FIELD OF INVENTION

The present invention relates to a method for assessing the fertilizing potential of sperm based on a measurement of longitudinal axis rotation of the sperm in a sample obtained from a test subject.

BACKGROUND OF THE INVENTION

Unwanted childlessness is a serious problem which affects approximately 80 million couples worldwide. In about 50% of the cases, male infertility is to blame for that circumstance. However, the causes of male infertility are extremely difficult to diagnose or are diagnosed unreliably. In about 30% of patients the causes remain unsolved and are qualified as "idiopathic infertility". This unsatisfactory situation is due to the lack of suitable and modern diagnostic methods:

Fertilization of an egg cell is the result of a complex behavioral choreography that must be performed by the successful spermatozoon during its passage through the female genital tract. In order to overcome the large distances (seen from the perspective of the sperm), navigational swimming is a crucial capability: if it is disturbed, fertilization is foredoomed to fail for the respective spermatozoon.

The ability of spermatozoa to navigate across the female genital tract is, however, not assessed by current rather rudimentary diagnostic methods used to assess male fertility. Thus it is not surprising that in many cases, the cause of male infertility or even the infertility perse are not detected. To assess the male fertility, a semen analysis (spermiogram) is routinely made, in which a sperm sample is examined under a microscope. The methods used to perform semen analysis date back to the 1960s and are still routinely used today in a largely unaltered form. Those methods encompass microscopic determination of some easily measurable parameters, such as the number of sperm, the portion of motile sperm, the portion of sperm which show a progressive motility or the portion of sperm with morphological abnormalities. With this phenomenological diagnosis only easily visible disorders of the sperm production and function can be diagnosed, such as low sperm counts, abnormal morphology, or severely impaired motility of the sperm. Computer-assisted sperm analysis (CASA) is the further development of the ocular evaluation, but it largely relies on the same characteristics of sperm motility as markers of infertility. However, in many cases of male infertility, the patient suffers from an unexplained dysfunction of the sperm, i.e. the fertilization fails although classical semen analysis and CASA yield parameters within normal limits. In those cases, the diagnostic process might be concluded with the very unsatisfactory diagnosis of idiopathic infertility. In fact, male infertility due to unexplained sperm dysfunction is severely underdiagnosed: the “normal” semen analysis often leads mistakenly to the conclusion that the unwanted childlessness of the couple rests on female-related factors. Unfortunately, the lack of diagnostic elucidation, however, prevents evidence-based therapy decisions in case of unwanted childlessness. In other words, a definite diagnosis is required to select a suitable therapy for assisted reproduction such as cycle optimization (CO), intrauterine insemination (IUI), in vitro fertilization (IVF), or intracytoplasm ic sperm injection (ICSI). Many couples in which the man experiences an unexplained sperm dysfunction go through the full spectrum of fertility treatment involving all of the mentioned procedures, from CO, through IUI and IVF, to ICSI. The duration of the treatment, the psychological burden, medical risk and, not least, costs increase with each new therapeutic approach. It is therefore crucial to match the current diagnostic repertoire to the functional complexity of the spermatozoa and to provide a diagnostic method which is able to provide the diagnosis of sperm dysfunction, i.e. male infertility, where none can be made so far.

SUMMARY

In view of the above mentioned problems, the method described herein partly closes the diagnostic gap in idiopathic male infertility due to unexplained sperm dysfunction. In that manner, the present method enables evidence-based therapy decisions which can optimize the course of treatment of patients who wish to have children but have not been able to conceive in a natural manner.

The method described herein enables determining the longitudinal axis rotation of spermatozoa, i.e. the rotation of spermatozoa around their longitudinal axis. The longitudinal axis rotation is crucial for the navigation of sperm across the oviduct towards the site of fertilization. It is known from mouse models that the longitudinal axis rotation is fundamental for the so-called rheotactic navigation behavior, in which the spermatozoa orient based on fluid flow in the fallopian tube. In the fallopian tube, the spermatozoa use physicochemical stimuli to navigate towards the egg cell in accordance with the fluid flow which continuously transports the fallopian-tube fluid in the direction of the uterus. The spermatozoa align their movement trajectory against (antiparallel to) the direction of the fluid flow, thus swimming upstream. This behavior is called rheotaxis. From experiments with mouse models, it is known that rheotactically inactive spermatozoa do not fertilize the egg - they fail to traverse the oviduct, do not reach the site of fertilization and, thus, cannot fertilize the egg. The forward motion of spermatozoa involves a rotation around their longitudinal axis induced by the flagellar beat. This rotation is crucial for the spermatozoa to undergo rheotaxis. Mouse spermatozoa which do not rotate around their longitudinal axis are rheotactically inactive and cannot fertilize the egg cell. Male mice suffering from this sperm dysfunction are infertile.

Longitudinal axis rotation, which may be seen as an essential mode of sperm movement, has not been measured in semen diagnostic applications so far.

