WO2008142396A1

WO2008142396A1 - Methods of assessing male fertility using a uridine marker

Info

Publication number: WO2008142396A1
Application number: PCT/GB2008/001715
Authority: WO
Inventors: Anthony Denis Maher; Prasad Patki; Michael D. Craggs; John Christopher Lindon; Jeremy Kirk Nicholson; Elaine Holmes
Original assignee: Anthony Denis Maher; Prasad Patki; Craggs Michael D; John Christopher Lindon; Jeremy Kirk Nicholson; Elaine Holmes
Priority date: 2007-05-18
Filing date: 2008-05-19
Publication date: 2008-11-27

Abstract

The present invention relates generally to field of human male fertility, and in particular, to the use of the presence or absence or amount of a uridine marker (e.g., uridine) in a seminal fluid sample from a human male subject as a means of assessing the subject's fertility status.

Description

METHODS OF ASSESSING MALE FERTILITY USING A URIDINE MARKER

RELATED APPLICATION

This application is related to United States patent application number 60/924,526 filed 18 May 2007, the contents of which are incorporated herein by reference in their entirety.

TECHNICAL FIELD

BACKGROUND

A number of publications are cited herein in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Each of these references is incorporated herein by reference in its entirety into the present disclosure, to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

Throughout this specification, including the claims which follow, unless the context requires otherwise, the word "comprise," and variations such as "comprises" and

"comprising," will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

It must be noted that; as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

Ranges are often expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent "about," it will be understood that the particular value forms another embodiment.

There are more than 50,000 people with spinal cord injury (SCI) in the U.K. and nearly 1300 new cases per year are reported. Out of all the SCI cases, 80% are males with an average age of 29 years (see, e.g., Stover et a!., 1987). Male infertility is a well recognised complication of SCI and not being able to father children has devastating psychological and emotional effects on their quality of life (see, e.g., Biering-Sorensen et al., 2001 ; Rutkowski et al., 1999). The fertility potential of sperm from semen samples of spinal cord injury (SCI) men is subnormal. This is either due to a qualitative defect in the sperm itself or a defect in its biochemical environment, that is, the seminal plasma.

The average age of SCI patient is 32.6 years and 80% of these injuries are seen in males (according to the National Spinal Cord Injury Database). Infertility affects nearly 90% of SCI men (see, e.g., Sonksen et al., 1992). Extreme impairment in sperm motility and viability rather than changes in sperm concentration dominate the unusual semen profile (see, e.g., Brackett et al., 2000). These changes take place within the first two weeks after spinal cord injury and may show improvement over the next six months, never achieving pre-injury status. Previous studies performed in order to understand the pathophysiology of the poor sperm quality have been inconclusive about the specific cause. Recently the sperm motility in sperm sample aspirated from vas deference was found to be better than sperm from ejaculated semen sample in SCI patient (see, e.g., Brackett et al., 2000). In an another study, seminal plasma of SCI men was found to inhibit sperm motility of normal men indicating that the seminal plasma in the SCI men could be the factor responsible for the poor sperm quality (Brackett et al., 1996).

Ejaculatory dysfunction and poor sperm quality are two main factors of infertility in SCI (see, e.g., Biering-Sorensen et al., 2001). While semen retrieval techniques have improved to overcome the former, investigations into the etiology of poor sperm quality have been various but inconclusive.

In a recently concluded study improvement in sperm morphology and forward progression by using repeated ejaculations has been reported (see, e.g., Hamid et al., 2006). However there is some evidence to suggest that poor sperm quality can be associated with the biochemical composition of the seminal plasma (see, e.g., Bracket et al., 1996; Bracket et al., 2000).

Secretions of the prostate and seminal vesicles in approximately a 40:60 ratio form seminal plasma. Their distinct compositions have already been characterised by using nuclear magnetic resonance studies (see, e.g., Lynch et al., 1994; Tomlins et al., 1998). The difficulties encountered in analyzing the complex dynamic biochemical matrix of semen may now be overcome by using the non-destructive high resolution NMR. SUMMARY OF THE INVENTION

One aspect of the present invention pertains to a method of assessing fertility status of a human male subject comprising a step of determining presence or absence of a uridine marker in a seminal fluid sample from said human male subject.

Another aspect of the present invention pertains to a method of diagnosis of infertility in a human male subject comprising a step of correlating absence of a uridine marker in a seminal fluid sample from said human male subject with infertility.

Another aspect of the present invention pertains to a method of determination of fertility in a human male subject comprising a step of correlating presence of a uridine marker in a seminal fluid sample from said human male subject with fertility.

Another aspect of the present invention pertains to a method of assessing fertility status of a human male subject comprising a step of determining a measure of a uridine marker in a seminal fluid sample from said human male subject.

Another aspect of the present invention pertains to a method of assessing fertility status of a human male subject comprising a step of comparing a measure of a uridine marker in a seminal fluid sample from said human male subject with a suitable control measure of said uridine marker, wherein said suitable control measure of said uridine marker is a measure of said uridine marker in a seminal fluid sample from a suitable control male of known fertility status, or an average of a measure of said uridine marker in seminal fluid samples from a population of suitable control males of same known fertility status.

Another aspect of the present invention pertains to a method of diagnosis of infertility in a human male subject comprising a step of correlating a reduced measure of a uridine marker in a seminal fluid sample from said human male subject, as compared to a suitable control measure of said uridine marker, with infertility, wherein said suitable control measure of said uridine marker is a measure of said uridine marker in a seminal fluid sample from a suitable fertile control male, or an average of a measure of said uridine marker in seminal fluid samples from a population of suitable fertile control males.

