samples without the transport buffer) and samples with buffer. Further, concentration and morphology was assessed at the end of 8 hours and no difference was observed between the control and the tested sample.
- the recovery buffer comprises sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dihydrate, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, Penicillin-G, Streptomycin, Phenol Red, bovine serum albumin, EDTA (ethylenediaminetetraacetic acid), HEPES (N-(2- Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)), and theophylline.
- experimental analysis was performed over a period of 30 days to check the recovery of motility in collected, transported and stored semen samples. Collected semen samples were transported to the laboratory in the transport buffer (comprising trehalose as one of the constituents). Semen analysis parameters like rapid progressive (RP), progressive motility (PM), non-progressive motility (NP) and immotility (IM) were assessed every hour, till 5 hours, at room temperature. The analysis was performed for the raw samples (i.e. samples without transport buffer) as well. A gradual decline of PM and NP and increase in IM was observed with time as indicated in the Table 2 below.
- RP rapid progressive
- PM progressive motility
- NP non-progressive motility
- IM immotility
- recovery buffer (comprising theophylline as one of the constituents) was added to all the samples.
- PM, NP, and IM values come closer to the initially recorded values at the 1 st hour.
- Table 3 A comparison of motility results between random samples and the samples tested in the laboratory using the protocol.
- FIG.l illustrates the average fraction sperm motility at 6th hour versus 0th hour in sample with buffer under different buffer storage conditions.
- Transport buffer stored at room temperature (RT), at 45°C (T45), and at 55°C (T55) were allowed to reach room temperature and mixed with semen sample for transport.
- RT room temperature
- T45 45°C
- T55 55°C
- motility was found to increase 25-30% as compared to transport buffer alone and about 78%-92% as compared to raw sample (control), showing that the recovery buffer effectively restores the motility.
- FIG.2 shows the linearly increasing mean fraction sperm motility in sample with transport buffer stored at different temperatures.
- FIG.3 shows the effect of incubation time on mean progressive motility of semen samples in transport buffer treated with recovery buffer after 5 hours.
- the recovery buffer for ejaculate semen samples is advantageous owing to the following reasons:
- the goal of this investigation is to improve the standards of semen analysis post transportation of semen to the laboratory from a collection site.
- the recovery buffer ensures that the semen and sperm parameters are not affected for further analysis.
- Another primary goal of this study is to development of non-liquid nitrogen based transportation of semen samples for invitro analysis without compromising the semen parameters such as motility, concentration, vitality and morphology.
- This product will give potential benefits for restoration of semen motility post transportation, without affecting optimal semen quality and sperm motility parameters.
- a few home sperm test kits or mail order kits collect sperm and use an algorithm to assess decline in motility over several hours- depending on the time of assessment. Some of them also use cryofreezing buffer or cryofreezing container for transport. They report semen generally after 24-48 hours. Most of these samples are mainly for men trying to freeze their semen for long term storage that is mainly used for IVF-ICSI and not for an assessment of a formal semen analysis that is needed for assessment of male factor infertility.
- motility decline varies from sample to sample and poor motility samples unless preserved to retain their motility will deteriorate over time and become zero motility at some point.
- Using an algorithm to predict original motility may give false reading or false positives.
- using an algorithm would be fraught with incorrect reporting.
- Algorithms derived motility decline are percentages which are fixed and does not vary from sample to sample which is needed if we are assessing semen sample. That is why restoring actual original motility with a recovery buffer is absolutely essential and much more beneficial over use of predictive algorithm.
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Abstract
The present invention relates to a recovery buffer for ejaculate semen samples that facilitates recovery/restoration/revival of the sperm motility in the samples that have been collected and transported at atmospheric temperature to the laboratory, thereby helping in the accurate prediction of the motility of the samples at the ejaculation time in semen analysis. The recovery buffer comprises sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dihydrate, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, Penicillin-G, streptomycin, phenol red, bovine serum albumin, EDTA (ethylenediaminetetraacetic acid), HEPES (N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)), and theophylline. Further, the present disclosure describes a process for preparation of the recovery buffer for ejaculate semen sample. Furthermore, the present disclosure describes a process of recovery/restoration/revival of sperm motility in collected and transported or stored semen samples by the addition of recovery buffer.
Description
RECOVERY BUFFER FOR SEMEN SAMPLE
TECHNICAL FIELD
[0001] This invention generally relates to a recovery buffer for ejaculate semen samples that facilitates recovery/restoration/revival of the sperm motility in the samples that have been collected and transported at atmospheric temperature to the laboratory, thereby helping in the accurate prediction of the motility of the samples at the ejaculation time in semen analysis. Particularly, when a considerable time span is required for the transport of the semen samples from the collection site to the laboratory, sperm motility may deteriorate leading to inaccuracies in semen analysis. This is prevented by addition of recovery buffer to the collected semen samples. The recovery buffer comprises sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dihydrate, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, Penicillin- G, phenol red, bovine serum albumin, EDTA (ethylenediaminetetraacetic acid), HEPES (N- (2-Hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid)) and theophylline. Further, the present disclosure describes a process for preparation of the recovery buffer for ejaculate semen sample. Furthermore, the present disclosure describes a process of recovery/restoration/revival of sperm motility in collected and transported or stored semen samples by the addition of recovery buffer. The recovery buffer not only helps a clinician to understand possible etiology for infertility but also to decide the suitable assisted reproductive technology (ART) to treat the patient, such as In-vitro fertilization (IVF) or Intracytoplasmic sperm injection (ICSI).
