IL196368A - Methods for analyzing semen - Google Patents

Description

Methods for analyzing semen M.E.S. Medical Electronic Systems Ltd.

C. 189026 196368/2 FIELD OF THE INVENTION This invention relates to semen analysis. The present application is a divisional application from Israel Patent Application No. 158949 filed May 24, 2001. IL 158949 relates to a sampling device for use in optically analyzing a biological fluid.

BACKGROUND OF THE INVENTION According to WHO statistics, 8-10% of all married couples consult medical professionals after failing to conceive. Over 40 million couples are currently being treated for infertility. Among these infertile couples, it is estimated that the infertility in 40% of the couples is due to male originating causes, and another 20% is due to combined male and female originating causes. Semen analysis is a major technique in evaluating male originating causes.

Standard semen analysis protocol involves the determination of at least three major semen parameters: 1. total sperm concentration (TSC); 2. percentage of motile sperm; and 3. percentage of normal sperm morphologies.

For all practical purposes, semen analysis, a key factor in human male fertility medicine, has not changed since the 1930's and is still done today by microscopic inspection. In fact, it is one of the very few remaining in vitro, body fluid analysis still performed almost solely via manual methods.

This manual methodology involves carefully observing the sperm cells, counting them to determine their concentration, classifying their motility, identifying their morphology, etc. This work requires high expertise, is very labor intensive and if done according to standard protocols, takes at least an hour per test.

Manual assessments are known to be quite inaccurate due to numerous sources of error. The main sources of error are: • Subj ectivity of the observer.

• The varying criteria used in the different labs and by different observers. · The large statistical errors due to the limited number of sperm analyzed.

The WHO manual (WHO laboratory manual for the examination of human semen and sperrn-cervical mucus interaction. 4th edition, Cambridge University Press, 1999) recommends observing not less than 200 sperm and classif ing the morphology and motility of each. This itself is an error introducing procedure due to the tediousness and time consuming nature of the task. In practice, 50 to 100 sperm cells at most are analyzed. Even if the observer introduces no errors, the statistical error alone reaches tens of percentages.

As a result of the above methodology, semen analysis test results are globally recognized to be highly subjective, inaccurate and poorly reproducible. Inter lab and inter technician variations are of such proportions that this issue is of major concern in male fertility medicine and the unresolved subject of discussion in the vast majority of symposiums, congresses and conventions on the subject In order to overcome these difficulties, medical iristrumentation companies have introduced dedicated computerized systems based on image analysis (CAS A -Computer Assisted Semen Analyzers). These systems require an extremely high quality image because all their results are based on image processing. Although these systems have attempted to replace manual analysis and establish industry accepted standards, they have not succeeded in either of these objectives.

The first objective could not be achieved because analysis results continue to be dependent on manual settings and on the different makes of equipment Replacing routine manual analysis is totally impracticable because the systems are extremely expensive, complex and difficult to use. The fact is that such systems are generally not found in routine semen analysis but have rather established their niche almost solely in research centers, university hospitals and occasionally in highly specialized fertility centers.

An additional approach for semen measurements is described in US Patent Nos. 4,176,953 and 4,197,450, whose entire contents are incorporated herein. These patents describe a method for measuring sperm motility using electro-optical means and an analog signal analyzer. A suspension of sperm cells is continuously examined in a predetermined field in order to detect variations in optical density by the motion of the sperm. An amplitude-modulated analog electrical signal is generated in response to the variations, and the peaks and valleys of this signal are counted over a predetermined time period to provide an abstract parameter termed Sperm Motility Index (SMI). This parameter is related to motility and gives readings which are proportional to the number of motile cells multiplied by their respective velocity.

An automatic sperm analyzer called the Sperm Quality Analyzer (SQA), which provides the SMI parameter, has been on the market for a number of years. The analyzer is used in the following manner: a sperm specimen is taken up by a disposable chamber which has a rubber bulb at one end to aspirate the sample, and a thin measuring compartment at the other end. After aspirating the sample, the measuring compartment is inserted into the SQA and the SMI of the sample is automatically determined. The SMI parameter, although useful in some applications, was not significantly accepted by the medical community as a viable alternative to the conventional microscopic semen measurements.

It is common knowledge that in some fields of veterinary fertility analysis, total sperm concentration (TSC), is evaluated by measuring optical turbidity of the specimen. The physical principle behind this approach is that sperm cells are more opaque than the surrounding seminal plasma, and absorption of a light beam by the specimen is therefore proportional to the TSC.