Another aspect of the present invention pertains to a method of determination of fertility in a human male subject comprising a step of correlating a similar measure of a uridine marker in a seminal fluid sample from said human male subject, as compared to a suitable control measure of said uridine marker, with fertility, wherein said suitable control measure of said uridine marker is a measure of said uridine marker in a seminal fluid sample from a suitable fertile control male, or an average of a measure of said uridine marker in seminal fluid samples from a population of suitable fertile control males. As will be appreciated by one of skill in the art, features and preferred embodiments of one aspect of the invention will also pertain to other aspect of the invention.

DETAILED DESCRIPTION

The inventors have determined that uridine in seminal fluid is a useful biomarkerfor fertility in human males, and more specifically, that reduced levels of uridine, or the absence of uridine, in seminal fluid is strong evidence of infertility in human males.

Uridine

Thus, the present invention relates generally to field of human male fertility, and in particular, to the use of the presence or absence or amount of a uridine marker (e.g., uridine) in a seminal fluid sample from a human male subject as a means of assessing the subject's fertility status.

Presence or Absence of a Uridine Marker

In one embodiment, the method comprises a further step of correlating presence of a uridine marker in a seminal fluid sample from said human male subject with fertility.

In one embodiment, the method comprises a further step of correlating absence of a uridine marker in a seminal fluid sample from said human male subject with infertility.

Another aspect of the present invention pertains to a method of determination of fertility in a human male subject comprising a step of correlating presence of a uridine marker in a seminal fluid sample from said human male subject with fertility. Uridine Markers

The term "uridine marker", as used herein, relates to uridine and other compounds that act as surrogates for uridine, for example: uridine and derivatives of uridine; metabolic products that result from the metabolism of uridine or derivatives of uridine; metabolic precursors that are metabolised to form uridine or derivatives of uridine; enzymes that act upon uridine or a derivative of uridine; enzymes that produce uridine or a derivative of uridine; etc.

Examples of "derivatives of uridine" include, but are not limited to, ethers (e.g., C_1-4aikyl ethers, e.g., methyl esters); carboxylic acid esters (e.g., C_1-4carboxylic acid esters, e.g., acetic acid esters); phosphoric acid esters (e.g., phosphates), including, for example, mono-phosphates (e.g., UMP)₁ di-phosphates (e.g., UDP), and triphosphates (e.g., UTP); sugar phosphoric acid esters, including, for example, UDP-glucose, UDP-galactose, UDP-glucoronic acid; and salts thereof.

In one embodiment, the uridine marker is uridine or a derivative of uridine; a metabolic product that results from the metabolism of uridine or a derivative of uridine; a metabolic precursor that is metabolised to form uridine or a derivative of uridine; an enzyme that acts upon uridine or a derivative of uridine; or an enzyme that produces uridine or a derivative of uridine.

In one embodiment, the uridine marker is an enzyme that acts upon uridine or a derivative of uridine; or an enzyme that produces uridine or a derivative of uridine.

In one embodiment, the uridine marker is a metabolic product that results from the metabolism of uridine or a derivative of uridine; or a metabolic precursor that is metabolised to form uridine or a derivative of uridine.

In one embodiment, the uridine marker is uridine or a derivative of uridine.

In one embodiment, the uridine marker is uridine; an ether, carboxylic acid ester, phosphoric acid ester, or sugar phosphoric acid ester of uridine; or a salt thereof.

In one embodiment, the uridine marker is uridine; or an ether, carboxylic acid ester, or phosphoric acid ester of uridine; or a salt thereof.

In one embodiment, the uridine marker is uridine. Human Male Subjects

The human male subject may be of any age, e.g., 15 to 70; 15 to 50; 18 to 70; 18 to 50.

Fertility Status

The human male subject has a fertility status of "fertile" or "infertile".

In one embodiment, fertile/fertility and infertile/infertility correspond to fertile/fertility and infertile/infertility as determined using the World Health Organisation (WHO) recommendations for sperm analysis.

Seminal Fluid Samples

For the avoidance of doubt, the seminal fluid sample is ex vivo, that is, the seminal fluid sample is not inside a human or animal body, for example, is not inside the body of the human male subject.

The seminal fluid sample is, or is derived from, seminal fluid.

In one embodiment, the seminal fluid sample is, or is derived from, seminal fluid plasma.

Presence or Absence of a Uridine Marker

Some aspects of the present invention rely upon the presence or absence of a uridine marker in a seminal fluid sample.

In one embodiment, presence of a uridine marker in a seminal fluid sample is presence at more than a de minimus level, for example, at or above a detection level.

In one embodiment, absence of a uridine marker in a seminal fluid sample is absence or presence at or below a de minimus level, for example, below a detection level.

In one embodiment, the detection level is actual detection level of the method or device used to detect and/or quantify the uridine marker.

Measure of a Uridine Marker

One aspect of the present invention pertains to a method of assessing fertility status of a human male subject comprising a step of determining a measure of a uridine marker in a seminal fluid sample from said human male subject. In one embodiment, the measure of a uridine marker is a concentration of a uridine marker (e.g., in the seminal fluid sample).

In one embodiment, the measure of a uridine marker is an amount of a uridine marker (e.g., in the seminal fluid sample).

In one embodiment, the concentration and/or amount is determined using an assay, for example, a conventional bench assay, for example, an assay employing chemical reagents and/or relying on chemical reactions.

In one embodiment, the concentration and/or amount is determined using a spectroscopic or spectrometric method.

In one embodiment, the measure of a uridine marker is a spectroscopic or spectrometric signal of, or associated with, the uridine marker. (For example, the spectroscopic or spectrometric signal may act as a surrogate for concentration and/or amount.)

Methods have been developed to specifically quantify nucleosides and nucleobases in complex mixtures (see, for example, Gao et al., 2007; Frycak et al., 2002).

Nuclear Magnetic Resonance (NMR) Spectroscopy

In one embodiment, the measure of a uridine marker is a nuclear magnetic resonance (NMR) signal of, or associated with, the uridine marker.