According to the invention, by measuring the longitudinal axis rotation, a diagnostic procedure is provided for the first time that indirectly, as a surrogate, evaluates the ability of sperm to navigate across the oviduct. In particular, the present method may uncover a sperm dysfunction and, thus, the cause of, so far, idiopathic male infertility. Additionally, an evidence-based optimization of the treatment strategy for assisted reproduction can be carried out on the basis of this parameter and application of ineffective fertility treatments with frustrating results for patients can be avoided. In the absence of longitudinal axis rotation, the cycle optimization and IUI as fertility treatments are unsuitable and doomed to failure.

Furthermore, the longitudinal axis rotation of sperm can be used as a biomarker for male infertility. Severely disturbed or absent longitudinal axis rotation most probably affects not only rheotaxis, but also other essential sperm functions, e.g. navigation via thermotaxis (orientation based on temperature differences) and chemotaxis (orientation based on differences in concentration of chemical stimuli) and/or the ability to penetrate through the protective vestments of the egg cell.

The present method for assessing the fertilizing potential of sperm is based on a determination of the longitudinal axis rotation of human spermatozoa in a microscopic procedure based on dark-field illumination. Expressed differently, the present method may be seen to be usable for assessing the suitability of sperm for fertilization of an oocyte. By modulating the illumination temporally or spatially, the longitudinal axis rotation of spermatozoa can be quantified. In particular, the method may be performed in an automated manner and it may be applied to a sample including several hundreds of spermatozoa, analyzing the sample on a timescale of less than a minute. With the inventive method it is also possible to distinguish longitudinal axis rotation from spurious artifacts that can occur due to certain phenotypes of sperm morphology and motility. Additionally, the inventive method integrates well with existing computer-assisted sperm movement analysis (CASA) systems. It may be conveniently implemented into regular existent microscopic setups by adding further optical elements to the existent dark-field microscope setup. However, those additional optical elements do not interfere with the regular use of the microscopic system but provide additional elements which may be used (switched on/off) when performing the method described herein.

The method of the present invention allows for an evidence-based fertility therapy which advantageously leads to a significant reduction in the medical risk for the (female) patients while at the same time increasing the success rates. Reproductive medicine centers that make use of the present method can optimize their success rates by adding this uncomplicated and easy to perform method into their diagnostic repertoire. On the other hand, CASA manufacturers can continue using existing devices and by application of relatively straightforward modifications they can manufacture a new generation of devices on that basis. Those new devices provide a substantial added value to users and in that manner exert a rather strong pressure to innovate on the users of existing devices which generally have rather long lifecycles.

In various embodiments a method for assessing the fertilizing potential of sperm is provided, wherein the method comprises measuring a rotation of at least one spermatozoon in a sperm sample around its longitudinal axis, the sperm being obtained from a male subject, wherein observing no rotation of the sperm is indicative that the sperm is not suitable for fertilization of an oocyte, in particular not suitable for fertilization of an oocyte in vivo; whereas observing rotation indicates that the sperm may be suitable for fertilization. In other words, the fertility of the test subject may be assessed based on the presence or absence of longitudinal axis rotation (will be also referred to as rotation in the following) of the spermatozoa in the sperm sample. For example, infertility may be diagnosed if none or a fraction of spermatozoa below a threshold are detected to carry out the rotation. The fertility of the test subject may be evaluated based on the ratio between spermatozoa that are rotating and spermatozoa that are not rotating during the observation of the sperm sample. It is noted that only if a spermatozoon rotates around its longitudinal axis it is qualified as “functional” or contributing to a positive assessment of its fertilizing potential. In particular, other kinds of motion which will be discussed in more detail below do not count as rotations. In the context of the assessment method disclosed herein, the lack of rotation of the sperm around its longitudinal axis is seen as a disqualifier for its suitability for fertilization in vivo. Contrastingly, an observed rotation of the sperm around its longitudinal axis is seen as a criterion indicating that the sperm might be able to fertilize. It is noted that the presence of rotation of the sperm around its longitudinal axis does not imply its fertility, since various other defects may compromise this characteristic.

The method disclosed herein may also be understood as one for determining the presence of a sperm dysfunction and, thus, a male fertility disorder. As explained above, the rotation (i.e. longitudinal axis rotation) may be seen as a crucial component of rheotaxis of the sperm. Therefore, an impairment of that rotational movement leads to an impairment of the rheotactic activity and, thereby, impairs the navigation of sperm across the oviduct. The present method, enabling analysis of the rotational movement of the spermatozoon around its longitudinal axis, thus determines a parameter which may be indicative of a sperm dysfunction and ensuing male fertility disorder. In order to detect the rotation of the spermatozoa, the sperm sample is observed under a microscope, preferably in a dark field microscope setting. Under a dark field microscope, the rotation may be observed with the naked eye as a small periodic contrast change occurring at the sperm head.

