GB2131972A - Channelled surface and cover for determining sperm quality - Google Patents

Abstract

A device for an objective determination of the quality of sperm, by measuring the concentration, motility and vitality which are the three main parameters of estimating the potential fertility of an ejaculate consists of a rectangular optical translucent solid surface having at least two, e.g. three, channels, each channel having an accurate depth ranging between 10 to 40 microns and wideness of about 1 mm, covered by a second rectangular optical translucent flat surface. The device can be successfully utilized for the determination of motility in the sperm of human beings as well as in animals. <IMAGE>

Description

SPECIFICATION A device for determining the sperm quality The present invention relates to a device for determining the quality of cells. More particularly the invention relates to a device for determining the sperm quality and specifically its concentration, motility, vitality and morphology.

As known, among the parameters to be considered for estimating the potential fertility of an ejaculate of either human or animal origin, the concentration and total number of normally moving sperm are of high importance. It is also known that sperm cells leaving the testes are inmotile, however during transit through the epididymis they develop the capacity for progressive motility. The most common method for evaluation of sperm motility involves direct observation through the microscope. The method involves calculating the sperm cell concentration based on a single or more determinations in haemacytometer counters. In a systematic study it was found that errors in the order of about 10% were found between duplicate pipettes.

The main disadvantages of this method are: its inaccuracy due to the rapid movement of the cells at high magnification, the tedious operation involved and its subjective approach. Mean differences of more than 20% in sperm concentration are to be expected between duplicate determinations even by the same person. Improvements in accuracy were later on suggested by filling of haemocytometers by means of interferometry. As a simple procedure for determining the numbers of moving and immotile sperm, first are counted the inmotile sperm cells and subsequently the whole lot is killed by a short heating up to 60 C and counting again.

In view of the drawbacks of using haemocytometer counting as such, regarding accuracy and consumption of time in multiple counting procedure, it has been suggested to use the cinematography technique, to record motility patterns for subsequent replay at speeds slow enough for evaluation of motility of human spermatozoon. However this technique is also very time consuming. A computer system has been developed (Computers and Biomedical Research 10, 127-38, 1977) for measuring the sperm velocities and for classifying them as motile or immotile. Movies of the sperm cells (filmed through a microscope) are read into the computer using a video camera and a video compressor interface. The velocity for each cell is calculated by multiplying the average distance moved between frames by the movie frame rate.Although equipment for this method is now even available on the market, the method is rather expensive.

It is mentioned therein that the method is also successfully applied for bovine sperm evaluation.

Laser and nuclear magnetic resonance have also been suggested for determining of sperm motility parameters. The value of these approaches is that they are quick to perform if the equipment is available. However, neither of these techniques is perfected to the required state for routine clinical use at this date. In particular the laser procedure shows considerable clinical promise for the future if the equipment can be simplified into a compact unit.

Recently it has been reported on a new chamber for multiple exposure photography method for an objective sperm motility evaluation. The chamber is constructed from two pieces of optical flat glass separated by tips of three metal pins located at the periphery of the lower piece. It is claimed that the chamber is only 10 microns deep, but still the very thin space enables the analysis of an undiluted specimen. Immobilized sperms from a pre-heated specimen are counted immediately after being applied to the chamber and their number within the area of the grid gives their concentration. The chamber is used together with a special still camera microphotography technique wherein a multiple exposure is achieved by a stroboscope. The inventor is claiming that the new chamber enables to get more accurate results but the price of said chamber is relatively high.

It is an object of the present invention to provide a simple method, utilizing an unexpensive device for measuring the concentration and motility of sperm cells. It is another object of the present invention to provide an unexpensive device which enables to measure in a single sampling at least two parameters which estimate the potential fertility of an ejaculate of either human or animal origin. It is yet another object of the present invention to provide a simple method, based on an unexpensive device which enables to obtain high accuracy and precision of the parameters determined for estimating the potential fertility.

The invention consists in an improvement of the method for an objective sperm motility evaluation by counting the sperm, being characterized by the fact that the chamber for testing the sperm consists of a rectangular optical translucent solid surface having at least two channels, each channel having an accurate depth ranging between 10 to 40 microns and wideness of about 1 mm. covered by a second rectangular optical translucent solid flat surface.