BACKGROUND [0002] Semen analysis is the cornerstone and the first step in the assessment of the male partner in a sub fertile couple. The seminal fluid comprises a mixture of secretions from the testes, epididymis, prostate, and seminal vesicles and the contribution from each of these glands varies by the interval of abstinence and the method used to obtain the semen samples. In men, semen samples collected by masturbation in the clinic may be of a lower quality than those collected at home.
[0003] Though seminal fluid has multitude of parameters, the assessment of sperm motility is extremely crucial as motility is dependent on the ATP activity of the sperm which deteriorates over time. Ideally, sperm motility is to be measured within an hour of ejaculation by the patient. This motility assessment is performed in most laboratories by visual
assessment under the microscope and quantified as percent total motility (progressive motility with non-progressive motility) and spermatozoa with no motility (immotile). Consequent to the fact that motility deteriorated over time rapidly, the semen collection and delivery should reach the lab within one hour. This necessitated many men to provide semen samples in the hospital/laboratory (due to inability to deliver the sample collected at home within an hour) and use the less than optimal conditions of the laboratory/hospital to collect the sample. Ejaculation is a mood driven, erotic thought driven process and many men needed a conducive environment to collect the sample and either failed to collect in hospital or ejaculated improperly delivering semen that was not their normal natural ejaculation. Additionally, many men needed their partner for ejaculation as many were not comfortable with masturbation or unaware of it or don't practice due to religious reasons and were seriously hampered to collect the sample both for diagnostic or treatment purpose in infertility. The standardization of the routine semen analyses (semen volume, sperm count, motility, and morphology) allows the comparison across laboratories as per WHO standard protocols.
[0004] The COVID-19 pandemic over the last year or more has further worsened the situation where men who are desirous to have children, have been denied access due to lockdown and male fertility is declining. Further , there is evidence now that COVID-19 may affect reproductive capacity in men, specifically impair semen parameters. As many aspects of clinical medicine and treatment moved online, male fertility assessment could not move online or remote testing, as there were technology challenges of motility impairment of semen samples if not reported within one hour.
[0005] With about 70 million cases of infertility worldwide, half of which are caused by male factors, sperm analysis is critical to determine male fertility potential. Conventional semen analysis methods involve complex and manual inspection with a phase contrast microscope and requires trained andrology laboratory personnel. Due to unavailability of rapid, convenient, and user-friendly semen analysis tools, many men do not seek medical evaluation, especially in resource-constrained settings. Furthermore, since conventional methods have to be conducted in the labs, many men are unwilling to be tested as a result of social stigma in certain regions of the world. One solution can be found in at-home sperm analysis, which allows men to test their semen without the hassle of going to and paying for a clinic.
[0006] Conversely, at-home analysis of male fertility is a cost effective, private and rapid solution to male fertility based inquiries, making it beneficial to men who are hesitant to
seek medical evaluation. Examples for such at-home analysis methods include kits/devices employing antibody reaction for color change, microscopic kits, centrifuge followed by smartphone application, microfluidic chip etc. Most at-home systems will provide rudimentary analysis of a sample but not a complete formal semen analysis, giving the person an idea of whether or not to pursue further testing.
[0007] In addition to home-based methods of sperm analysis, mail-in sperm analysis kits in which users collect a sample at home and then send it to the provider for analysis are also available. Such a Home Collection Kit contains a shipping container that is able to maintain semen quality enough for a proper evaluation within 26 hours and usually has an addition of a cryoprotectant which decreases motility and not an ideal method for an accurate formal semen analysis. It also includes a preservative tube, a pipette, a biohazard bag, ice packs and cooling gels to keep the sample cold, and labels to ship it back.
[0008] Although at-home, paper-based, and microfluidic sperm analysis products are a step ahead of the traditional methods for semen analysis, they still have many limitations. The primary issue is the fact that currently, non-conventional sperm analysis methods are best used only for indicating whether a user should or should not pursue further testing. Most can only provide information on one or a few parameters at a time which also is reported in 2-4 days and not a comprehensive semen analysis as evaluated in the laboratory today and do not replace the need for further laboratory testing. There is also research underway and few kits available overseas which use algorithms to define the motility percentage decline over time. However , these are fraught with danger in intrinsically poor motility samples and these samples cannot be accurately reported. In brief, none of these non-conventional sperm analysis methods provides an accurate motility and are only used as screening tests or used for semen cryofreezing.
[0009] While semen testing with any of the above technologies can be helpful, only having data on some parameters, but not all parameters, can lead to false positives or false negatives in evaluation of male infertility, since sperm can be simultaneously considered normal in one characteristic, but abnormal in another. A single parameter does not define whether an individual is fertile or infertile, but whether or not a natural pregnancy occurs within a year does. As a result, these methods are not yet a replacement for lab analysis. Formal confirmation from a fertility specialist is still recommended even after the use of a home-based test, which can actually delay getting a full clinical evaluation. Semen analysis from an andrology laboratory is much more detailed and can provide information on many more parameters simultaneously.