According to an embodiment, the method may include measuring the frequency of the rotation of the at least one sperm around its longitudinal axis. Preferably, for each spermatozoon of the sample, for example for each spermatozoon in the sample which is able to rotate, the frequency of its rotation may be determined. The frequencies may be analyzed and the suitability for fertility of the subject may be assessed on that basis. For example, the spermatozoa may be divided in different classes, according to their frequencies of rotation. Each spermatozoon in the sample may be classified according to the frequency of its rotation. Alternatively or in addition, at least one threshold rotation frequency may be used to subdivide the spermatozoa in the sample into at least two groups, accordingly. For example, a threshold rotation frequency may be determined below which a corresponding spermatozoon is classified as dysfunctional. The at least one threshold rotation frequency may be determined from a patient study, for example. In particular, rotational kinematics of the sperm of the tested subject, such as the rotation frequency, may be compared to rotational kinematics of sperm samples from fertile subjects.

According to an embodiment of the method, the rotation of the sperm sample containing the at least one sperm may be observed by observing the sperm sample in a first light field which corresponds to a dark field illumination, wherein the rotation of at least one sperm may be observed by a periodically blinking pattern in the first light field. The periodic blinking may be generated by the movement of the head of the spermatozoon in the light field which varies the amount of scattered light which reaches the microscope objective. Specifically, the sperm sample may be observed under a dark-field illumination microscope. The analysis of the sperm sample under the dark-field microscope may be performed in an automated manner, by employing an imaging device (e.g. a camera) and using an algorithm which is configured to detect blinking patterns in the images acquired by the imaging device. In general, the method may include an automated and quantitative analysis of the rotation of at least one spermatozoon, preferably of a multitude of spermatozoa contained in a sperm sample.

For the purpose of sperm analysis according to the method disclosed herein, an atypical configuration of the dark field microscope may be used. When the microscope illumination is configured correctly as described below, the rotation of the head of the spermatozoon becomes visible as a periodic blinking of the sperm head with high contrast. By simple tracking of the head of a spermatozoon over short periods of time, the presence of rotation and, in particular, the rotation frequency can be calculated from the movement of the sperm head. By using image processing to process images recorded by the imaging device, a population of several hundred spermatozoa can be observed and their motions analyzed, for example in terms of their rotation frequency, in a short period of time, e.g. in a few minutes, and in an automated manner. The data obtained from such an analysis may be compared with data obtained from spermatozoa of healthy subjects. In doing so, patients in which the rotation of the sperm is disturbed may be identified.

According to an embodiment of the method, the intensity of the first light field may be adjusted such that an image of the at least one sperm in the first light field acquired by an imaging device is not saturated. For that purpose, the intensity of the light source generating the first light field may be appropriately adjusted. The intensity of the first light field may be adjusted such that none of the pixels of the recording imaging device, e.g. a CCD camera, is saturated during a period of time during which the spermatozoa are observed under the microscope. That period of time may have a duration of a few seconds to 1 minute, for example. The intensity of the first light field may be adjusted such that none of the spermatozoa present in the sperm sample, independent of its overall orientation and in particular independent of the alignment of its head, generates a light signal which leads to a saturated image of the head. Such a configuration of the first light field is advantageous because then the intensities of the observed spermatozoa are dependent on the orientation of the sperm head. Saturation may be avoided by analyzing the histogram of the acquired image or video signal and by adjusting the intensity of the first light field accordingly. Such a configuration of the dark field microscope for analyzing motility of spermatozoa in a sperm sample is rather unusual. Usually, the intensity of the microscope is adjusted to generate saturated images of the spermatozoa in order to be able to track their trajectories. In other words, in ordinary motility analysis, a blinking pattern is rather undesired and avoided by choosing a sufficiently high intensity of the illumination light. This is understandable, since from the perspective of the standard semen analysis method the blinking nature is a disturbing effect when a spermatozoon is to be tracked to evaluate its swim trajectory. According to the present invention, however, saturation in the detection channel based on the first light field is avoided, because the presence or absence of blinking (i.e. variation of the brightness of the respective spermatozoon in the acquired images) is the crucial information which is detected and used to determine whether a spermatozoon is rotating or not. When the intensity of the first light field is set such that saturation is avoided in the acquired images, the rotation of the sperm head becomes visible as a periodic blinking of the sperm head with high contrast. By simply tracking a spermatozoon over a short period of time, its rotational movement can be analyzed and, for example, its rotation frequency can be calculated, from the temporal variation of the brightness of the sperm head.