In one embodiment, the measure of a uridine marker is a nuclear magnetic resonance (NMR) signal at a chemical shift of, or associated with, the uridine marker.

In one embodiment, the nuclear magnetic resonance (NMR) signal is, or is related to, signal intensity (e.g., peak height).

In one embodiment, the nuclear magnetic resonance (NMR) signal is, or is related to, integrated signal intensity (e.g., peak area).

The chemical shifts and splitting patterns of uridine markers (e.g., uridine) are known from the literature and from databases of NMR spectra of authentic material.

Examples of ¹H NMR chemical shifts that are suitable for the detection and/or quantification of uridine include: the chemical shifts of the resonances from one or both of the two olefinic protons of uridine, for example, at or near 7.87 and 5.89 ppm, and showing mutual spin coupling of or near 8.07 Hz; and the chemical shift of the resonance from the anomeric proton of the ribose ring of uridine, for example, at or near 5.92 ppm, and showing a spin coupling to the ribose-2 proton of or near 4.04 Hz.

Mass Spectrometry (MS)

In one embodiment, the measure of a uridine marker is a mass spectrometry (MS) signal of, or associated with, the uridine marker.

In one embodiment, the mass spectrometry (MS) signal is, or is related to, signal intensity (e.g., peak height).

In one embodiment, the mass spectrometry (MS) signal, or is related to, integrated signal intensity (e.g., peak area).

The mass spectrometry patterns of uridine markers (e.g., uridine) are known from the literature and from databases of mass spectra of authentic material.

The molecular formula of uridine is C₉H₁₂N₂O₆ and so it has a nominal molecular weight of 244. Uridine may therefore be detected, for example, at m/z 243 (in negative ion mode) or at m/z 245 (in positive ion mode). It may also be possible to detect uridine via the sodium adduct (Uridine+Na⁺) at m/z 267 or the potassium adduct (Uridine+K⁺) at m/z 283.

Methods Employing Controls

One aspect of the present invention pertains to a method of assessing fertility status of a human male subject comprising a step of determining a measure of a uridine marker in a seminal fluid sample from said human male subject, the method further comprising a step of comparing said measure of a uridine marker in said seminal fluid sample from said human male subject with a suitable control measure of said uridine marker.

The "suitable control measure of said uridine marker" is of the same data type (e.g., NMR peak intensity) as the "measure of a uridine marker in a seminal fluid sample from said human male subject".

In one embodiment, said suitable control measure of said uridine marker is a measure of said uridine marker in a seminal fluid sample from a suitable control male of known fertility status, or an average of a measure of said uridine marker in seminal fluid samples from a population of suitable control males of same known fertility status. A suitable control male is a male subject that is matched, or closely matched, with the human male subject according to one or more of the following criteria: age, health, medical history, race, lifestyle (e.g., non-smoker), etc.

A population of suitable control males relates to a group of one or more suitable control males, preferably two or more suitable control males.

In one embodiment, said suitable control male of known fertility status is a suitable fertile control male, and said population of suitable control males of same known fertility status is a population of suitable fertile control males.

Thus, in one embodiment, said suitable control measure of said uridine marker is a measure of said uridine marker in a seminal fluid sample from a suitable fertile control male, or an average of a measure of said uridine marker in seminal fluid samples from a population of suitable fertile control males.

In one embodiment, the method further comprises a step of correlating a reduced measure of said uridine marker in said seminal fluid sample from said human male subject, as compared to said suitable control measure of said uridine marker, with infertility.

In one embodiment, said reduced measure of said uridine marker, as compared to said suitable control measure of said uridine marker, is indicated by a ratio of said measure of said uridine marker to said suitable control measure of said uridine marker that is below a threshold value for fertility.

In one embodiment, the method further comprises a step of correlating a similar measure of said uridine marker in said seminal fluid sample from said human male subject, as compared to said suitable control measure of said uridine marker, with fertility.

In one embodiment, said similar measure of said uridine marker, as compared to said suitable control measure of said uridine marker, is indicated by a ratio of said measure of said uridine marker to said suitable control measure of said uridine marker that is above a^ threshold value for fertility.

In one embodiment, the threshold value for fertility is 0.5. In one embodiment, the threshold value for fertility is 0.4. In one embodiment, the threshold value for fertility is 0.3. In one embodiment, the threshold value for fertility is 0.2. In one embodiment, the threshold value for fertility is 0.1. In one embodiment, the threshold value for fertility is 0.05. In one embodiment, said suitable control male of known fertility status is a suitable infertile control male, and said population of suitable control males of same known fertility status is a population of suitable infertile control males.

Thus, in one embodiment, said suitable control measure of said uridine marker is a measure of said uridine marker in a seminal fluid sample from a suitable infertile control male, or an average of a measure of said uridine marker in seminal fluid samples from a population of suitable infertile control males.

In one embodiment, the method further comprises a step of correlating an increased measure of said uridine marker in said seminal fluid sample from said human male subject, as compared to said suitable control measure of said uridine marker, with fertility.

In one embodiment, the method further comprises a step of correlating a similar measure of said uridine marker in said seminal fluid sample from said human male subject, as compared to said suitable control measure of said uridine marker, with infertility.

Methods of Assessing Fertility Status

One aspect of the present invention pertains to a method of assessing fertility status of a human male subject comprising a step of comparing a measure of a uridine marker in a seminal fluid sample from said human male subject with a suitable control measure of said uridine marker, wherein said suitable control measure of said uridine marker is a measure of said uridine marker in a seminal fluid sample from a suitable control male of known fertility status, or an average of a measure of said uridine marker in seminal fluid samples from a population of suitable control males of same known fertility status.