For example, U.S. Patent No. 4,632,562 discloses a method of measuring sperm density by measuring the optical absorbance of a sperm containing sample and relating the absorbance output signal to the density by using at least three summing channels. The disclosed method is intended for use in artificial insemination in the cattle breeding industry, and measures the optical absorbance in the range of 400-700 urn.

This technology however, has not and could not be adopted for human use for the following reasons: (1) Human sperm concentrations in the normal range (and even in higher than normal cases), are more than an order of magnitude lower than in most of their veterinary counterparts - where this technology has been adopted. (2) Human cases are treated even when sperm concentrations are far below their normal levels. This of course is not the case for animals. Infertile animals are l o normally culled - in any case, they are not treated for infertility. (3) TSC in humans is a parameter, which in itself, is totally insufficient for fertility investigations, and microscopic analysis is in any case required for all the other data in the standard semen analysis protocol. To a large degree, this also holds for veterinary applications. This fact made optical absorption measurements superfluous, and no real effort has been invested in this field.

There is thus a need for a simple, objective technique for measuring TSC in human semen.

According to the "WHO manual, sperm motility assessment (considered by most to be the most important single semen parameter) can be carried out manually 20 using a grid system under the microscope or, alternatively, by use of CASA.

CASA provides some advantages over manual methods. However, accuracy and provision of quantitative data are totally dependent on precise semen preparation techniques and instrument settings. These factors (high expertise and sophisticated environment) along with the prohibitive cost of such instrumentation, 25 rule out for all practical purposes their application for routine semen analysis.

U.S. Patent No. 4,896,966 discloses a motility scanner for characterizing the motion of sperm, bacteria and particles in fluid. The scanner comprises an optical system including a coUimating lens, condensing lens, imaging lens and a pair of reflecting elements, a source of illumination, radiation sensing means, signal - 5 - 196368/2 processing means, and display means. The imaging lens has a useful depth of field at its object plane of at least about 0.2 mm.

U.S. Patent No. 4,857,735 discloses a spectrophotometer incorporating a pulsed light emitting diode as a source of radiation. The light emitting diode emits substantially monochromatic light thus negating the need for a separate wavelength control. The spectrophotometer may incorporate a pair of light emitting diodes for performing bichromatic spectrophotometric determinations. The light emitting diodes are pulsed with a duty cycle and pulse amplitude such that it is possible to obtain a higher amplitude pulse than the light emitting diode could sustain at a continuous voltage input level.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for measuring TSC.

It is another object of the invention to provide a system for the determination of semen parameters.

In a first aspect of the invention, there is provided a method for measuring the total sperm concentration (TSC) in a sample. The method comprises (i) placing the sample in a transparent container between a synchronically pulsed light source and a photodetector; and (ii) measuring the optical absorbance of the sample in the range of 800-1000 nm, the TSC of the sample being proportional to the absorbance.

The method of the invention provides an objective measurement of TSC which is not dependent on image analysis, and which can measure human TSC. However, the method may also be used to measure animal TSC.

In a second aspect of the invention, there is provides an optical system for measuring the total sperm concentration (TSC) in a sample comprising: (i) a synchronically pulsed light source which emits light in the range of 800-1000 nm; (ii) a synchronically pulsed photodetector; and - 6-10 - 196368/2 (iii) a transparent sample holder positioned between said light source and photodetector.

Passages of the description which are not within the scope of the claims do not constitute part of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS In order to understand the invention and to see how it may be carried out in practice, preferred embodiments will now be described, by way of non-limiting examples only, with reference to the accompanying drawings, in which: Fig. 1 is block diagram illustrating one embodiment of the method of measuring TSC according to the invention; Fig. 2 is a perspective top view of one embodiment of a sampling device according to the invention; Fig. 3 is a partial side sectional view of the device of Fig. 2; Fig. 4 is a sectional view of the separating valve rotated 90° from the view of Fig. 3; Fig. 5 is a schematic illustration of a system for semen analysis according to > one embodiment of the invention; Fig. 6 is a flow chart mustrating an algorithm for calculating the MSC; Fig. 7 is a flow chart illustrating an algorithm for calculating the average velocity; Fig. 8 illustrates a typical analog signal of motile sperm as a function of ) time; Fig. 9 is a correlation curve of the MSC with average analog signal; and Fig. 10 is a block d agram wustrating one embodiment of a video visualization system according to the invention .