As explained above, sperm rotation (again, around the longitudinal axis) can be observed as blinking of the sperm head when the intensity of the illumination of a conventional dark field microscope is dimmed to an unusually low level. When the illumination intensity is set correctly, the rotation of the sperm head may be observed as a variation, e.g. a periodic variation, of the intensity with high contrast. This technically simple illumination strategy underlying the invention makes it possible to precisely measure rotation of the spermatozoa, e.g. to determine the presence of rotation or to measure the rotation frequencies, in the sperm sample. This microscopic setup has been already proven to deliver sufficient results during studies with healthy volunteers. For clinical examination studies of patient samples, however, the present method may be further improved in order to determine in a reliable manner whether a spermatozoon is rotating or not. Due to impaired or disturbed sperm motility and rotation, abnormal movements of the sperm head which cannot be seen to correspond to rotation can generate periodic intensity variations in dark field microscopy which are similar to rotation and may thus be misinterpreted as rotation in the sense of this application. For example, this "apparent rotation" could be observed when motile spermatozoa

- do not move along the surface of the observation chamber (xy plane), as is usually the case, but change their position periodically in the z-direction, leaving and re entering the focal plane of the dark field illumination in a recurring manner,

- rock back and forth (or side to side) by 180°, but do not rotate, i.e. do not fully rotate by 360°,

- have deformed heads that trigger large changes in the reflection intensity even with minimal rocking movements.

Those abnormal movement patterns which can potentially falsify obtained results can be identified and taken into account by spatial or temporal modulation of the dark field illumination.

Accordingly, in an embodiment, the method may further include observing the sperm sample in a second light field, the second light field having an illumination characteristic different from the first light field, wherein during observation of the sperm sample the first light field and the second light field are provided in an alternating manner. In this mode of observation, the sperm sample is observed using one microscope and the first light field and the second light field are both provided with respect to that microscope. Thus, the second light field may also correspond to a dark field illumination. In particular, the first light field and the second light field may be generated by the same light source or light generating elements which may be switched between different illumination patterns. The first light field may differ from the second light field with respect to its spectral configuration, its spatial configuration, its temporal configuration or any combination of those aspects. Providing the light fields in an alternating manner may be understood to correspond to an illumination of the sperm sample during which either the first light field is provided or the second light field is provided. The periods of time during which each of the light fields is provided may be the same or different.

The first light field may correspond to a dimmed dark field illumination in which saturation is avoided. It may be also referred to as the roll channel. The second light field may correspond to a conventional dark field illumination and may be also referred to as the control channel. The intensity of the second light field may be adjusted (e.g. by adjusting the light source and/or optical elements generating the second light field) so that the pixels of the imaging device receiving light from the sperm heads during observation are in saturation. As a result, the intensities in this detection channel are independent of the orientation of the sperm heads and, consequently, of the rotation of the sperm heads. In the roll channel, rotation of the spermatozoa still leads to a blinking (flashing) of their heads. Blinking or flashing may correspond to a more or less periodic variation of the brightness of the image of the spermatozoon. The two detection channels have in common that they are sensitive to the position of the sperm head relative to the focal plane of the microscope. Non- rotational movements of the sperm head through the focal plane lead to correlated, e.g. synchronous, blinking in both the roll channel and the control channel. In that manner, apparent rotation may be reliably differentiated from genuine rotation, i.e. rotation about the longitudinal axis of the spermatozoon. Genuine rotation does not generate correlated blinking in both channels. Depending on the illumination pattern in each of the detection channels, genuine rotation may be distinguished from an up and down motion by the phase difference between the two observation channels. According to an embodiment of the method, the first light field and the second light field may be switched at a switching frequency which corresponds to the frame rate of the imaging device used for acquiring images of the sperm sample. In general, the switching frequency may lie in the range of at least 50 Hz, of at least 60Hz or more. Choosing the switching frequency to lie in the range of the frame rate (image acquisition rate) of the imaging device may be advantageous in that it leads to a pseudo-simultaneous acquisition of information in both the roll channel and the control channel. It is noted that at very high frame rates, e.g. frame rates substantially higher than 100 the switching does not necessarily have to be synchronous with the frame rate, but can be chosen at a fraction of the frame rate. For example, at a frame rate of 200 Hz, switching between the two light fields may take place every two frames, i.e. at a rate of 100 Hz.

According to an embodiment of the method, the spatial illumination patterns of the first light field and of the second light field may be radially symmetric. A radially symmetric light field may be understood as one which is invariant with regard to light propagation along any axis emanating from the center of the light source generating the light field (but naturally bound by the radiation angle of the light source). In those embodiments, the illumination patterns of the first light field and of the second light field may be the same.

According to an embodiment of the method, the intensity of the second light field may be adjusted such that a portion of an image of the head of the at least one sperm acquired by the imaging device is saturated, as mentioned above. The second light field may be adjusted such that the head of the at least one sperm, in particular of the sperm heads in the sperm sample, remain saturated, as long as the head of the spermatozoon remains substantially within the focal plane of the microscope. This is in particular the case for spatially symmetric illumination patterns which can be used to effectively exclude up and down movements of the sperm head in z-direction (i.e. perpendicular to the focal plane) from being erroneously qualified as rotation around the longitudinal axis.