In particular preferably, the depth of the channels is to be selected in a range of between 10 to 20 microns. The depth of the channel in the range of between 10 to 40 microns and preferably 10 to 20 microns enables a free linear movement of the sperm from human or animal origin.

The sperm is introduced into the calibrated channel by an applicator from one edge of the rectangular surface filling the channel. The surface of the channel is covered by a flat, rectangular optical translucent surface, having a heavy frame which will prevent spoiling of the sperm as well as removal of the cover slide. The chamber is subsequently placed on a microscope in which the ocular is already scaled in microns so that the counted area of the chamber is very accurately determined. This scaling can also be done on the chamber itself or on the rectangular cover surface.

According to a most preferred embodiment of the present invention, the chamber will contain three channels which enab [ e to determine simultaneously from one single sample, the three main parameters of estimating the potential fertility of. an ejaculate. These parameters are as follows: (a) Concentration of sperm.

(b) Percentage of motile and inmotile sperm.

(c) Vitality of the sperm.

The determination of the above three parameters from one single sample is of most important advantage considering the fact that the ejaculate is rather non-homogeneous from its nature.

Furthermore, in view of the viscosity of the ejaculate, errors in these determinations cannot be avoided.

The entire method utilizing the new device according to the present invention is very simple to be carried out and is characterized by the following further advantages: - The chamber is very unexpensive and can be easily manufactured.

- The chamber can be onetime used and disposable.

- The chamber can be used by any laboratory.

- Dilution of the sample is generally unnecessary for human ejaculate when the concentration of sperm is in the normal range.

- The chamber can be successfully utilized in research laboratories to determine the influence of various compounds on the motility of sperm (e.g. influence of caffeine, kallikrein, etc.).

- The chamber can be utilized for human ejaculates as well as for animal ones.

- The results obtained compare very favourable with those obtained by using the expensive known cells. Moreover in view of the possibility of determining several parameters in one single sample, the results are more accurate and precise, their interpretation being much more reliable than in the known cell when several samples have to be analysed for determining the various parameters.

The results can be interpretated by a computer and translated into the standard units for each case.

In case that a dilution of the sample is necessary, it is possible to repeat the determination from the same diluted sample.

The simplest method for counting the motile sperm is by a common microscope. Of course any type of microscope with various condensors and dark field can be successfully utilized, the accuracy of the counting being independent from the chamber developed according to the present invention. Since the sperm is harmed by the production of hydrogen peroxide in aerobic metabolism and under illumination, it is suggested to reduce the contact of any ejaculate with air as much as possible and to protect it from light as well.

As known the percentage of moving sperm and their mean velocity is influenced markedly by the temperature. Therefore the counting of the moving sperm should be determined at a constant temperature in a biological range as known by a person versed in the art.

In addition to the microscope any other method for determining the motile sperm such as multiple exposure microphotography using an electronic pulser or stroboscope can be also utilized, the advantages of the chamber according to the present invention being also present in these methods.

The chamber developed according to the present invention is schematically illustrated in the attached sheet wherein Figs. 1 and 2 are presented. Fig. 1 is a perspective view in which a, b and c represent the three channels in which the ejaculate for determining the three parameters is introduced. Fig. 2 is also a perspective view, in which d, e and f are the cover surfaces, which also should be optical translucent and inert towards the ejaculate. The chamber itself can be made from any solid material which is optical translucent, on which the required channels can be produced. An absolute requirement of the chamber is its inertness towards the sperm, examples of materials from which it can be produced being teflon (RTM), glass, polycarbonate etc. It can also be envisaged to utilize any solid surface covered by inert dyes.

The entire method is very simple and can be carried out even by an unskilled person. The applied sperms into the channels are uniformly distributed and monolayered and are thus observed in one focal plane, eliminating blurring. Once the sample was applied into the channel, the analysis can be started after a few minutes of equilibrium. Mobility of the observed sperm is free, frictionless and horizontal monolayered, with no vertical component, which might affect the measurement accuracy. This is contrary to the known method of motility determination. Also as known the seem is a non-homogeneous fluid, fact which further adds to the inaccuracy of the results obtained, by the prior art methods.