DETAILED DESCRIPTION OF THE INVENTION Example 1 As stated above, the automatic optical measurement of TSC in human semen samples as opposed to animal samples has been hampered in the past due to the low concentration of sperm cells. This, together with the high background electronic and optical noise due e.g. to seminal plasma variability has prevented the application of methods routinely used in veterinary fertility analysis. The method of the present invention comes to overcome these obstacles by combining the following features: (i) the sample is placed in a transparent container between a synchronically pulsed light source and a synchronically enabled photodetector. The use of a synchronically pulsed light source and photodetector enables the distinction of sperm cells at low concentrations over electronic noise levels. (ii) measuring the optical absorbance of the sample in the range of 800-1000 urn. It has been found that measuring the absorbance in the near infrared region provides the optimal conditions for obtaining strong absorption by sperm cells and low absorption by seminal plasma. Preferably the measured range is 850-950 nm. Most preferably, the range is 880-900 nm.

By using the method of the invention, the TSC of a sample may be determined as a function of the absorbance. Although the method of the invention is preferably used with samples of human semen or human sperm, it may also be used with animal semen and animal sperm, preferably after appropriate dilution.

An example of an optical system using one embodiment of the method of the invention is illustrated in Fig. 1. The system, indicated generally by the numeral 2, comprises a light source 4, a photodetector 6 and a sample holder 8 interposed therebetween. A preferred light source may be a fast-switching synchronically pulsed light emitting diode (LED) which emits light in the near infrared region. The light source may be controlled by a light intensity controller 10 which in turn is regulated by a modulator 12. The photodetector is capable of detecting synchronically pulsed light. The photodetector transmits the measured analog signals to a demodulator 14, which is also regulated by the modulator 12, and from there to output 16 of the signal in digital form.

The beam path through the sample is preferrably vertical. The length of the beam path through the sample is generally between 5 and 15 mm, preferably 10 mm. The sample holder must be fully transparent to light waves in the near infra-red region of between 800 and 1000 nm. The plastic material from which the sample holder is made must be totally non toxic to sperm cells. A preferred material is polystyrene PG-79. The sample holder should preferably be designed to totally prevent penetration and forming of air bubbles in the sample, which interefere with the optical measurement.

By using the method of the invention, TSC detection levels down to appr. 2 million cells/ml. have been achieved. This level already indicates extreme semen pathology.

Example 2 Fig. 2 illustrates one embodiment of a sampling device 20 according to the invention, for use in measuring semen. The device comprises an anterior optical viewing section 22, a posterior aspirating section 24 and an intermediate air exclusion section 26.

The optical viewing section 22 comprises a thin measuring chamber 28 and a thick measuring chamber 30. The thin chamber is used to measure SC and/or for visualization, while the thick chamber is used to measure TSC. In this way, multiple parameters can be measured simultaneously using the same sampling device and sampling step.

The aspirating section 24 comprises a cylinder 32 and a plunger 34 slidingly inserted therein. These parts match each other and function as in a standard syringe. This section serves for the aspiration of the semen sample into the measuring chambers The air exclusion section 26 comprises a separating valve 36 for separation of the measuring chambers from the cylinder volume after filling. The aspirator, thin measuring chamber, thick measuring chamber and air exclusion section are all in fluid communication.

An adapter 38 in the form of a rectangular rail extends along one side of the device 20 and serves for the correct sHding in and aHgmng of the device upon insertion into an optical instrument by which the sample is evaluated. It also provides the mechanical support and stability required for precision electro-optical measurements.

The parts of the device may be seen more clearly in Fig. 3. The thin measuring chamber 28 is an internal cavity having an upper 40 and a lower 42 parallel transparent wall through which the optical beam may pass. The distance - 14 -between the walls is in the range of 100-500 microns, preferably 250-350 microns, most preferably approximately 300 microns, in the later case, the volume of liquid in the chamber is approximately 25 μΐ. The anterior end 44 of the chamber has an aperture through which the sample may be drawn into the device. In the illustrated embodiment, the chamber is approximately 4 mm wide.

The thin measuring chamber serves for evaluation of sperm motility and may be positioned between a light source e.g. opposite the lower wall 42 and a photodetector e.g. opposite the upper wall 40. It will be understood that the light source and photodetector may also be positioned on the opposite sides of the chamber. A Ιϊ ί beam is transmitted through the chamber containing a semen sample. The detector on the other side of the chamber registers optical density variations caused by moving sperm cells. The optical density variations are translated into an electrical signal by the photo-detector which is then routed to the electronic circuits to be filtered, digitized and processed so as to indicate the MSC. The thin measuring chamber may also be used with a video visualization system, as will be further explained below.

