WO2022264162A1

WO2022264162A1 - Transport buffer for semen sample

Info

Publication number: WO2022264162A1
Application number: PCT/IN2022/050520
Authority: WO
Inventors: Satya Srini VASAN; Sheeba LOBO; Vani V
Original assignee: Vasan Satya Srini; Lobo Sheeba; Vani V
Priority date: 2021-06-15
Filing date: 2022-06-03
Publication date: 2022-12-22

Abstract

The present invention relates to a transport buffer for ejaculate semen sample that facilitates transportation of the semen sample at atmospheric temperature to the laboratory, thereby enabling accurate semen analysis and reporting. 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 transport buffer helps in detection of abnormal semen parameters and in turn suggests a clinician regarding possible etiology for infertility diagnosis. Further, the present disclosure describes a process for preparation of the transport buffer for ejaculate semen sample.

Description

TRANSPORT BUFFER FOR SEMEN SAMPLE

TECHNICAL FIELD

[0001] This invention generally relates to a transport buffer for ejaculate semen sample that facilitates transportation of the semen sample at atmospheric temperature to the laboratory, thereby enabling accurate semen analysis and reporting. In particular, the transport buffer comprisessodium 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 transport buffer helps in detection of abnormal semen parameters and in turn suggeststo a clinician regarding possible etiology for infertility diagnosis. In this way, it helps a clinician to decide a suitable assisted reproductive technology (ART) to treat the patient, such as In-vitro fertilization (IVF) or Intracytoplasmic sperm injection (ICSI) Further, the present disclosure describes a process for preparation of the transport buffer for ejaculate semen sample.

BACKGROUND

[0002] Semen analysis is the cornerstone and the first step inthe 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 theATP 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 and collection variability. As such, a centralized andrology laboratory with most advanced equipment for evaluation can minimize inter-observer variability and, collection variability thus, improve the diagnostic utility of the test results.

[0015] Accordingly, in view of the rising number of male patients seeking infertility treatment, there is a need for an efficient transport buffer for semen that will preserve sperm motility for sufficient duration for it to be transported to a laboratory for a 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 transport buffer for ejaculate semen sample, 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 trehalose.

[0017] In an embodiment of the present disclosure, the transport 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 to 0.06 grams of streptomycin, about 0.15-0.25 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 25-40 grams of 0.20M to 0.35M trehalose.

[0018] The present disclosure relates to a process for preparation of a transport 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, the process for preparing 250 ml of the transport buffer for ejaculate semen sample comprises the steps of: a) taking 150 ml of Milli-Q water in a 500 ml glass beaker with stirrer-bar, placed onto a magnetic stirrer; b) adding buffer components namely 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; and add trehalose gradually into it 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.

[0020] The present disclosure further relates to a kit to facilitate collection and transportation of semen sample, wherein the kit comprises a transport buffer vial; container for collection of sample; a box to pack the container; labels to indicate the patient’s name, date and time of collection; and an instructions manual.

[0021] In an embodiment of the present disclosure, the kit comprises the transport 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 0.001-0.003 grams of phenol red, about 0.04 to 0.06 grams of streptomycin, 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 25-40 grams of 0.05M-0.35M trehalose.

[0022] Furthermore, the present disclosure relates to a method of collecting and transporting semen sample with a kit, wherein the kit comprises a transport buffer vial; a container for collection of samples; a box to pack the container; labels to indicate the patient’s name, date and time of collection; and an instructions manual, 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 trehalose, and wherein the method comprises the steps of: a) labelling the vials with name, date and time of collection of the semen sample; b) collecting the semen sample in the container; c) dispensing and mixing 2 ml of the transport buffer into the sample directly . d) closing the container with screw caps or stoppers; e) packaging the container in box provided ; and f) transporting the collected semen sample by courier or other means.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The examples herein will be better understood from the following detailed description with reference to the drawings, in which:

[0024] FIG.1 illustrates the average motility in semen samples stored for a period of 5 hours in room temperature (RT) stored transport buffer and treated with recovery buffer at the 6^th hour.

[0025] FIG.2 illustrates the average motility in semen samples stored for a period of 5 hours in 45°C stored transport buffer treated with recovery buffer at the 6th hour.

[0026] FIG.3 illustrates the average motility in semen samples stored for a period of 5 hours in 55°Cstored transport buffer and treated with recovery buffer at the 6th hour.

[0027] FIG.4 illustrates the average fraction sperm motility at 6^th hour versus 0^th hour in sample with buffer under different buffer storage conditions.