[0010] At-home sperm analysis is a valuable tool for determining fertility potential, especially for couples struggling with infertility, as well as vasectomy patients. Men who are reluctant to seek conventional clinical testing due to high cost, long wait time, inconvenience, or social stigma might be more willing to use home-based sperm analysis kits, which overcome those problems. With these kits, men are able to rapidly evaluate their fertility potential with ease at a low-cost from the comfort and privacy of their own homes, unlike the traditionally used methods (these only provide a rough estimate of semen quality and are used as a guide to fertility).
[0011] Hence, new approaches to sperm analysis are needed to overcome the challenges of current technology and provide comprehensive information of all seminal parameters as presently done in the lab.
[0012] There exist several barriers to in-office semen analysis, including physical access to laboratories, difficulty in providing a fresh sample through masturbation, and the current COVID-19 pandemic, which has mandated less physician-patient contact. Current at- home test kits are best suited for screening and identifying men who should seek professional workup. They also require considerable processing by the patient, which may lead to inaccurate results.
[0013] The ability to collect samples from home and transport accurately to the andrology laboratory for a full-fledged semen analysis testing has numerous clinical and patient implications, including reducing patient anxiety, improving convenience, and potentially the ability to see higher levels of patient compliance. Several areas for which at- home sperm collection and formal laboratory testing can be valuable include the initial workup of infertility as a potential tool to identify whether an individual requires standard semen analysis or if additional investigations are required, post vasectomy testing, and routine testing that is required after vasostomy, vasoepididymostomy, or varicocelectomy.
[0014] Further, another clinically important implication of at-home testing is the potential for better standardization of results. Routine semen analysis is fraught with inter observer variability. As such, a centralized location for evaluation can minimize inter-observer variability and, thus, improve the diagnostic utility of the test results. However, when there is a considerable time lapse during the transport of the semen samples from a collection site (for example, residence of the individual) to a diagnostic laboratory, sperm motility may deteriorate leading to inaccuracies in semen analysis.
[0015] Accordingly, in view of the deterioration of sperm motility in collected and transported/stored semen samples which may lead to inaccuracies in semen analysis, there is a
need for an efficient recovery buffer that will recover/restore the motility, enabling accurate prediction of the motility of the samples at the ejaculation time, and thereby facilitating comprehensive analysis of semen.
SUMMARY
[0016] It is an object of the present disclosure to mitigate, alleviate or eliminate one or more of the above-identified deficiencies and disadvantages in the prior art and solve at least the above-mentioned problem. In view of the foregoing, an embodiment herein provides a recovery buffer for ejaculate semen sample which facilitates recovery of sperm motility in collected, stored or transported semen sample at atmospheric temperature, wherein the recovery buffer comprises sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dihydrate, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, Penicillin-G, streptomycin, phenol red, bovine serum albumin, EDTA (ethylenediaminetetraacetic acid), HEPES (N-(2-
Hydroxy ethyl) piperazine-N'-(2-ethanesulfonic acid)) and theophylline.
[0017] In an embodiment of the present disclosure, the recovery buffer at a volume of 1000 ml comprises about 4-7 grams of sodium chloride, about 0.2-0.4 grams of potassium chloride, about 0.03- 0.08 grams of magnesium sulphate heptahydrate, about 0.03-0.06 grams of potassium dihydrogen phosphate, about 0.2-0.4 grams of calcium chloride dihydrate, about 1.5-2.5 grams of sodium bicarbonate, about 0.4-0.6 grams of glucose, about 0.025-0.04 grams of sodium pyruvate, about 3.00- 4.00 ml of sodium lactate, about 0.065-0.080 grams of Penicillin-G, about 0.04-0.06 grams of streptomycin, about 0.001-0.003 grams of phenol red, about 3.5-5.5 grams of bovine serum albumin, about 0.04-0.06 grams of EDTA (ethylenediaminetetraacetic acid), about 0.15-0.40 grams of HEPES (N-(2 -Hydroxy ethyl) piperazine-N'-(2-ethanesulfonic acid)) and about 5.0-10.0 grams of 0.02-0.08M theophylline .
[0018] Further, the present disclosure relates to a process for preparation of a recovery buffer for ejaculate semen sample comprising the steps of: a) taking Milli-Q water;b) adding buffer components gradually into the water with gentle stirring;c) adding bovine serum albumin (BSA) slowly and taking care to avoid frothing, to result in the buffer; d) checking if the pH of the buffer is 7.2-7.8 and osmolality of the buffer is 270-290 mOsmols/Kg water; e) making the volume upto a desired level with Milli-Q water; f) sterilizing the buffer by passing through sterile filters, disposable filter units; and
g) storing the buffer in sterile storage bottles at 4°C until use.