The thick measuring chamber 30 has an upper 46 and a lower 48 transparent wall through which an optical beam may pass. The distance between the walls is in the range of 0.5-3 cm, preferably 0.8-1.2 cm, most preferably approximately 1 cm. The approximate volume held by the thick compartment in the latter case would be approximately 0.5 ml.

This chamber serves for electro-optical absorption measurements of sperm concentration. A light beam, which may be the same or different from that of the thin chamber 28, is trarismitted through the upper and lower walls of the chamber and detected by a photo-detector. The chamber volume should be completely filled with a sperm sample in order to avoid inaccuracies due to air bubbles. The attenuation of the light beam as it passes through the chamber is proportional to the sperm concentration. The light beam intensity is measured after passing through the chamber and translated to units of TSC by electronic means. The order of the chambers in the sampling device may be exchanged.

The cylinder 32 is in fluid communication with the two measuring chambers 28 & 30, so that by drawing the plunger 34, fluid is drawn into the chambers. This method of aspiration allows large sample volumes to be aspirated into the device. In order to prevent air bubbles from remaining in the measuring chambers, a separating valve 36 is interposed between the cylinder and the measuring chambers, and is in fluid communication with them. The valve is shown in detail in Fig. 4 and comprises a piston 50 slidingiy held in a valve housing 52. A connecting bore 54 connecting between the measuring chamber 30 and the cylinder 32 passes through the piston 50.

When the valve is in the upper position, there is a connection between the measuring chambers and the aspirating cylinder. Pressing the valve down breaks that connection and ensures that no air remains in the measuring chambers where the samples are measured and no leakage will occur even when there is a temperature variation. This technique is equivalent to positive displacement since air is excluded from the measured fluid volumes (except at the anterior end 44). This design enables working with samples of virtually all viscosities, while at the same time preventing leakage and the penetration of air bubbles into the specimen volumes to be analyzed.

Although the means for excluding air from the measuring chambers has been exemplified by a separating valve, other means may also be used, such as a positive displacement pipette All parts of the device may be manufactured from any material which is not toxic to the measured cells. Preferably, the material is relatively cheap, such as plastic materials, so that the device can be disposable. An example of a polymer which may be used to produce the device is polystyrene PG79. The separating valve, cylinder and piston may be made from polypropylene. The thin measuring compartment is by far the most toxi-sensitive part of the device due to the very high area to volume ratio of the seminal liquid in that section.

In order to aspirate a sample into the device 20, the tip 44 of the thin measuring compartment 28 is dipped approximately 5 mm deep into the semen sarnple, which is then aspirated into the device past the separating valve 36. Only app. 0.6cc are required for a complete filling of the device. The separating valve is then pushed down, and the device may be inserted into an optical measuring apparatus.

Example 3 As mentioned above, detenriination of the MSC according to the invention requires the generation of a voltage signal which is proportional to the MSC. Fig. 5 shows one embodiment of a system for semen analysis capable of generating such a 10 signal.

An optical capillary 100 having a rectangular cross-section is used to hold a semen sample 102. The capillary 100 is illuminated with an incident light beam 105 produced by a light source 110. The capillary 100 has an optical path of 300 um through which the light beam 105 passes. After passing through the capillary, the scattered beam 106 is collimated by a round aperture 108 having a diameter of 70 μτη. The collimated beam 107 impinges upon a photodetector 115. The photodetector 115 produces an analog voltage signal 120 proportional to the intensity of the beam 107. The analog signal varies in time due to the motility of the sperm in the semen sample 102, as shown for example in Fig. 8. The analog signal 120 is inputted to an analog-to-digital converter 125 that samples the analog signal 120 at a rate of e.g. 8000 Hz and generates a digital output signal 128. The digital output signal may be stored in a memory 130. Sperm motion in the sample 102 leads to a modulation in the intensity of the beam 107, which in turn affects the analog signal 120 and digital signal 128.

A processor 135 is configured to carry out an analysis of data stored in the memory 130 in order to produce an analysis of the semen sample 102. The results of the analysis may be displayed on any display device such as a CRT screen 140 of a personal computer 145, or on an internal LCD screen 148 of the measuring device. - 17 - Fig. 6 shows a flow chart diagram for one embodiment of an algorithm for calculating the MSC as carried out by e.g. tfie processor 135 of Fig. 5, in accordance with the invention.