According to an embodiment the method may include determining that the observed spermatozoon performs a (genuine) rotation if i) the intensity of the head portion of the sperm observed in in the first light field shows a substantially periodic variation (e.g. resembling a blinking pattern), and ii) the intensity of the head portion of that same sperm observed in the second light field remains substantially constant. Those requirements may be checked for when two spatially symmetric light fields are used.

According to an embodiment of the method, the spatial illumination pattern of the first light field may be different from the spatial illumination pattern of the second light field. For example, one light field, e.g. the first light field, may have a radially symmetric light pattern, while the other light field, e.g. the second light field, may have a point symmetric light pattern or correspond to a one-sided illumination. According to an embodiment of the method, the second light field may correspond to a portion of the first light field (or vice versa), wherein the second light field corresponds a one-sided illumination of the sperm sample. According to an embodiment of the method, the first light field and the second light field may be substantially point-symmetric. This may be the case for each light field taken alone or for a unified light pattern which is formed when both light fields are considered in combination. In the latter case, both light fields provide one-sided illumination and correspond to mirrored illumination patterns. In general, various illumination patterns may be chosen, where both light fields may have the same illumination pattern or a different illumination pattern, with the first light field having a lower intensity (to avoid saturation) than the second light field (to achieve saturation).

When asymmetric, one-sided illumination, is used for one of the light fields for illuminating the sperm sample under the microscope, the relation between the measured intensity and the angle of the sperm head becomes more pronounced and is different from the case in which a light field with a symmetric illumination pattern is used. By alternating images acquired with a radially symmetric light field with images acquired with a one-sided light field of a dark field illumination microscope, the two different channels mentioned above may be obtained. The intensity in each of the channels depends on the rotation angle of the sperm head in a different manner.

From the ratio of the intensities acquired in the two detection channels information about the rotation of the sperm head may be obtained. In particular, that ratio may be evaluated to distinguish between a full rotation and a rocking motion. In addition, however, both detection channels are sensitive with respect to the position of the sperm head relative to the focal plane of the microscope. A similar advantage may be achieved by alternating two light fields, each one providing one-sided illumination, from opposite directions, i.e. by alternating a one-sided illumination from the left side with a one-sided illumination from the right side.

Technically, the first light field and the second light field which overall provide the spatially modulated dark field illumination may be obtained from LED light sources which emit light of orthogonal polarizations. Polarizing filters with polarization axes aligned perpendicular to one another, each filter covering at least a portion of the emitted light which generates the respective light field, may be used and switched alternately in order to switch between at least two illumination patterns. Alternatively, the different illumination patterns may be generated using an LCD placed in the light path on which different patterns are displayed in order to provide a “stencil” for light.

According to an embodiment of the method, the subject may be a mammal. The mammal may be selected to be a human, a farm animal or a zoo animal, for example. The farm animal may be male cattle, a male sheep, a male horse, a male goat or a male pig.

According to an embodiment, the method may further comprise subjecting a sperm sample obtained from the subject to further fertility test in case a rotation of the at least one sperm of the subject around its longitudinal axis has been measured. The screening of a sperm sample for the presence of rotation of the sperm heads may correspond to a first step of a multi-step examination aimed at assessing the fertility of the subject. This may be advantageous since observing no rotation of the at least one sperm is indicative that the sperm is not suitable for fertilization of an oocyte and thus further futile testing of the sperm of the subject may be avoided.

According to further embodiments, an in vitro method for determining infertility of a male subject is provided, the method comprising the steps of: a) detecting the rotation of the sperm in a sperm sample around their longitudinal axis, the sperm being obtained from a male subject; and b) determining the suitability of sperm for fertilization of an oocyte in vivo based on the rotation/motility detected in step (b). In the context of this method, measuring or observing no rotation of the sperm is indicative that the sperm is not suitable for fertilization of an oocyte, whereas measuring or observing rotation indicates that the sperm may be suitable for fertilization. Observing of no rolling in a sperm sample or a percentage of sperm with normal rotational motility in a sperm sample below a predetermined reference value is indicative for infertility of said subject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 A illustrates how longitudinal axial rotation of a spermatozoon manifests itself in recorded images using an illumination scenario featuring temporal modulation thereof.

FIG. 1 B illustrates how rocking back and forth of a spermatozoon manifests itself in recorded images using the illumination scenario of FIG. 1A.

FIG. 1C illustrates how periodical changes in the z-position of a spermatozoon manifests itself in recorded images using the illumination scenario of FIG. 1A.

FIG. 2A illustrates how longitudinal axial rotation of a spermatozoon manifests itself in recorded images using an illumination scenario featuring spatial modulation thereof.

FIG. 2B illustrates how rocking back and forth of a spermatozoon manifests itself in recorded images using the illumination scenario of FIG. 2A.

FIG. 2C illustrates how periodical changes in the z-position of a spermatozoon manifests itself in recorded images using the illumination scenario of FIG. 2A.