[0028] FIG.5 illustrates the mean fraction sperm motility insample with transport buffer stored at different temperatures.

[0029] FIG.6 illustrates the effect of incubation time on mean progressive motility of semen samples treated with recovery buffer after 5 hours. The samples here include raw semen sample, and samples with RT, 45°C and 55°C transport buffers.

DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention relates to a transport buffer for ejaculate semen samples that facilitates transportation of the semen sample at atmospheric temperature to the laboratory, thereby enabling accurate semen analysis and reporting. The transport buffer comprises a buffering system composed of a HEPES and Sodium Bicarbonate combination and does not require the use of a CO2 incubator. This buffer contains, salts and ions, energy substrates, buffers and the antibiotics i.e., Penicillin-G and streptomycin. It contains protein components and does not require protein supplement. This buffer is synthetic (bicarbonate- buffered), aseptically processed/prepared according to standard operating protocols, which have been validated to meet a sterility assurance level. The transport buffer helps in detection of abnormal semen parameters and in turn suggests a clinician regarding possible etiology for infertility diagnosis. The transport buffer helps in the retention of semen parameters especially, motility for more than six hours which gives an option to evaluate it under appropriate laboratory conditions, thereby, not only helping 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) and Intracytoplasmic sperm injection (ICSI).

[0031] In particular, 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. Further, the present disclosure describes a process for preparation of the transport buffer for ejaculate semen sample.

[0032] In an embodiment of the present disclosure, 1000 ml of the transport buffer comprises about 4-7 gramsof 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 0.001-0.003 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-Hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid)) and about 25- 40 grams of 0.05 - 0.35 M trehalose

[0033] Male infertility is a 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 be a marker of overall 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).

[0034] 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, a semen transportation buffer for efficient semen quality and sperm viability preservation is not available. Hence, the present disclosure provides a transport buffer for semen sample that allows accurate assessment of sperm quality in the laboratory. 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.

[0035] Another embodiment of the present disclosure relates to a kit comprising a transport buffer with a collection container, a vial of transport buffer, a pasteur pipette, labels to indicate the patient’s name, date and time of collection, a box to pack the container, and an instructions manual. The transport buffer from the vial is directly is added to the semen sample in the container, after which the container is tightly closed.

[0036] A person who wishes to get his semen sample tested in a laboratory, without physically visiting a laboratory may employ the kit by labelling the container with name, date and time of collection, using the container to collect the sample, dispensing and mixing 2 ml of the transport buffer provided in the vial directly into the sample , closing the container with screw caps/stoppers, packaging the container in the box provided and ordering a transport of the same to the laboratory via courier or by other means of transport. The empty transport buffer vial (after the content is mixed with semen sample) is discarded. [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 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. Table 1: Composition of Transport Buffer

[0039] In another embodiment of the present disclosure, the transport 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 bufferpreparation:

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 mih) 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 transport 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. CaCb is dissolved in 5 mL of H2O separately and added to the solution. 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 the transport buffer till 8 hours of duration. The below procedure was followed:

Collection of informed consented semen samples

Addition of 2 ml of the transport buffer to the ejaculate (immediately without waiting for liquefaction).

- Preliminary testing of all parameters of semen testing - Volume, Ph, Pus cells, Vitality, RBC, debris, Concentration, Motility (in accordance with WHO 5th Manual and draft 6th manual, Morphology) with staining.

[0043] 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.

[0044] 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.

[0045] In order to arrive at accurate results, a recovery buffer is 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.

[0046] Table 2: Composition of RecoveryBuffer

[0047] In an embodiment of the present disclosure, experimental analysis was performed over a period of 45 days to check the recovery of motility in collected, transported and stored semen samples. The protocol that was followed involved collection of semen samples and transportation to the laboratory in the transport buffer (comprising trehalose as one of the constituents). This was followed by semen analysis of parameters like rapid progressive (RP), progressive motility (PM), non-progressive motility (NP) and immotility (IM) assessment 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. At the 6th hour, recovery medium (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. This is further elaborated in the below Examples.