Methods of Diagnosis of Infertility

One aspect of the present invention pertains to a method of diagnosis of infertility in a human male subject comprising a step of correlating a reduced measure of a uridine marker in a seminal fluid sample from said human male subject, as compared to a suitable control measure of said uridine marker, with infertility, wherein said suitable control measure of said uridine marker is a measure of said uridine marker in a seminal fluid sample from a suitable fertile control male, or an average of a measure of said uridine marker in seminal fluid samples from a population of suitable fertile control males.

In one embodiment, the threshold value for fertility is 0.5. In one embodiment, the threshold value for fertility is 0.4. In one embodiment, the threshold value for fertility is 0.3. In one embodiment, the threshold value for fertility is 0.2. In one embodiment, the threshold value for fertility is 0.1. In one embodiment, the threshold value for fertility is 0.05.

Methods of Determination of Fertility

One aspect of the present invention pertains to a method of determination of fertility in a human male subject comprising a step of correlating a similar measure of a uridine marker in a seminal fluid sample from said human male subject, as compared to a suitable control measure of said uridine marker, with fertility, wherein said suitable control measure of said uridine marker is a measure of said uridine marker in a seminal fluid sample from a suitable fertile control male, or an average of a measure of said uridine marker in seminal fluid samples from a population of suitable fertile control males.

In one embodiment, said similar measure of said uridine marker, as compared to said suitable control measure of said uridine marker, is indicated by a ratio of said measure of said uridine marker to said suitable control measure of said uridine marker that is at or above a threshold value for fertility.

Subjects with Spinal Cord Injury (SCI)

In one embodiment, the human male subject has or had a spinal cord injury.

In this context, a spinal cord injury (SCI) is functional damage or trauma to the spinal cord that results in a loss or impaired function of the musculoskeletal system (sensory and motor), the genitourinary system, and/or the gastrointestinal system. Common causes of damage are trauma (road traffic accident, gunshot, falls, sports injuries, etc.) or disease (Transverse Myelitis, Polio, Spina Bifida, Friedreich's Ataxia, etc.). The spinal cord does not have to be physically severed in order for a loss of functioning to occur. In most people with SCI₁ the spinal cord is intact, but the cellular damage to it results in a loss of functioning. It is possible for a person to have vertebral fracture without spinal damage and still have varying degrees of spinal dysfunction. Usually the dysfunction is at or below the level of injury, which is designated as a vertebral level such as T 10 (i.e., thoracic 10 vertebrae). This assessment is based on the American Spinal Injury Association (ASIA) protocol of clinical testing. The "duration of injury" is the time since injury.

In one embodiment, the human male subject has or had a spinal cord injury at or below thoracic 10 vertebrae.

In one embodiment, the human male subject has or had a spinal cord injury for a duration of injury of at least six months.

Reporting

In one embodiment, the method (e.g. method of assessing fertility status; method of diagnosis of infertility; method of determination of fertility) further comprises a subsequent step of reporting the result (e.g., reporting a determined fertility status; reporting a diagnosis of infertility; reporting a determination of fertility), for example, to a supervising medical technician, to the human male subject, etc.

The step of reporting may be done directly (e.g., face to face) or otherwise (e.g., by post; by email; by text message; by accessing an internet website; etc.).

Automation

In one embodiment, a part of, or all of, the method (e.g. method of assessing fertility status; method of diagnosis of infertility; method of determination of fertility) is automated.

For example, a suitable computer may be operationally linked to a suitable device (e.g., an NMR machine, a MS machine, etc.) for determining the presence or absence of, or determining a measure of, a uridine marker, that is configured to receive, optionally process, and analyse a seminal fluid sample from the human male subject. Use in Other Methods

The method (e.g. method of assessing fertility status; method of diagnosis of infertility; method of determination of fertility) may form part of a larger method, for example, a method of screening a population of human male subjects, e.g., for fertility status.

Thus, one aspect of the present invention pertains to a method of screening a population of human male subjects on the basis of fertility status that includes a method (e.g., a method of assessing fertility status; a method of diagnosis of infertility; a method of determination of fertility) as described herein.

Kits

One aspect of the present invention pertains to a kit of parts comprising: (a) means (e.g., an assay device) for determining the presence or absence of, or determining a measure of, a uridine marker; and (b) instructions for using said means in accordance with a method (e.g., a method of assessing fertility status; a method of diagnosis of infertility; a method of determination of fertility) as described herein.

EXAMPLES

The following examples are provided solely to illustrate the present invention and are not intended to limit the scope of the invention, as described herein.

Methods

This study was performed as part of a wider fertility study at the Spinal Research Centre at the Royal National Orthopaedic Hospital (RNOH) in Stanmore, and with full approval of the Joint Research and Ethical Committee of the RNOH Trust.

Male patients, aged 18 to 50, with a spinal cord injury at or above the level of the tenth thoracic vertebra with a duration of injury of greater than 6 months were recruited for the study. Urine, plasma, and semen (obtained via vibro-ejaculation) from spinal cord injury (SCI) patients were collected from 11 men with SCI above T10 of 6 months or more duration, and from 9 age-matched healthy fertile volunteers. (In cases of SCI injuries below this T10, semen collection would require electro-ejaculation because vibro-ejaculation is unsuccessful in majority of the patients.) (This approach ensures an acceptable success with vibro-ejaculation, since the majority of SCI men are out of spinal shock within the first 6 months after injury.) Corresponding samples were obtained from a healthy control group of males aged 18 to 50 with proven fertility_, status (i.e. males who had fathered a child before the study date by natural conception).

Sperm characteristics were noted in a part of each semen sample using World Health Organisation (WHO) criteria.

Assessment of sperm quality was carried out using the World Health Organisation (WHO) recommendations for sperm analysis. Part of the semen was snap-frozen in liquid nitrogen within 5 minutes of collection using vibro-ejaculation and stored for later NMR analysis.

NMR analysis was carried out using Brucker instruments with 600 MHz and 800 MHz field strength, which were preferred for higher sensitivity and resolution.