In step 200, the digital signal 128 of Fig. 5 is digitally filtered in order to remove high and low frequencies that are not relevant to the dominant frequency of the signal, which is determined by the motility characteristics of the semen sample 102. This is done in order to optirnize the signal to noise ratio. The DC component of the signal 128 is also removed. For human sperm samples, for example, the optimal relevant frequency range was found to be between 5 and 30 Hz. In step 205, digital samples having an absolute value below a first predetermined threshold, which may be deiennined empirically, are excluded. In step 210 the same threshold value is subtracted from ail remaining samples.

In step 215, a waveform selection procedure is carried out to discard all waveforms due to artifacts such as from non-relevant cells, etc. A preferred embodiment of waveform selection with human sperm is to eliminate all waveforms not satisfying the following criteria: Minimum height - 10 millivolts.

Minimum width— 37.5 milliseconds.

Maximum width - 500 milliseconds.

Minimum rise/fall time - 2.5 milliseconds.

The correct definition (and detection) of the beginning and end of sperm associated waveforms are defined as those where significant changes of waveform direction occur. The time difference between two such points defines the time width of a given wave. The manner of selection may be understood by way of example with reference to Fig. 8 (not drawn to scale), which shows the amplitude of the analog signal (120 in Fig. 5) as a function of time. The threshold 302 is determined empirically to provide optimal linearity between the output signal and the microscopically measured MSC. The waveforms that are used for the calculation of MSC are labeled 304, 305, 306 and 307. The other waveforms have - 18 -been rejected for various reasons: 308 because its peak is less than the threshold; 310 because it is too wide; and 312 because it is too narrow.

In step 220 of Fig. 6 the absolute value of all selected samples is calculated, and in step 225, the average a of the absolute values is calculated. In step 230, the MSC of the sample 102 is calculated based upon the average a. For example, it was found that the dependency of MSC on a can be described by a linear equation of the form: MSOcta where a is an empirically derived constant. In a preferred embodiment, the dependency of MSC on a may be described by a quadratic equation of the form: MSC=Aa2+Ba With reference to Fig. 9, a specific human sperm sample was analyzed in accordance with the invention. It was found that the dependency of MSC on a could be described by the following algebraic expression: MSC = 0.0047a2 + 0.869a (I) A good linear correlation was found to exist for small values of a. Using formula (I), the correlation factor (r) for fresh sperm over the entire range was > 0.98.

Analysis of treated semen samples with varying viscosity was also performed using thawed samples, washed sperm, diluted samples (both in 3% Sodium Citrate and Test Yolk buffer) as well as with samples containing up to 20% glycerol having artificially raised viscosity. It was found that varying sample viscosity (and therefore sperm velocity), did not significantly affect the correlation between MSC and average signal ("r" in all case remained above 0.96).

Using centrifugal enrichment techniques, a very wide range of motile human sperm concentrations were measured (up to 250 Mml). No significant saturation was found. The slight non-linearity at the highest ranges is easily corrected by a simple second-degree polynomial correction - given above.

Analysis of bovine semen was also carried out and correlation factors between bovine MSC and identically averaged signals (same methodology as for humans) provided similarly excellent results. It is to be noted however, that bovine semen has to be diluted prior to measurements. This is due to their MSC being typically an order of magnitude above that of human.

Example 4 As explained above, the average velocity is a function of SMI and MSC. With reference to Fig. 7, the SMI is calculated in step 235. This may be done, for example, as disclosed in U.S. Patent No. 4,176,953, or using an SQA analyzer. In step 240 the MSC is calculated by any known method. In a preferred embodiment, MSC is calculated by the algorithm of the invention (see Example 3 above). In step 245 the average velocity AV is calculated using an algebraic expression involving the ratio SMI MSC. In one embodiment AV is calculated using the algebraic expression: f SM. Λ . f SMI r SM AV = 0.001 + 0.1 .89 MSC) MSC J + 0 V MSC In step 250 the results are displayed on the display device 145 or 148 (Fig. 5).