[0048] EXAMPLE 1:

COMPARABILITY OF MOTILITY BETWEEN RANDOM SAMPLES AND SAMPLES USED IN THE TAB FOR TESTING AND VALIDATING THE EFFICIENCY OF THE TRANSPORT AND RECOVERY BUFFERS

[0049] 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 in transport buffer and treated with the recovery buffer using the aforementioned protocol. It was observed that the results of the said two sets of samples were almost similar (rate of recovery). The results are tabulated as given below:

[0050] Table 3:A comparison of motility results between random samples and the samples tested in the laboratory using the protocol (rate of recovery)

[0051] EXAMPLE 2:

STABILITY OF TRANSPORT BUFFER AT VARIOUS TEMPERATURES

According to a further embodiment of the present disclosure, buffer stability was tested at room temperature, 45°C and 55°C. As it is well understood about the storage condition of buffer from retrospective studies, higher temperature is known to destabilize the buffer. Hence to understand the stability of the buffer, functional capability (retaining sperm motility) of the buffer was set as a one of the parameter. In order to decrease the time in assessing the stability and shelf life of 6 months, it was decided to accelerate in the destabilization condition at various high temperatures and analyze sperm motility retaining capacity of the buffer. With this it is possible to establish the mathematical model which can determine stable shelf life of the buffer at given temperature.

[0052] The control and samples which were taken for the analysis are as follows: Control samples/raw samples without buffer (room temperature): motility assessment every hour till 6 hours

Samples with buffer stored at room temperature (RT)

Samples with buffer stored at 45°C temperature (T45)

Samples with buffer stored at 55°C temperature (T55)

[0053] FIG. 1 shows the average motility in semen samples stored for a period of 5 hours in transport buffer at room temperature (RT) and treated with recovery buffer at the 6th hour. Significant change was noticed in progressive motility after incubating the samples for certain period of time. Samples were converted from normal to asthenozoospermic. No Significant change was seen for non-progressive and rapid motility. Hence progressive motility data was considered for the analysis. Sample stored at room temperature in transport buffer alone can retain 45%-55% of motility as compared to zero hour in raw sample. With the addition of theophylline at 6th hour, motility was found to increase by about 25-30% as compared to transport buffer alone and 60-70% as compared to raw sample (control).

[0054] FIG. 2 shows the average motility in semen samples stored for a period of 5 hours in45°C-transport buffer (T45) and treated with recovery buffer at the 6th hour. Sample stored at 45°C-transport buffer alone can retain about 55%-65% of motility as compared to zero hour in raw sample. With the addition of theophylline at 6th hour, motility was found to increase by about 25-30% as compared to transport buffer alone and 75-85% as compared to raw sample (control).

[0055] FIG. 3 shows the average motility in semen samples stored for a period of 5 hours in 55°C-transport buffer (T55) and treated with recovery buffer at the 6th hour. Sample stored at 55°C-transport buffer alone can retain about 65%-75% of motility as compared to zero hour in raw sample. With the addition of theophylline at 6th hour motility was found to increase 25-30% as compared to transport buffer alone and 85-93% as compared to raw sample (control).

[0056] FIG.4 shows the average fraction sperm motility at 6th hour versus 0th hour in sample with buffer under different buffer storage conditions. On an average, sample stored at room temperature buffer(RT), at 45°C-buffer (T45), and at 55°C-buffer (T55) 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 75% as compared to raw sample (control), showing the efficiency of the transport buffer at various temperature and also that the recovery buffer effectively restores the motility.

[0057] In the buffer stability assessment at various temperatures it was seen that the percentage of sperm motility in the samples were efficiently maintained/retained. It was observed that the percentage of sperm motility in the samples were maintained/retained in all the samples at room temperature, 45°C, and 55°Cbuffers.

[0058] 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. 5 shows the mean fraction motility for sample with transport buffer stored at different temperatures (RT, T45, T55).

[0059] EXAMPLE 3:

EFFECT OF INCUBATION TIME ON MOTILITY IN THE COLLECTED AND

STORED SEMEN SAMPLES

[0060] The optimum condition to store any buffer is in the refrigerated conditions since room temperature (RT) and above may induce microbial contamination as well as structurally destabilize the protein. Hence, the original idea was to identify the stability of the buffer at accelerated temperatures. Therefore, buffer was stored at RT, 45°C and 55°C for 36 days and its impact on the motility of the sperm was estimated at different time points of the storage period. The transport buffer stored at different temperatures (RT, 45°C and 55°C) were allowed to attain room temperature and then used for the transportation of semen samples. Experimental analysis was performed to assess the recovery of motility in collected, transported and stored semen samples, wherein the semen samples were stored with buffers of three different temperatures namely at RT, 45°C and 55°C. The protocol that was followed involved collection of semen samples and aliquoting it in to four parts. Three parts were transported to the laboratory in the transport buffer of RT, 45°C and 55°C (comprising trehalose as one of the constituents) and another part as it is (raw sample).