FIG. 3A illustrates how longitudinal axial rotation of a spermatozoon manifests itself in recorded images using an illumination scenario featuring spatial modulation thereof.

FIG. 3B illustrates how rocking back and forth of a spermatozoon manifests itself in recorded images using the illumination scenario of FIG. 3A.

FIG. 3C illustrates how periodical changes in the z-position of a spermatozoon manifests itself in recorded images using the illumination scenario of FIG. 3A.

FIG. 4A illustrates the experimental setup based on dark-field microscopy.

FIG. 4B shows images of a single sperm obtained via dark-field microscopy. FIG. 4C shows images of a sperm population obtained via dark-field imaging (upper half of the figure) and the corresponding variation of brightness of the sperm heads (lower half of the figure).

FIG. 4D shows an exemplary distribution of rotation frequencies among freely swimming sperm.

FIG. 4E illustrates the experimental setup for the laser-based optical tweezer.

FIG. 4F shows bright-field images of an optically trapped sperm cell.

FIG. 4G shows representative time courses of the rotation frequency of trapped sperm cells.

FIG. 4H shows an exemplary distribution of rotation frequencies in trapped sperm. FIG. 41 shows image series of trapped sperm cells.

DETAILED DESCRIPTION OF THE INVENTION

In the following figures, it will be described how the rotation of a spermatozoon can be distinguished from disturbed or impaired rotation which is indicative of male infertility. It is once again noted that the term “rotation” of a spermatozoon, as used herein, is used for the natural (or genuine) behavior of a spermatozoon where its head rotates or gyrates around its longitudinal axis, the axis being substantially aligned with the direction of motion caused by the rotation. In contrast thereto, a lateral or vertical motion of the head of the spermatozoon where the trajectory of the head is not circular because the head does not rotate around the longitudinal axis of the spermatozoon will be referred to as rocking (motion) or dysfunctional rotation. In the latter case, the motion of the head of the sperm is rather an oscillatory motion which is indicative of an impairment of the motility of the spermatozoon and the fertility of the male subject.

FIG.1 A illustrates how longitudinal axial rotation of a spermatozoon manifests itself in recorded images using an illumination scenario featuring temporal modulation thereof. The temporal modulation of the illumination may include switching of a light source, such as an LED, between two intensities. The illustration is of qualitative nature and focuses on one spermatozoon to explain the influence of its motion on the recorded images. In FIG.1 A, a first detection channel 1 and a second detection channel 2 are shown. Each of the two detection channels is described from two different perspectives. Even though the description is based on the usage of two light fields and, consequently, two detection channels, using only one detection channel (e.g. the first detection channel 1) may already provide sufficient results for a determination of a rotation of a spermatozoon in an analyzed sperm sample. If both detection channels 1 , 2 are used, the corresponding light fields can be generated alternately by the same light source, for example. Furthermore, the switching between the first light field and the second light field, i.e. the switching between the first detection channel 1 and the second detection channel 2, may take place synchronous with the rate at which the images (frames) are recorded by the imaging device which may be attached to or may replace the microscope objective.

In a first series of pictograms 3 in the first detection channel 1 , a spermatozoon, in particular its head portion 5, is shown together with the first illumination light field 6 and a scattered light field 7. In each of the pictograms 3, a different orientation of the head 5 is shown. The perspective of the first series of pictograms 3 is indicated by a first coordinate system 9. The sperm sample is usually provided on a carrier which is assumed to be oriented in the xy plane while the z-axis corresponds to the optical axis of the dark field microscope. Particularly, the xy plane may correspond to the plane defined by the microscope slide and/or the microscope stage on which the microscope slide containing the sperm sample is placed for analysis. The first illumination light field 6 impinges on the microscope slide. As well known, in dark field microscopy the illumination is configured such that it does not directly reach the objective lens of the microscope, but only light that is scattered by the analyzed sample - here the spermatozoa in the sperm sample - into the objective lens can be observed. . The scattered light field 7 represents light scattered by the head portion 5 of the sperm.

In a second series of pictograms 4 in the first detection channel 1, the spermatozoon in particular its head portion 5, is shown as viewed through the microscope (the spermatozoon head 5 is explicitly labelled with a reference sign only in the last one of the five pictograms in the second series of pictograms 4). In other words, the second series of pictograms 4 illustrates an idealized image of the head 5 of a spermatozoon as observed through the microscope. This perspective is indicated by a second coordinate system 10 which is rotated by 90° with respect to the first coordinate system 9. Therefore, the view presented in the second series of pictograms 4 may be seen to substantially correspond to a top view of the sperm sample while the view presented in the first series of pictograms 3 substantially corresponds to a side view thereof. Each of the pictograms in the second series of pictograms 4 correlates with the pictogram arranged directly above from the first series of pictograms 3. This correlation may be understood as a temporal correlation. In other words, an orientation of the head 5 of the spermatozoon, as shown in any of the pictograms in the first series of pictograms 3, produces or translates into a corresponding microscope image depicted below in the second series of pictograms 4.