Urine, plasma, and remaining semen samples were analysed using 600 MHz and 800 MHz ¹H NMR spectroscopy. For all samples, a water-suppressed 1 D spectrum was obtained, and for plasma and semen samples, additional spectra edited on the basis of r₂-relaxation times (showing predominantly low molecular weight metabolites) and diffusion coefficients (showing mainly high molecular weight metabolites) were acquired. The data were analysed using advanced chemometric techniques, described in more detail below.

Results

Significant differences were seen in the ¹H NMR-visible metabolite profiles of all biofluids from the SCI patients when compared to the control group.

Urine from SCI patients had lower levels of W-methyl nicotinic acid, while in plasma, altered distributions of low density and very low-density lipoproteins, increased acetyl signals from glycoproteins, and decreased glucose were observed.

Significant differences were seen in the metabolic profiles of the semen samples, including the absence of uridine in all but one of the SCI patients, altered amino acid distribution, and increased signals from acetyl groups of glycoproteins.

Corresponding significant differences were observed in the sperm characteristics such as motile sperms (23.9% and 87%), forward progression (34% and 79.4%), normal sperm morphology (21.5% and 62.7%) in SCI men and healthy fertile volunteers respectively. Also, different types of coagulation and liquefaction abnormalities were indicated. Decreased prostate derived enzyme activity, high protein/peptide content, and reversal of the normal prostate and seminal vesicle contribution ratio were also prominent.

Chemometric Analysis

Seminal fluid poses several challenges for successful metabolic profiling. Variations in the salt content, metabolic cation content, and pH can potentially introduce peak position variation across samples. Small total sample volume, especially for patients with spinal cord injuries, also imposes limitations.

In this study, optimised sample preparation methods and optimised NMR parameters have been determined for the metabolic profiling of seminal fluid.

Seminal fluid is a complex and dynamic mixture of proteins, peptides, amino acids, sugars, and other small molecules and metabolites. Several pulse sequences are available to observe molecules with different physio-chemical properties.

A standard 1D NMR pulse program that can be used to observe all molecules simultaneously is the well-established 1 D version of the NOESY pulse sequence that has been widely applied to metabolic profiling of biofluids. Usually the 1 D pulse sequence for plasma or urine would be centred at the water resonance and acquired to a resolution of 64k data points with a spectral width of 20 ppm. However, seminal fluid contains many proteins with broad resonances such that even at 20 ppm the parts of the spectrum furthest from the water resonance are not pure noise. This makes base-lining difficult, especially automated base-lining. Also, in the past there have been limitations on the number of data points collected in NMR spectra because of storage space on computer disks; now it is possibly to collect 128k resolution spectra, store them, and perform pattern recognition methods on them.

1 D NMR spectra were recorded for a seminal fluid sample at several different sweep widths (SWs). With SW = 20 ppm, the spectra do not appear to reach 0 at the parts of the spectrum furthest from the centre. As the SW is increased, baseline noise becomes apparent. At higher values of SW (up to 40 ppm) the spectra become increasingly noisy, as the FID is acquiring more noise. It was therefore concluded that for seminal fluid the following parameters are recommended: SW = 30 ppm and TD = 128k.

Temperature. Most biofluids are analysed in metabonomic publications at a temperature of 300 K. Differences between 1 D NMR spectra recorded at 300K and 310K were examined. Differences were seen in many parts of the spectra where resonances had changed chemical shifts and intensities. In one part of the spectrum (near δ 5.8), a large, broad resonance seen at 300 K was seen to be significantly reduced and revealed a doublet. A comprehensive assignment of metabolic resonances in has been published (see, e.g., Lynch et al. 1994) for seminal fluid at "ambient probe temperature", defined as 292 K in another publication by the same group (Nicholson et al., 1995). It was therefore concluded that for seminal fluid, it is recommended that the spectra be recorded at 310 K.

CPMG. The Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence is ideal for selectively observing smaller molecules in a biofluid with a high dynamic range of molecular sizes, such as plasma or seminal fluid. With d20 = 400 μs, the broad protein resonances are significantly attenuated when L1 is increased from 1 to 128. If d20 is increased to 1000 μs and L1 to 304, even more attenuation is seen, but the signal-to-noise ratio is significantly decreased as well. Therefore, a trade-off must be made between broad peak attenuation and signal-to-noise ratio. It was therefore concluded that for seminal fluid the following parameters are recommended: L1 = 128 and D20 = 400 μs (which gives 2nτ = 102.4 ms).

Diffusion Editing. Diffusion-edited pulse sequences can be used to selectively observe larger molecules such as proteins and macromolecular complexes in seminal fluid. Using a diffusion time (called "big delta") of 100 ms and diffusion pulse length time (called "little delta") of 5 ms, it was found that 75% gradient strength was ideal to suppress sharp peaks from smaller, rapidly diffusion molecules.

Effects of Dilution. Seminal fluid is viscous. Increased viscosity can increase transverse relaxation times, thereby broadening peaks. The sample was originally diluted 2:1 in a 50 mg/mL D₂O solution. This was then split and diluted again 1 :2 in either 30:70 D₂O:H₂O solution or in 0.2 M phosphate buffer containing TSP (1 mM) and sodium azide (0.1% (v/v), pH = 7.4.

It was observed that dilution of the sample in 30:70 D₂O:H₂O decreased the linewidth of many peaks, probably because of the decrease in viscosity leading to an increase in T₂ relaxation. When the same sample was diluted in 0.2 M phosphate buffer, many chemical shifts had changed, and there was a very large increase in pulse lengths (from 9.25 μs up to 15 μs), and shimming was difficult.