Example 5 One embodiment of a video visualization subsystem (WS) which may be used with the analyzing system of the invention is illustrated in Fig. 10. A semen sample 300 is placed before a diffused, phase contrasted iUuniinator 305. The sample may be held in a standard laboratory slide or smear, or may be held in a sampling device according to the invention. Light from the illuminator 305 passes through the sample 300 and through a switchable dual lens system 310, preferably with amplifications of 20 and 40. The amplified light is then conveyed to a miniature CCD video camera 315. The resulting image may be displayed on a built-in internal viewing screen 320 or on external displaying means 325 such as PCs, screens, printing devices, etc. - 20 - In a preferred embodiment, the WS is built around the sampling device of the invention, and particularly the thin measuring compartment. The object of this feature is that no extra preparations will be necessary to incorporate this function to the normal testing procedure. One simply takes the semen filled device on which the automated test is performed and inserts it -as such, into the viewing port. However, the WS is not limited to use with the sampling device of the invention, and may be used with standard laboratory slides or smears.

The front end of the WS is similar to that of the microscope. Two objective lenses are selectable for optimizing magnification and field of view, according to the application (x20 or x40). However, instead of the eyepieces of the microscope, the image from the objective is conveyed to a miniature CCD video camera. The size of the CCD (diagonal) is 6mm. The viewing screen is a 100mm LCD. This provides a video amplification of app. 17. This in effect gives a potential overall amplification of 340 or 680. Although amplification factors of only 200 and 400 are required, this set up is selected so that the above amphfication could be reached in a much smaller construction. This is desirable e.g. for a compact and robust desk-top unit (decreasing the specified image distance decreases the amplification to what is required).

The lenses and their magnification set-up may be selected so that the "Working distance" (from object to lenses) can be varied to enable scanning throughout the whole depth of the thin measuring compartment (e.g. 300 microns). This is opposed to normal microscopic viewing which does not require such scanning, because the object is normally enclosed in a slide which is just 20 microns deep and the whole depth can be viewed without scanning orrefocusing.

As mentioned above, an overall amplification factor of 200 or 400 may be selected. An amplification of 400 will be the choice when it is necessary to identify non-spermic cells (white blood cells, round cells, etc.), as well as to investigate and evaluate various morphological pathologies of sperm cells (agglutinations, immature cells, sperm head or tail defects, etc.). An amplification of 200 will be preferable for cell counting - irrespective of whether they are sperm or others. The lower amplification provides a larger field of view (4 times larger) and thereby improved counting statistics. The possibility of freezing images greatly enhances both applications.

In order to facilitate cell counts and acquire a truly quantitative result using the WS, in a preferred embodiment a calibrated grid may be charted directly on the LCD viewing screen. The grid comprises 2 cm squares which are equivalent to a pre-amplification size of 0.1mm in the semen filled measuring compartment (amplification factor of 200). This approach precludes the very difficult task of precisely charting a minute grid on the measuring compartment itself. The latter expensive solution is incorporated in the Mackler Counting Chamber as well as some other hemacytometers - precluding their use as disposables. In the present invention this is unnecessary and the WS allows the grid to be a part of the viewing screen.

The WS may be useful in the following applications: (a) Measuring very low sperm concentrations. (b) Identifying foreign cells in the semen (other than sperm cells). (c) Manual morphology analysis according to any selected criteria. (d) Vasectomy efficacy validation. (e) Diagnosing Azoospermia. (f) On the spot comparison of computerized results with visual analysis. (g) Providing hard copy "Snap shots" of immobilized images of various semen layers. The immobilization is achieved by electronic freezing of the images.

Claims (8)

- - CLAIMS:

1. A method for measuring the total sperm concentration (TSC) in a sample comprising: (i) placing said sample in a transparent container between a synchronically pulsed light source and a photodetector; and (ii) measuring the optical absorbance of said sample in the range of 800-1000 nm, the TSC of the sample being proportional to said absorbance.

2. A method according to Claim 1 wherein the absorbance is measured in the range of 850-950 nm.

3. A method according to Claim 2 wherein the absorbance is measured in the range of 880-900 nm.

4. A method according to Claim 1 wherein said sample comprises human semen.

5. A method according to Claim 1 wherein said sample comprises human sperm.

6. A method according to Claim 1 wherein said sample comprises animal semen.

7. A method according to Claim 1 wherein said sample comprises animal sperm.

8. An optical system for measuring the total sperm concentration (TSC) in a sample comprising: (i) a synchronically pulsed light source which emits light in the range of 800-1000 ran,; (ii) a synchronically pulsed photodetecter; and (iii) a transparent sample holder positioned between said light source and photodetector. For the Applicants,