As can be taken from the second series of pictograms 4 in the first detection channel 1 , the rotation of the head 5 of the spermatozoon indicated in the first series of pictograms 3 causes a variation of the shape and of the brightness of its image. In particular, the second series of pictograms 4, seen in repeated continuation, correspond to a flashing image of the head 5 of the spermatozoon, i.e. an image of the head 4 which becomes bright (see middle pictogram) and dimmed/dark (see first and second pictogram) in cycles. A motion pattern as depicted in the first series of pictograms 3 in the first detection channel 1 may be particularly caused by a spermatozoon moving (swimming around) on the microscope slide, i.e. in the xy plane. Therefore, by observing the variation of the shape and/or the brightness of the image of a spermatozoon in a sperm sample may provide sufficient information to determine whether it is rotating or not. As mentioned earlier, the intensity of the first illumination light field 6 may be adjusted such that the image of the head 5 is not saturated, independent of the actual orientation of the head 5. In that manner, every differential movement of the head 5 may be correlated with a corresponding variation of the brightness of the image of the head 5.

As noted above, in order to obtain better results (e.g. faster and more reliable results), the second detection channel 2 may be used. The second detection channel 2 which is based on a second illumination light field 8 is described in FIG. 1 A in analogy to the first detection channel 1 with the same elements being assigned the same reference numbers. In this example, the second illumination light field 8 has the same illumination pattern as the first illumination light field 6. However, as mentioned earlier, the intensity of the second illumination light field 8 may be adjusted such that the image of the head 5 of the spermatozoon is saturated throughout its movement. Consequently, independent of the actual orientation of the head 5 of the spermatozoon, the image thereof is constantly saturated and thus has a constant level of brightness, as indicated by the pictograms in the second series of pictograms

4 in the second detection channel 2. It is noted that the variation of brightness is closely correlated with the amount of light scattered by the spermatozoon, in particular its head portion 5, which impinges on the objective lens of the microscope and may be detected. The variation of the imaged shape of the head 5 is substantially the same when both illumination light fields 6, 8 have the same illumination pattern.

By correlating the images obtained from the first detection channel 1 and the second detection channel 2, it may be concluded more reliably whether the spermatozoon is rotating or performing movements which cannot be qualified as rotation. In the exemplary scenario of FIG.1 A, the variation of brightness of the images of the head 5 of the spermatozoon in the first detection channel 1 corresponds to the blinking pattern mentioned above. In contrast thereto, no blinking pattern can be observed in the images acquired in the second detection channel 2 where the image of the head

5 has a substantially constant brightness. From the blinking of the image of the head 5 of the spermatozoon observed in the first detection channel 1 and from the static brightness level of the image of the head 5 of the spermatozoon in the second detection channel 2 it may be concluded that the spermatozoon is not performing rocking movements along the z-axis but is either performing rotations or is rocking sideways. In other words, the second detection channel 2 may be used to distinguish vertical (i.e. along the z-axis) rocking motion of the spermatozoon from the other two motion patterns, i.e. (genuine) rotation and lateral or sideways oriented (i.e. in the xy- plane) rocking. The circular arrow 11 is used to indicate that the movement pattern of the spermatozoon illustrated in FIG.1 A corresponds to a rotation.

FIG.1 B illustrates how a dysfunctional rotation of a spermatozoon manifests itself in recorded images. Flere, the dysfunctional rotation is indicated by a horizontal double arrow 12 with a slight bend. In this scenario it is assumed that the head 5 of the spermatozoon is rocking back and forth or from side to side (sideways) without performing rotations. In FIG.1 B only the microscope images, i.e. the second series of pictograms 4, as acquired in the first detection channel 1 and in the second detection channel 2, are shown. Since the nature of the pictograms presented in FIG.1 B is the same as in FIG.1 A, the same reference signs are used.

Comparing the images of the head 5 the spermatozoon from FIG.1 B to the ones from FIG.1 A it may be seen that while the image of the head 5 remains saturated in the second detection channel 2, the variance of brightness in the first detection channel 1 is heavily reduced. The lateral rocking motion of the head 5, during which the head 5 substantially remains in the focal plane, generates a very soft variation of brightness. In the images of the head 5 in the first detection channel 1 a flashing pattern, as illustrated by the second series of pictograms 4 in Fig.1 A, cannot be observed.

FIG.1 C illustrates how a further dysfunctional rotation of a spermatozoon manifests itself in recorded images. Flere, the dysfunctional rotation is indicated by a vertical double arrow 13. In this scenario it is assumed that the head 5 of the spermatozoon is rocking up and down along the z-axis (i.e. vertical rocking) without performing rotations. In FIG.1 C, in analogy to FIG.1 B, only the microscope images, i.e. the second series of pictograms 4, as acquired in the first detection channel 1 and in the second detection channel 2 are shown. Since the nature of the pictograms presented in FIG.1 C is the same as in FIG.1 A and 1 B, the same reference signs are used.