Seminal fluid is renowned for its high buffering capacity, and has a pH of approximately 7.7 (see, e.g., Owen et al., 2005). Thus for metabonomic studies, it is recommended to dilute seminal fluid 1 in 4 using 30:70 D₂O:H₂O supplemented with 10 mg/mL TSP. The pH should be checked and adjusted to 7.4 +/- 0.1 using concentrated HCI or NaOH if necessary. Effect of a freeze-thaw cycle. In many studies, samples are collected, delivered, and analysed at different times. Sometimes interesting samples are re-analysed using 2D methods and the like in order to assign unknown peaks. Therefore, it is important to bear in mind the effect of a freeze-thaw cycle on seminal fluid. It has previously been reported that the effect of freeze-thaw cycle is minimal with respect to the ¹H NMR observable metabolites in seminal fluid (see, e.g., Averna et al., 2005).

In the study described herein, spectra recorded for the same sample after one freeze- thaw cycle showed almost no visible differences between the spectra, in agreement with previous findings.

Effect of addition of TSP. In order to investigate the effect of adding TSP as a chemical shift reference, a ¹H NMR spectrum was acquired from a seminal fluid sample, which then had a small amount of TSP in D₂O added to it, and another spectrum acquired. Minor differences were observed in the ¹H NMR spectrum after addition of TSP; however, there were no major changes in terms of the disappearance of peaks or induced changes in chemical shift. TSP also helps with frequency-dependent phase correction for these spectra. Therefore, it was concluded that for seminal fluid, a small amount of TSP should be added.

Pattern Recognition. Each sample was thawed at room temperature for 48 hours (to allow all enzymatic reactions to come to equilibrium), then diluted 2:1 in a solution of 50 mg/mL TSP in D₂O. After centrifuging for 5 minutes at 1600Og, 300 μL of supernatant was diluted 1 :3 in D₂O and centrifuged at 1600Og for 5 minutes. 550 μL was transferred to a 5 mm NMR tube for analysis.

All experiments were performed using a Bruker NMR spectrometer equipped with a cryoprobe operating at 800 MHz.

Three experiments were performed on each sample: (1 ) 1 D (first increment of a 2D NOESY pulse sequence). Mixing time = 100 ms; relaxation delay = 2 s; time domain = 128k; number of scans = 16.

(2) CPMG. Total echo time 2τn = 409.6 ms (number of loops = 256, T = 800 μs); number of scans = 64.

(3) Diffusion edited. Spectral editing on the basis of diffusion coefficient was achieved using a pulse sequence employing bipolar pulse pair sine-shaped gradient pulses and a longitudinal eddy-current delay. "Big delta" = 75 ms; "little delta" = 5 ms; gradient strength = 75% of 53 G/cm; number of scans = 64.

In all experiments, continuous wave irradiation was applied at the frequency of the water resonance during the relaxation delay (and the mixing time for the 1 D spectra). Results from 1D (NOESY) Spectra.

¹H NMR spectra were recorded for seminal fluid samples from infertile male patients (that had suffered spinal cord injury) and fertile male controls. It was noted that there are many peaks in each spectrum, and that these peaks are of varying multiplicities, and that many peaks overlap. Analysis of complex spectra such as these is greatly facilitated by chemometric techniques such as PCA and O-PLS-DA.

PCA. Principal components analysis (PCA), on mean centred data, revealed few systematic trends in the data, mostly due to the dominant glycerol peak at 3.21 ppm.

O-PLS-DA. An O-PLS-DA model was constructed from the ¹H NMR data from the seminal fluid samples in order to identify metabolites that are significant in the discrimination of the groups. The analysis determined O-PLS coefficients for the model (after computing 3 orthogonal components, Q²Yhat = 0.64) as a function of chemical shift. Variables (metabolites) that are significant in the discrimination of the groups were identified. Some specific regions of the plot that were significant in discriminating are summarised in the following table.

Results from CPMG Spectra.

PCA. Principal components analysis (PCA) revealed no systematic variation in the spectra. O-PLS-DA. An O-PLS-DA model was constructed from the CPMG data from the seminal fluid samples in order to identify metabolites that are significant in the discrimination of the groups. The analysis determined O-PLS coefficients for the model as a function of chemical shift. Variables (metabolites) that are significant in the discrimination of the groups were identified. Some specific regions of the plot that were significant in discriminating are summarised in the following table.

Uridine, in particular, was identified as very significant in the discrimination of the groups.

The foregoing has described the principles, preferred embodiments, and modes of operation of the present invention. However, the invention should not be construed as limited to the particular embodiments discussed. Instead, the above-described embodiments should be regarded as illustrative rather than restrictive, and it should be appreciated that variations may be made in those embodiments by workers skilled in the art without departing from the scope of the present invention.

REFERENCES

A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. Each of these references is incorporated herein by reference in its entirety into the present disclosure, to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

Averna, T.A., et al., 2005, "A decrease in H-1 nuclear magnetic resonance spectroscopically determined citrate in human seminal fluid accompanies the development of prostate adenocarcinoma", J. Urology. Vol. 173, No. 2., pp. 433-438. Biering-Sorensen, F., et al., 2001 , "Sexual function in spinal cord lesioned men,"

Spinal Cord, Vol. 39, No. 9, pp. 455-470. Brackett, N. L., et al., 1996, "Seminal plasma of spinal cord injured men inhibits sperm motility of normal men," J. Urol.. Vol. 155, No. 5, pp. 1632-1635. Brackett, N. L., et al., 2000, "Sperm motility from the vas deferens of spinal cord injured men is higher than from the ejaculate," J. Urol., Vol. 164 (3 Pt 1), pp. 712-715. Frycak. P., et al., 2002, "Atmospheric pressure ionization mass spectrometry of purine and pyrimidine markers of inherited metabolic disorders", J. Mass Spec, Vol. 37,