[0019] In an embodiment of the present disclosure, aprocess for preparing 250 ml of the recovery buffer comprises the steps of: a) taking 150 ml of Milli-Q water in a 500 ml glass beaker with a stirrer-bar, placed onto a magnetic stirrer; b) adding buffer components gradually into the water with gentle stirring, wherein the buffer components are sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dehydrate, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, Penicillin-G, streptomycin, phenol red, EDTA (ethylenediaminetetraacetic acid), HEPES (N-(2-Hydroxyethyl) piperazine-N'-(2- ethanesulfonic acid)), c) adding bovine serum albumin (BSA) slowly and taking care to avoid frothing, to result in the buffer; then add theophylline gradually into the water with gentle stirring; d)making the volume up to 250 ml with Milli-Q water in a volumetric flask; e) checking if the pH of the buffer is 7.2-7.8 and osmolality of the buffer is 270-290 mOsmols/Kg water; f) sterilizing the buffer by passing through 0.2 pm sterile filters, disposable filter units; and g) storing the buffer in sterile storage bottles at 4°C until use.
[0020] Furthermore, the present disclosure relates to a kit to facilitate recovery of sperm motility in collected, stored or transported semen sample, wherein the kit comprises a recovery buffer vial; a pasteur pipette to measure out 2ml of the recovery buffer and dispense the same into the sample; and an instructions manual, wherein the recovery buffer comprises sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dihydrate, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, Penicillin-G, streptomycin, phenol red, bovine serum albumin, EDTA (ethylenediaminetetraacetic acid), HEPES (N-(2-Hydroxyethyl) piperazine-N'-(2- ethanesulfonic acid)), and theophylline.
[0021] In an embodiment of the present disclosure, the recovery buffer at a volume of 1000 ml comprises about 4-7 grams of sodium chloride, about 0.2-0.4 grams of potassium chloride, about 0.03- 0.08 grams of magnesium sulphate heptahydrate, about 0.03-0.06 grams of potassium dihydrogen phosphate, about 0.2-0.4 grams of calcium chloride dehydrate, about 1.5-2.5 grams of sodium bicarbonate, about 0.4-0.6 grams of glucose, about 0.025-0.04 grams of sodium pyruvate, about 3.00- 4.00 ml of sodium lactate, about 0.065-0.080 grams of Penicillin-G, about 0.04-0.06 grams of streptomycin, about 0.001-0.003 grams of phenol red,
about 3.5-5.5 grams of bovine serum albumin, about 0.04-0.06 grams of EDTA (ethylenediaminetetraaceticacid), about 0.15-0.40 grams of HEPES (N-(2 -Hydroxy ethyl) piperazine-N'-(2-ethanesulfonic acid)) and about 5.0-10.0 grams of 0.02-0.08 M theophylline
[0022] The present disclosure relates to a method of recovering sperm motility in collected, stored or transported semen samples by measuring 2 ml of a recovery buffer and dispensing to a sample, wherein the recovery buffer comprises sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dihydrate, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, Penicillin- G, streptomycin, phenol red, bovine serum albumin, EDTA (ethylenediaminetetraacetic acid), HEPES (N-(2-Hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid)) and theophylline, and wherein the sample comprises semen and transport buffer.
[0023] In an embodiment of the present disclosure, the semen samples are transported in a transport buffer comprising sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dihydrate, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, Penicillin-G, streptomycin, phenol red, bovine serum albumin, EDTA (ethylenediaminetetraaceticacid), HEPES (N-(2- Hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid)), and trehalose.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] The examples herein will be better understood from the following detailed description with reference to the drawings, in which:
[0025] FIG.l illustrates the average fraction sperm motility at 6th hour versus 0th hour in three sets of semen samples stored at different buffer storage conditions, i.e. room temperature, at 45°C and at 55°C.
[0026] FIG.2 shows the effect of incubation time on mean progressive motility of semen samples in transport buffer stored at three different temperatures (room temperature, at 45°C and at 55°C) and treated with recovery buffer after 5 hours.
[0027] FIG.3 illustrates the mean fraction sperm motility insample with transport buffer stored at different temperatures.