[0061] Thereafter, the samples were categorized into 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°Ctransport buffer and incubated at room temperature sample treated with 55°Ctransport buffer and incubated at room temperature This was followed by semen analysis of parameters like rapid progressive (RP), progressive motility (PM), non-progressive motility (NP) and immotility (IM) assessment every hour, till 5 hours. A gradual decline of PM and NP and increase in IM was observed with time. At the 6th hour, recovery medium (comprising theophylline as one of the constituents) was added to all the samples. Upon analysis of the samples, it was observed that the PM, NP, and IM values were restored.FIG. 6 illustrates the effect of incubation time on mean progressive motility of semen samples in transport buffer, treated with recovery buffer after 5 hours. [0062] EXAMPLE 4:

COMPARISON OF THE EFFECT OF TRANSPORT BUFFER ON SPERM MOTILITY AND THE EFFECT OF NIDACON ON SPERM MOTILITY

Nidacon is an optimized solution for protecting human sperm from the damaging effects of freezing and thawing. The solution contains osmotically active ingredients to reduce intracellular water, and cryoprotectants to reduce injury caused by ice crystal formation. The effect of transport buffer on sperm motility in semen samples was compared with that of nidacon. One set of semen samples were treated with the transport buffer (comprising 0.2M- 0.35Mtrehalose) and another set of semen samples were treated with Nidacon. Both the sets were stored at room temperature and analysed for PM, NP and IM every hour for a period of 5 hours.

The results were compared against control samples.

The following results were observed:

[0063] Table 4: A comparison of motility results between samples treated with transport buffer and samples treated with Nidacon.

[0064] From the above results, it was observed that the motility values were better for the samples treated with the transport buffer when compared to the motility values for samples treated with Nidacon. [0065] In yet another embodiment of the present invention, the transport 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 transportation process and to ensure that the semen and sperm parameters should not get affected for further analysis. 2. This product will give potential benefits for transportation of semen without affecting optimal semen quality and sperm motility parameters.

3. It is user friendly, works at extreme temperatures of Indian summer (tested upto 55°C) as well both for storage and during transport.

4. It will ensure that sperm viability and motility (i.e. percentage of motility) are maintained until 8 hours. It provides for accurate semen analysis without needed for re testing.

5. It is not single or two parameter semen assessment as the methods available today but a full comprehensive semen analysis

6. It is a boon for patients with inability to masturbate, people who collect sample only at night, people who collect semen sample only with sexual activity and people who hesitate to come to semen testing centers due to stigma attached to it, in India.

7. This method helps us to overcome the limitations involved in semen collection protocols and transportation in ART centers (WHO, 2010).

8. Most importantly, this method has potential implications in sterile collection and transportation of semen for diagnostic or research purposes or assisted reproduction.

9. 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.

[0066] 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

We Claim:

1. A transport buffer for ejaculate semen sample, 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 trehalose.

2. The transport buffer as claimed in claim 1, wherein the transport 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 to 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 25-40 grams of 0.05M to 0.35M trehalose.

3. A process for preparation of a transport buffer for ejaculate semen sample comprising 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.

4. The process as claimed in claim 3, wherein the process for preparing 250 ml of the transport buffer for ejaculate semen sample comprises the steps of: a) taking 150 ml of Milli-Q water in a 500 ml glass beaker with stirrer-bar, placed onto a magnetic stirrer; b) adding buffer components namely 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; and add trehalose 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 collection and transportation of semen sample, wherein the kit comprises a transport buffer vial;a container for collection of sample;a box to pack the container; labels to indicate the patient’s name, date and time of collection; and an instructions manual.

6. The kit as claimed in claim 5, wherein the transport 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 0.001-0.003 grams of phenol red, about 0.04 to 0.06 grams of streptomycin, 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 25-40 grams of 0.05M-0.35M trehalose.

7. A method of collecting and transporting semen sample with a kit, wherein the kit comprises a transport buffer vial ; a container for collection of samples ; a box to pack the container; labels to indicate the patient’s name, date and time of collection; and an instructions manual, 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 trehalose, and wherein the method comprises the steps of: a) labelling the vials with name, date and time of collection of the semen sample; b) collecting the semen sample in the container; c) dispensing and mixing 2 ml of the transport buffer into the sample directly. d) closing the container with screw caps or stoppers; e) packaging the container in box provided ; and f) transporting the collected semen sample by courier or other means.