No. 12, pp. 1242-1248. Gao, J. L, et al., 2007, "Qualitative and quantitative analyses of nucleosides and nucleobases in Ganoderma spp. by HPLC-DAD-MS", J. Pharrn. Biomed. Anal., in press (available online from 20 March 2007). Hamid, R., et al., 2006, "Effects of repeated ejaculations on semen characteristics following spinal cord injury", Spinal Cord, Vol. 44, No. 6, p. 369-373. Lynch, MJ. , et al., 1994, "Ultra high field NMR spectroscopic studies on human seminal fluid, seminal vesicle and prostatic secretions," J. Pharm. Biomed. Anal., Vol. 12,

No. 1 , pp. 5-19. Nicholson, J. K., et al., 1995, "750-MHz H-1 and H-1-C-13 NMR-Spectroscopy of Human

Blood-Plasma", Anal. Chem.. Vol. 67, No. 5, pp. 793-811. Owen, D.H., et al., 2005, "A review of the physical and chemical properties of human semen and the formulation of a semen simulant", J. Androloαv. Vol. 26, No. 4, pp. 459-469. Rutkowski, S. B., et al., 1999, "A comprehensive approach to the management of male infertility following spinal cord injury." Spinal Cord. Vol. 37, No. 7, pp. 508-514. Sonksen et al., 1992, "Fertility in Men with Spinal-Cord or Cauda-Equina Lesions",

Seminars in Neurology. Vol. 12, No. 2., pp. 106-114.

Stover, S. L., et al., 1987, "The epidemiology and economics of spinal cord injury," Paraplegia. Vol. 25, No. 3, pp. 225-228.

Tomlins, A.M., et al., 1998, "High resolution 1 H NMR spectroscopic studies on dynamic biochemical processes in incubated human seminal fluid samples," Biochim.

Biophvs. Acta. Vol. 1379, No. 3, pp. 367-380.

Claims

1. A method of assessing fertility status of a human male subject comprising a step of determining a measure of a uridine marker in a seminal fluid sample from said human male subject.

2. A method according to claim 1 , wherein said method further comprises a step of comparing said, measure of a uridine marker in said seminal fluid sample from said human male subject with a suitable control measure of said uridine marker.

3. A method according to claim 2, wherein said suitable control measure of said uridine marker is a measure of said uridine marker in a seminal fluid sample from a suitable control male of known fertility status, or an average of a measure of said uridine marker in seminal fluid samples from a population of suitable control males of same known fertility status.

4. A method according to claim 2, wherein said suitable control measure of said uridine marker is a measure of said uridine marker in a seminal fluid sample from a suitable fertile control male, or an average of a measure of said uridine marker in seminal fluid samples from a population of suitable fertile control males.

5. A method according to claim 4, further comprising a step of correlating a reduced measure of said uridine marker in said seminal fluid sample from said human male subject, as compared to said suitable control measure of said uridine marker, with infertility.

6. A method according to claim 5, wherein said reduced measure of said uridine marker, as compared to said suitable control measure of said uridine marker, is indicated by a ratio of said measure of said uridine marker to said suitable control measure of said uridine marker that is below a threshold value for fertility.

7. A method according to claim 4, further comprising a step of correlating a similar measure of said uridine marker in said seminal fluid sample from said human male subject, as compared to said suitable control measure of said uridine marker, with fertility.

8. A method according to claim 7, wherein said similar measure of said uridine marker, as compared to said suitable control measure of said uridine marker, is indicated by a ratio of said measure of said uridine marker to said suitable control measure of said uridine marker that is at or above a threshold value for fertility.

9. A method according to claim 2, wherein said suitable control measure of said uridine marker is a measure of said uridine marker in a seminal fluid sample from a suitable infertile control male, or an average of a measure of said uridine marker in seminal fluid samples from a population of suitable infertile control males.

10. A method according to claim 9, further comprising a step of correlating an increased measure of said uridine marker in said seminal fluid sample from said human male subject, as compared to said suitable control measure of said uridine marker, with fertility.

11. A method according to claim 9, further comprising a step of correlating a similar measure of said uridine marker in said seminal fluid sample from said human male subject, as compared to said suitable control measure of said uridine marker, with infertility.

12. A method of assessing fertility status of a human male subject comprising a step of comparing a measure of a uridine marker in a seminal fluid sample from said human male subject with a suitable control measure of said uridine marker, wherein said suitable control measure of said uridine marker is a measure of said uridine marker in a seminal fluid sample from a suitable control male of known fertility status, or an average of a measure of said uridine marker in seminal fluid samples from a population of suitable control males of same known fertility status.

13. A method of diagnosis of infertility in a human male subject comprising a step of correlating a reduced measure of a uridine marker in a seminal fluid sample from said human male subject, as compared to a suitable control measure of said uridine marker, with infertility, wherein said suitable control measure of said uridine marker is a measure of said uridine marker in a seminal fluid sample from a suitable fertile control male, or an average of a measure of said uridine marker in seminal fluid samples from a population of suitable fertile control males.

14. A method according to claim 13, wherein said reduced measure of said uridine marker, as compared to said suitable control measure of said uridine marker, is indicated by a ratio of said measure of said uridine marker to said suitable control measure of said uridine marker that is below a threshold value for fertility.

15. A method of determination of fertility in a human male subject comprising a step of correlating a similar measure of a uridine marker in a seminal fluid sample from said human male subject, as compared to a suitable control measure of said uridine marker, with fertility, wherein said suitable control measure of said uridine marker is a measure of said uridine marker in a seminal fluid sample from a suitable fertile control male, or an average of a measure of said uridine marker in seminal fluid samples from a population of suitable fertile control males.

16. A method according to claim 15, wherein said similar measure of said uridine marker, as compared to said suitable control measure of said uridine marker, is indicated by a ratio of said measure of said uridine marker to said suitable control measure of said uridine marker that is at or above a threshold value for fertility.

17. A method according to any one of claims 1 to 16, wherein said measure of said uridine marker or each of said measures of said uridine marker is concentration of said uridine marker.