DETAILED DESCRIPTION OF THE INVENTION
[0028] The present invention relates to a recovery buffer for ejaculate semen samples that facilitates recovery/restoration/revival of the sperm motility in the samples that have been
collected and transported at atmospheric temperature to the laboratory, thereby helping in the accurate prediction of the motility of the samples at the ejaculation time in semen analysis. Particularly, during the transport of the semen samples from the collection site to the laboratory when there is a considerable lapse in time span, the sperm motility may deteriorate leading to inaccuracies in semen analysis. This is prevented by addition of recovery buffer to the collected semen samples. The recovery buffer comprises sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dihydrate, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, Penicillin- G, Streptomycin, phenol red, bovine serum albumin, EDTA (ethylenediaminetetraacetic acid), HEPES (N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)) and theophylline. Further, the present disclosure describes a process for preparation of the recovery buffer for ejaculate semen sample. Furthermore, the present disclosure describes a process of recovery/restoration of sperm motility in collected and transported/stored semen samples by the addition of recovery buffer. [0029] In an embodiment of the present disclosure, 1000 ml of the recovery buffer comprises about 4-7 grams of sodium chloride, about 0.2-0.4 grams of potassium chloride, about 0.03- 0.08 grams of magnesium sulphate heptahydrate, about 0.03-0.06 grams of potassium dihydrogen phosphate, about 0.2-0.4 grams of calcium chloride dehydrate, about 1.5-2.5 grams of sodium bicarbonate, about 0.4-0.6 grams of glucose, about 0.025-0.04 grams of sodium pyruvate, about 3.00- 4.00 ml of sodium lactate, about 0.065-0.080 grams of Penicillin-G, about 0.04-0.06 grams of streptomycin, about 0.001-0.003 grams of phenol red, about 3.5-5.5 grams of bovine serum albumin, about 0.04-0.06 grams of EDTA (ethylenediaminetetraacetic acid), about 0.15-0.40 grams of HEPES (N-(2 -Hydroxy ethyl) piperazine-N'-(2-ethanesulfonic acid)) and about 5.0-10.0 grams of 0.02-0.08 M theophylline. [0030] Male infertility is common cause of infertility with a strong impact on the psychology and physiology of couple. It can be due to several reasons including abnormal semen parameters. Men with impaired semen parameters have an increased mortality rate in the years following an infertility evaluation suggesting semen quality may provide a marker of health. Hence, the transport buffer of the present disclosure helps to promote efficient semen testing by transporting semen efficiently for semen analysis without affecting sperm motility parameters and viability, which in turn has potential therapeutic application in assessing infertility associated with male factor (Oligospermia, asthenospermia and teratospermia).
[0031] Semen analysis is the cornerstone for the assessment of the male partner in a sub fertile couple. Semen analysis is the first step in identifying male factor infertility.
Compared to many other tests used in the assessment of the infertile couple, semen analysis has been standardized throughout the world. Standardized methods of semen analysis are available, however, semen transportation medium for efficient semen quality and sperm viability preservation is not available. Hence, the present disclosure provides a buffer for semen sample that allows accurate assessment of sperm quality. Measures of semen quality are used as surrogate measures of male fertility in clinical andrology, reproductive toxicology, epidemiology, and risk assessment. This could also be very useful to study the impact of COVID-19 on reproductive health by remotely collecting samples.
[0032] In an embodiment, the present disclosure describes a process of recovery/restoration/revival of sperm motility in collected and transported/stored semen samples by the addition of recovery buffer.
[0033] A person who wishes to get his semen sample tested in a laboratory, without physically visiting a laboratory may employ a transport buffer to facilitate preservation of the sample collected at his residence, and may then transport the sample to the laboratory via courier or by other means of transport. The transport buffer comprises sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dihydrate, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, Penicillin-G, streptomycin, phenol red, bovine serum albumin, EDTA
(ethylenediaminetetraacetic acid), HEPES (N-(2-Hydroxyethyl)piperazine-N'-(2- ethanesulfonic acid)) and trehalose.
[0034] Post transportation of the sample, which may take about 4-5 hours in general, during which there could be a decline in the motility of the sample, there is a necessity of a method to restore/recover/revive the sperm motility in the collected and transported samples. This issue is resolved by the addition of a recovery buffer to the samples. [0035] Another embodiment of the present disclosure relates to a kit comprising a recovery buffer vial; a pasteur pipette to measure out 2ml of the recovery buffer and dispense the same into a sample; and an instructions manual, wherein the sample comprises semen mixed with 2ml of a transport buffer.
[0036] The process of recovery/restoration/revival of sperm motility in collected and transported/stored semen samples involves the application of the kit, wherein the recovery buffer is measured out and dispensed to the collected and transported/stored semen samples.
[0037] In the following detailed description, reference is made to the accompanying drawings that form a part hereof, and in which is shown by way of illustration specific embodiments, which may be practiced. These embodiments are described in sufficient detail
to enable those skilled in the art to practice the embodiments, and it is to be understood that other embodiments may be utilized without departing from the scope of the embodiments. The invention is described in connection with such embodiments, but the invention is not limited to any embodiment. While only certain features of the invention have been illustrated and described herein, many modifications and changes will occur to those skilled in the art.
[0038] In an embodiment of the present disclosure, the recovery buffer comprises sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dihydrate, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, Penicillin-G, Streptomycin, phenol red, bovine serum albumin, EDTA (ethylenediaminetetraacetic acid), HEPES (N-(2-Hydroxyethyl)piperazine-N'-(2- ethanesulfonic acid)), and theophylline.
[0039] In another embodiment of the present disclosure, the recovery buffer for semen samples is prepared by the following procedure:
[0040] Materials required
Requirements of research resources, all reagents, buffer components and buffer preparation equipment are listed.
Requirements for buffer preparation:
1. Buffer components and other reagents are acquired from reputed vendors: Sigma Chemical Co.., St Louise, MO, USA; In-Vitrogen, USA or any other local vendors.