Since the chamber is so simple and unexpensive, it is possible to heat the area surrounding the channel (about 60 C) and thus to count directly the motile sperm. In this case the chamber should be made from a suitable resistant material to withstand the heating temperature.

The chamber according to the present invention can be also utilized in determining the fertility of animal beings. In an investigation of the hemocytometer technique applied to human semen, it was reported that 55% of the variation between duplicate determinations by the same technician were due to sampling and pipetting error and 45% to chamber and counting error.

The most encountered method for motility determination of animal ejaculates is by optical density measurements. This gives only a range of concentration of sperm in ejaculate and therfore is known to be very inaccurate. In the following Table 1 are given the data (concentration, and size) of the sperm of various animals.

Table 1: Characterization of the spermatozoa from the various animals.

Sperm concen- Sperm dimensions (in microns) ml of tration total Head dimensions The animal ejaculate (milion/ml) length length width thick- tail ness Bull 2-10 300-1000 65 9 4 1 44 Ram 0.7-2.0 2000-5000 65 9 4 1 44 Stallion 30-800 30-800 58 7 4 2 42 Boar 150-500 25-300 57 8 4 1 38 Dog 2-15 1000-9000 60 7 4 1 44 Rabbit 0.4-6 100-2000 56 9 1 1 39 Chicken 0.05-1 30-11000 100 15 4 0.5 80 Turkey 0.2-0.5 3600-13000 100 15 4 0.5 80 Human: 2-6 50-150 55 5 4 3 45 It appears from the above data that the sizes are of the same order of magnitude and thus the chamber can be utilized successfully also for motility determinations of the sperm in these animals, being much more accurate and reliable than the normal used methods.

The chamber according to the present invention, containing three channels was experimented with human ejaculate and the results of sperm concentration, motility and vitality were determined. The results are summarized in Table 2. The results obtained with the standard hemocytometer on the concentration are given for compairison.

Table 2: Comparison between the standard hemocytometer method for estimation of human sperm concentration, motility and vitality and the results obtained by the new chamber according to the present invention: The present invention hemocytometer method sperm conc.

depth (millions motility concentration motility No. (microns)* per ml) (%) millions/ml (%) 1 12.6+0.9** 35+3 11#2 28*** 15 2~ 7.3*0.7 65*8 9#1 28 15 3 13.7*0.8 39*2 43*2 28 15 4 11.1*1.0 51*6 45*4 28 15 5 7.7+0.7 70*6 34#2 34 55 6 12.6*0.9 8*1 42 + 3 7 45 7 7.3*0.7 11#1 37#3 7 45 8 13.7*0.8 7#1 # 28#2 # 7 45 Determined by the Israel Standard Institute and represent average of four measurements.

Mean f Standard error of 10 measurements.

Estimation of the same sample by the routine method.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications, and this patent is intended to cover any variation, uses or adaptations of the invention and including such departures from the present disclosure as come within known or customary practice in the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as fall within the scope of the invention. Although the invention has been in particular referred for determining the quality of spermatozoon, it is clear that it can be also applied for determining the quality of other cells wherein similar problems might be encountered.

Claims (11)

1. A device for an objective determination of the quality of sperm, which comprises a chamber possessing a rectangular optical translucent solid surface having at least two channels, each channel having an accurate depth ranging between 10 to 40 microns and wideness of about t mm, covered by a second rectangular optical translucent solid flat surface.

2. A device according to Claim 1, wherein said chamber has three channels.

3. A device according to Claim 2, wherein the depth of each channel is between 10 to 20 microns.

4. A device according to Claim 1, wherein the quality of sperm is determined according to the motility evaluation.

5. A device according to Claim 4, wherein said evaluation is determined by counting the sperm.

6. A device according to Claim 5, wherein said counting is done by a microscope.

7. A device according to Claim 5, wherein said counting is performed by multiple exposure microphotography.

8. A device according to Claim 6, wherein the ocular in said microscope is already sealed.

9. A device according to Claim 1, wherein said chamber is made from a material selected from the group consisting of glass, teflon, plycarbonate or a solid material coated by an inert dye.

10. A device according to Claim 1, wherein said sperm is of human origin.

11. A device according to Claim 1, wherein said sperm is of animal origin.