18. A method according to any one of claims 1 to 16, wherein said measure of said uridine marker or each of said measures of said uridine marker is amount of said uridine marker.

19. A method according to any one of claims 1 to 16, wherein said measure of said uridine marker or each of said measures of said uridine marker is a spectroscopic or spectrometric signal of, or associated with, said uridine marker.

20. A method according to any one of claims 1 to 16, wherein said measure of said uridine marker or each of said measures of said uridine marker is a nuclear magnetic resonance (NMR) signal of, or associated with, said uridine marker.

21. A method according to any one of claims 1 to 16, wherein said measure of said uridine marker or each of said measures of said uridine marker is a nuclear magnetic resonance (NMR) signal at a chemical shift of, or associated with, the uridine marker.

22. A method according to any one of claims 1 to 16, wherein said measure of said uridine marker or each of said measures of said uridine marker is a nuclear magnetic resonance (NMR) signal at a chemical shift of, or associated with, the resonances from one or both of the two olefinic protons of uridine (for example, at or near 7.87 and 5.89 ppm).

23. A method according to any one of claims 1 to 16, wherein said measure of said uridine marker or each of said measures of said uridine marker is a nuclear magnetic resonance (NMR) signal at a chemical shift of, or associated with, the anomeric proton of the ribose ring of uridine (for example, at or near 5.92 ppm).

24. A method according to any one of claims 20 to 23, wherein said nuclear magnetic resonance (NMR) signal is, or is related to, signal intensity (e.g., peak height).

25. A method according to any one of claims 20 to 23, wherein said nuclear magnetic resonance (NMR) signal is, or is related to, integrated signal intensity (e.g., peak area).

26. A method according to any one of claims 1 to 16, wherein said measure of said uridine marker or each of said measures of said uridine marker is a mass spectrometry (MS) signal of, or associated with, the uridine marker.

27. A method according to any one of claims 1 to 16, wherein said measure of said uridine marker or each of said measures of said uridine marker is a mass spectrometry (MS) signal at an m/z value of, or associated with, the uridine marker.

28. A method according to any one of claims 1 to 16, wherein said measure of said uridine marker or each of said measures of said uridine marker is a mass spectrometry (MS) signal at an m/z value of, or associated with, the negative ion of uridine (e.g., m/z 243).

29. A method according to any one of claims 1 to 16, wherein said measure of said uridine marker or each of said measures of said uridine marker is a mass spectrometry (MS) signal at an m/z value of, or associated with, the positive ion of uridine (e.g., m/z 245).

30. A method according to any one of claims 26 to 29, wherein said mass spectrometry (MS) signal is, or is related to, signal intensity (e.g., peak height).

31. A method according to any one of claims 26 to 29, wherein said mass spectrometry (MS) signal, or is related to, integrated signal intensity (e.g., peak area).

32. A method of assessing fertility status of a human male subject comprising a step of determining presence or absence of a uridine marker in a seminal fluid sample from said human male subject.

33. A method according to claim 32, comprising a step of correlating presence of a uridine marker in a seminal fluid sample from said human male subject with fertility.

34. A method according to claim 32, comprising a step of correlating absence of a uridine marker in a seminal fluid sample from said human male subject with infertility.

35. A method of diagnosis of infertility in a human male subject comprising a step of correlating absence of a uridine marker in a seminal fluid sample from said human male subject with infertility.

36. A method of determination of fertility in a human male subject comprising a step of correlating presence of a uridine marker in a seminal fluid sample from said human male subject with fertility.

37. A method according to any one of claims 1 to 36, wherein said uridine marker is: uridine or a derivative of uridine; a metabolic product that results from the metabolism of uridine or a derivative of uridine; a metabolic precursor that is metabolised to form uridine or a derivative of uridine; an enzyme that acts upon uridine or a derivative of uridine; or an enzyme that produces uridine or a derivative of uridine.

38. A method according to any one of claims 1 to 36, wherein said uridine marker is an enzyme that acts upon uridine or a derivative of uridine; or an enzyme that produces uridine or a derivative of uridine.

39. A method according to any one of claims 1 to 36, wherein said uridine marker is a metabolic product that results from the metabolism of uridine or a derivative of uridine; or a metabolic precursor that is metabolised to form uridine or a derivative of uridine.

40. A method according to any one of claims 1 to 36, wherein said uridine marker is uridine or a derivative of uridine.

41. A method according to any one of claims 1 to 36, wherein said uridine marker is uridine; an ether, carboxylic acid ester, phosphoric acid ester, or sugar phosphoric acid ester of uridine; or a salt thereof.

42. A method according to any one of claims 1 to 36, wherein said uridine marker is uridine; an ether, carboxylic acid ester, or phosphoric acid ester of uridine; or a salt thereof.

43. A method according to any one of claims 1 to 36, wherein said uridine marker is uridine.

44. A method according to any one of claims 1 to 38, wherein said seminal fluid sample, or each of said seminal fluid samples, is, or is derived from, seminal fluid plasma.

45. A method according to any one of claims 1 to 39, wherein said human male subject has or had a spinal cord injury.

46. A method according to any one of claims 1 to 40, wherein said human male subject has or had a spinal cord injury at or below thoracic 10 vertebrae.

47. A method according to any one of claims 1 to 41 , wherein said human male subject has or had a spinal cord injury for a duration of injury of at least six months.

48. A method according to any one of claims 1 to 47, wherein the method further comprises a subsequent step of reporting a determined fertility status, or reporting a diagnosis of infertility, or reporting a determination of fertility.

49. A method according to any one of claims 1 to 48, wherein a part of, or all of, the method is automated.

50. A method of screening a population of human male subjects on the basis of fertility status that includes a method according to any one of claims 1 to 49.

51. A kit of parts comprising: (a) means for determining the presence or absence of, or determining a measure of, a uridine marker; and (b) instructions for using said means in accordance with a method according to any one of claims 1 to 49.