2. Autoclaved Stainless steel spatulas
3. Embryo culture grade (MilliQ) sterile water (sensitivity: 18 MW-cm at 25°C).
4. Culture-washed, autoclaved glass vessels or sterile disposables: 150 ml capacity bottles; 500 ml beakers; 100 ml measuring cylinders; 100 or 250 ml volumetric flasks
5. Culture tubes (4 and 15 ml)
6. Eppendorf tubes
7. Sterilization filter (0.2 pm) units
8. Buffer storage glass bottles (100 ml, 250 ml, 1L)
9. Magnetic stir bars and stirrers.
10. Ph Meter
11. Osmometer
[0041] Buffer Preparation:
The standard buffer composition of recovery buffer is comprehensively described in Table 1 above. 250 -500 ml (desired volume) of the buffer is prepared on the day before the experiment. 150 ml of Milli-Q water is taken in a 500 ml glass beaker with stirrer-bar, placed onto a magnetic stirrer. To this, the required buffer components are slowly added one by one (refer to Table 1) into water with gentle stirring. It is to be noted that 60% syrup of sodium lactate has a large osmotic effect and must be measured very accurately. CaChis dissolved in 5 mL of EbO separately and added to the solution. . Thereafter, BSA is slowly added at the end and care is taken to avoid frothing. pH and osmolality of the buffer (pH: 7.2-7.8; osmolality: 270-290 mOsmols/Kg water) is checked. The volume is made up to 250 ml with Milli-Q water in a volumetric flask. The buffer is sterilized by passing through 0.2 pm sterile filters, disposable filter units and stored in sterile storage bottles. The buffer is stored at 4°C until use.
[0042] In yet another embodiment of the present disclosure, experiments were performed to assess sperm motility parameters in semen samples with a transport buffer till 8 hours of duration. The below procedure was followed:
[0043] Semen samples were collected after informed consent. The samples were divided into four parts. Three parts were transported to the laboratory in 2ml of transport
buffer stored at RT, 45°C and 55°C (comprising trehalose as one of the constituents) and another part as it is (raw sample). The transport buffer comprises sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dihydrate, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, Penicillin- G, streptomycin, phenol red, bovine serum albumin, EDTA (ethylenediaminetetraacetic acid), HEPES (N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)), and trehalose.
[0044] Thereafter, the samples were labelled/categorized as four groups: raw sample or sample without the transport buffer, sample treated with RT transport buffer and incubated at room temperature , - sample treated with 45°C -transport media and incubated at room temperature sample treated with 55°C transport media and incubated at room temperature Preliminary testing of all parameters of semen testing was carried out, such as Volume, Ph, Pus cells, Vitality, RBC, debris, Concentration, Motility (in accordance with WHO 5th Manual and draft 6th manual, Morphology) with staining. [0045] Motility was assessed subsequently every hour, as it is only parameter that deteriorates for both Control samples (i.e. samples without the transport buffer) and samples with buffer. Further, concentration and morphology was assessed at the end of 8 hours and no difference was observed between the control and the tested sample.
[0046] Further, it was observed that at the end of 8 hours there was consistently a maximum 12% deterioration in motility. The deterioration started after 3 hours and deteriorated by 3% every hour.
[0047] In order to maintain accurate results, the recovery buffer was added to semen samples received after 3 hours which helps in restoring the sperm motility in the collected samples, and hence accurately predicts the motility of the samples at the ejaculation time. The recovery buffer comprises sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dihydrate, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, Penicillin-G, Streptomycin, Phenol Red, bovine serum albumin, EDTA (ethylenediaminetetraacetic acid), HEPES (N-(2- Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)), and theophylline.
[0048] EXAMPLE 1:
A METHOD TO CHECK THE RECOVERY OF MOTILITY IN COLLECTED. TRANSPORTED AND STORED SEMEN SAMPLES
[0049] In an embodiment of the present disclosure, experimental analysis was
performed over a period of 30 days to check the recovery of motility in collected, transported and stored semen samples. Collected semen samples were transported to the laboratory in the transport buffer (comprising trehalose as one of the constituents). Semen analysis parameters like rapid progressive (RP), progressive motility (PM), non-progressive motility (NP) and immotility (IM) were assessed every hour, till 5 hours, at room temperature. The analysis was performed for the raw samples (i.e. samples without transport buffer) as well. A gradual decline of PM and NP and increase in IM was observed with time as indicated in the Table 2 below. At the 6th hour, recovery buffer (comprising theophylline as one of the constituents) was added to all the samples. Upon analysis of recovery buffer - treated samples, it was observed that the PM, NP, and IM values come closer to the initially recorded values at the 1st hour.
[0050] The experiment was carried out using transport buffer incubated at other temperatures as well, such as, at 45°C and 55°C. It was found that change in temperature did not hamper the restorative property of the recovery buffer. In fact, high temperature incubated buffers have shown to increase the motility retaining capacity of the buffer. The experiment was repeated every day consecutively for the next 29 days and consistent results were recorded. Table 2 below shows the semen analysis results of the raw and tested samples recorded on the first day. A similar pattern in the results was observed for the rest of the days. Table 2: Semen analysis results of raw and tested semen samples every hour for a period of 6 hours at different temperatures
[0051] EXAMPLE 2:
COMPARABILITY OF MOTILITY BETWEEN RANDOM SAMPLES AND SAMPLES USED TN THE LAB FOR TESTING AND VALIDATING THE EFFICIENCY OF THE TRANSPORT AND RECOVERY BUFFERS
[0052] To check for reproducibility of results, semen samples were collected from random patients who visited the diagnostic center. The samples were assessed for motility at the time of collection, stored in the transport buffer for 6-8 hours, treated with recovery buffer (comprising theophylline) and once again assessed for motility. The results were compared with the motility results of samples collected at home, transported to the laboratory using the transport buffer and then treated with recovery buffer. It was observed that the results of the said two sets of samples were almost similar. The results are tabulated as given below:
[0053] Table 3: A comparison of motility results between random samples and the samples tested in the laboratory using the protocol.
[0054] FIG.l illustrates the average fraction sperm motility at 6th hour versus 0th hour in sample with buffer under different buffer storage conditions. Transport buffer stored at room temperature (RT), at 45°C (T45), and at 55°C (T55) were allowed to reach room temperature and mixed with semen sample for transport. On an average, transport buffer stored at different temperatures can retain about 55%-65% of motility as compared to zero hour in raw sample. With the addition of recovery buffer (comprising theophylline at 6th hour), motility was found to increase 25-30% as compared to transport buffer alone and about 78%-92% as compared to raw sample (control), showing that the recovery buffer effectively restores the motility.
[0055] Surprisingly, increase in the buffer storage temperature, has shown to increase the motility retaining capacity of the buffer. It is interesting to note that improvement in the motility of stored sample at various temperature is linearly increasing with the storage temperature condition. FIG.2 shows the linearly increasing mean fraction sperm motility in sample with transport buffer stored at different temperatures.
[0056] FIG.3 shows the effect of incubation time on mean progressive motility of semen samples in transport buffer treated with recovery buffer after 5 hours.
[0057] In yet another embodiment of the present invention, the recovery buffer for ejaculate semen samples is advantageous owing to the following reasons:
1. The goal of this investigation is to improve the standards of semen analysis post transportation of semen to the laboratory from a collection site. The recovery buffer ensures that the semen and sperm parameters are not affected for further analysis.
2. Another primary goal of this study is to development of non-liquid nitrogen based transportation of semen samples for invitro analysis without compromising the semen parameters such as motility, concentration, vitality and morphology.
3. This product will give potential benefits for restoration of semen motility post transportation, without affecting optimal semen quality and sperm motility parameters. A few home sperm test kits or mail order kits collect sperm and use an algorithm to assess decline in motility over several hours- depending on the time of assessment. Some of them also use cryofreezing buffer or cryofreezing container for transport. They report semen generally after 24-48 hours. Most of these samples are mainly for men trying to freeze their semen for long term storage that is mainly used for IVF-ICSI and not for an assessment of a formal semen analysis that is needed for assessment of male factor infertility. As it is known, motility decline varies from sample to sample and poor motility samples unless preserved to retain their motility will deteriorate over time and become zero motility at some point. Using an algorithm to predict original motility may give false reading or false positives. Similarly, if intrinsically sample has zero sperm, using an algorithm would be fraught with incorrect reporting. Algorithms derived motility decline are percentages which are fixed and does not vary from sample to sample which is needed if we are assessing semen sample. That is why restoring actual original motility with a recovery buffer is absolutely essential and much more beneficial over use of predictive algorithm.
4. It will ensure that sperm viability and motility (i.e. percentage of motility) are restored/recovered in transported and stored semen samples. It provides for accurate semen analysis without the need for re-testing.
5. The method described in the present disclosure is not a single or two parameter semen assessment such as the methods available today, but a full comprehensive semen analysis.
6. Most importantly, this method has potential implications in sterile collection and transportation of semen for diagnostic or research purposes or assisted reproduction. 7. This product will not have any adverse impact on semen parameters and reference values as set by the WHO. The breadth and scope of the invention is not limited to the exemplary embodiments described above. The invention should be defined only in accordance with the following claims and their equivalents.
[0058] The foregoing description of the specific examples will so fully reveal the general nature of the examples herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific examples without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed examples. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the examples herein have been described in terms of preferred examples, those skilled in the art will recognize that the examples herein can be practiced with modification within the spirit and scope of the embodiment.
Claims
1. A recovery buffer for ejaculate semen sample that facilitates recovery of sperm motility in collected, stored or transported semen sample at atmospheric temperature, wherein the transport buffer comprises sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dihydrate, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, Penicillin-G, streptomycin, phenol red, bovine serum albumin, EDTA (ethylenediaminetetraacetic acid), HEPES (N-(2- Hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid)), and theophylline.
2. The recovery buffer as claimed in claim 1, wherein the recovery buffer at a volume of 1000 ml comprises about 4-7 grams of sodium chloride, about 0.2-0.4 grams of potassium chloride, about 0.03- 0.08 grams of magnesium sulphate heptahydrate, about 0.03-0.06 grams of potassium dihydrogen phosphate, about 0.2-0.4 grams of calcium chloride dehydrate, about 1.5-2.5 grams of sodium bicarbonate, about 0.4-0.6 grams of glucose, about 0.025-0.04 grams of sodium pyruvate, about 3.00- 4.00 ml of sodium lactate, about 0.065-0.080 grams of Penicillin-G, about 0.04-0.06 grams of streptomycin, about 0.001-0.003 grams of phenol red, about 3.5-5.5 grams of bovine serum albumin, about 0.04-0.06 grams of EDTA (ethylenediaminetetraacetic acid), about 0.15-0.40 grams of HEPES (N-(2 -Hydroxy ethyl) piperazine-N'-(2-ethanesulfonic acid)), and about 5.0-10.0 grams of 0.02-0.08M theophylline.
3. A process for preparation of a recovery buffer for ejaculate semen sample comprising the steps of: a) taking Milli-Q water; b) adding buffer components gradually into the water with gentle stirring; c) adding bovine serum albumin (BSA) slowly and taking care to avoid frothing, to result in the buffer; d) checking if the pH of the buffer is 7.2-7.8 and osmolality of the buffer is270-290 mOsmols/Kg water; e) making the volume upto a desired level with Milli-Q water; f) sterilizing the buffer by passing through sterile filters, disposable filter units; and g) storing the buffer in sterile storage bottles at 4°C until use.
4. The process as claimed in claim 3, wherein the process for preparing 250 ml of the recovery buffer comprises steps of: a) taking 150 ml of Milli-Q water in a 500 ml glass beaker with a stirrer-bar, placed onto a magnetic stirrer; b) adding buffer components gradually into the water with gentle stirring, wherein the buffer components are sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dihydrate, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, Penicillin-G, streptomycin, phenol red, EDTA (ethylenediaminetetraacetic acid), HEPES (N-(2-Hydroxyethyl) piperazine-N'-(2- ethanesulfonic acid)) c)making the volume up to 250 ml with Milli-Q water in a volumetric flask; d) adding bovine serum albumin (BSA) slowly and taking care to avoid frothing, to result in the buffer; then add theophylline gradually into the water with gentle stirring; e) checking if the pH of the buffer is 7.2-7.8 and osmolality of the buffer is 270-290 mOsmols/Kg water; f) sterilizing the buffer by passing through 0.2 pm sterile filters, disposable filter units; and g) storing the buffer in sterile storage bottles at 4°C until use.
5. A kit to facilitate recovery of sperm motility in collected, stored or transported semen sample, wherein the kit comprises a recovery buffer vial; a pasteur pipette to measure out 2ml of the recovery buffer and dispense the same into the sample; and an instructions manual, wherein the recovery buffer comprises sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dihydrate, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, Penicillin-G, streptomycin, phenol red, bovine serum albumin, EDTA (ethylenediaminetetraacetic acid), HEPES (N-(2- Hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid)), and theophylline.
6. The kit as claimed in claim 5, wherein the recovery buffer at a volume of 1000 ml comprises about 4-7 grams of sodium chloride, about 0.2-0.4 grams of potassium chloride, about 0.03- 0.08 grams of magnesium sulphate heptahydrate, about 0.03-0.06 grams of potassium dihydrogen phosphate, about 0.2-0.4 grams of calcium chloride dehydrate, about 1.5-2.5 grams of sodium bicarbonate, about 0.4-0.6 grams of glucose, about 0.025-0.04 grams of sodium pyruvate, about 3.00- 4.00 ml of sodium lactate, about 0.065-0.080 grams of Penicillin-G, about 0.04-0.06 grams of streptomycin, about 0.001-0.003 grams of phenol red,
about 3.5-5.5 grams of bovine serum albumin, about 0.04-0.06 grams of EDTA (ethylenediaminetetraacetic acid), about 0.15-0.40 grams of HEPES (N-(2 -Hydroxy ethyl) piperazine-N'-(2-ethanesulfonic acid)), and about 5.0-10.0 grams of 0.02-0.08M theophylline.
7. A method of recovering sperm motility in collected, stored or transported semen samples by measuring 2 ml of a recovery buffer and dispensing to a sample, wherein the recovery buffer comprises comprising sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dihydrate, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, Penicillin-G, streptomycin, phenol red, bovine serum albumin, EDTA (ethylenediaminetetraacetic acid), HEPES (N-(2- Hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid)), and theophylline, and wherein the sample comprises semen and transport buffer.
8. The method as claimed in claim 7, wherein the semen samples are transported in a transport buffer comprising sodium chloride, potassium chloride, magnesium sulphate heptahydrate, potassium dihydrogen phosphate, calcium chloride dihydrate, sodium bicarbonate, glucose, sodium pyruvate, sodium lactate, Penicillin-G, streptomycin, phenol red, bovine serum albumin, EDTA (ethylenediaminetetraacetic acid), HEPES (N-(2- Hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid)), and trehalose.
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WO2021055170A1 (en) * | 2019-09-17 | 2021-03-25 | Longhorn Vaccines And Diagnostics, Llc | Multipurpose compositions for collecting and transporting biological material |
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WO2005116081A2 (en) * | 2004-05-24 | 2005-12-08 | Genvault Corporation | Stable protein storage and stable nucleic acid storage in recoverable form |
CN109628380B (en) * | 2018-12-25 | 2020-12-25 | 深圳韦拓生物科技有限公司 | Human body external receptor semen and preparation method thereof |
WO2021055170A1 (en) * | 2019-09-17 | 2021-03-25 | Longhorn Vaccines And Diagnostics, Llc | Multipurpose compositions for collecting and transporting biological material |
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