From the images of the spermatozoon shown in FIG.1 C it may be seen that a vertical rocking of the head 5 of the spermatozoon leads to flashing in the second detection channel 2. The flashing observed in the second channel 2 is indicative of the head 5 of the spermatozoon moving out of and moving back into the focal plane of the microscope. Generally, when the spermatozoon head 5 is substantially arranged within the focal plane of the microscope, a maximum amount of scattered light may potentially reach the microscope objective. Therefore, movements of the head 5 of the spermatozoon out of the focal plane of the microscope lead to larger intensity variations than movements of the head 5 of the spermatozoon which substantially take place within the focal plane. Further generally, when the motion of the spermatozoon remains within the focal plane of the microscope, the amount of illumination light scattered into the objective lens depends on the orientation of the spermatozoon. Additionally, it may be seen that the shape of the observed spermatozoon in both detection channels 1 , 2 is similar. In fact, the images of the spermatozoon in both detection channels 1 , 2 only differ with regard to scaling - in this example the head 5 of the spermatozoon appears round all the time. This is not the case for a rotating spermatozoon (see FIG.1 A) or for the sideways rocking spermatozoon (see FIG.1B).

Comparing the images of the spermatozoon in each of the first series of pictograms 3 in each of the first channels 1 for the different motion patterns, a discrimination between different patterns of motion, such s rotation (FIG.1 A) and rocking or disturbed rotation (FIGS.1B and 1C), may be made based on the variation of the brightness, i.e. variation of the intensity of the light scattered by the spermatozoon, and further optionally based on the variation of the shape of its image. In particular, a flashing image of the spermatozoon may be seen as indicative of a rotating spermatozoon. Additionally, by evaluating the second detection channel 2, a more reliable statement can be made with regard to the motion state of the spermatozoon. As discussed with reference to the second series of pictograms 4 in FIG.1C, a flashing pattern in the second detection channel 2 indicates abnormal movement of the spermatozoon which does not correspond to rotation. Therefore, a stable image of the spermatozoon (with regard to light intensity) in the second detection channel 2 may be seen as a necessary requirement for a rotating spermatozoon. The second detection channel 2 in Figs. 1A-1C is used to rule out rocking movements of the head 5 of the spermatozoon out of (e.g. perpendicular to) the focal plane as the cause of the blinking pattern in the first detection channel. It is further noted that in the scenarios depicted in FIGS.1A-1C, both the first illumination light field 6 and the second illumination light field 8 are radially symmetric.

FIGS.2A-2C illustrate the detection of the kinematics of a spermatozoon using a first detection channel 1 which is based on radially symmetric illumination and a second detection channel 2 which is based on a radially asymmetric illumination. The intensity of the illumination in both detection channels 1 , 2 is chosen such that the image of the head 5 of the spermatozoon is not saturated and therefore rotations of the head 5 of the spermatozoon lead to a change of intensity of the images. In the exemplary scenario illustrated in FIGS.2A-2C, the second illumination light field 8 corresponds to a one sided illumination of the sperm sample. In particular, the illumination pattern of the second illumination light field 8 may correspond to a portion of the illumination pattern of the first illumination light field 6, for example to one half of the illumination pattern of the first illumination light field 6 or to an even smaller segment thereof. Except for this difference, FIGS.2A-2C correspond to FIGS.1A-1C with regard to the meaning of the elements shown therein and the order of analyzed motion patterns of the spermatozoon. That is, the results shown in FIG.2A are based on a rotating spermatozoon, whereas the results shown in FIGs.2B and 2C are based on a spermatozoon with a disturbed rotation. Therefore, the same elements are labelled with the same reference numbers and will not be described in detail again.

Due to the same nature of the first illumination light field 6 in FIGS.2A-2C and FIGS.1A-1C, the images of the spermatozoon in the first detection channel 1 (first series of pictograms 3) are the same such that the statements made above with regard to the conclusions which may be derived therefrom apply here as well.

Since the second illumination light field 8 provides an asymmetric, one-sided illumination of the sperm sample, the relation between the measured intensity and the angle of the sperm head 5 is different from, in particular much stronger than in the case of symmetric illumination. In the case of a vertically rocking sperm (FIG.2C), a variation of brightness in the form of a synchronous blinking pattern can be observed in the first and second detection channel 1 , 2.. In addition, the image of the sperm head 5 substantially maintains its overall geometrical shape; only its scaling varies over time. A laterally rocking sperm (FIG.2B) manifests itself in a blinking image of the sperm head 5 in both second detection channels 1 , 2, wherein the amplitude of the variation of the intensity of the blinking pattern in the first detection channel 1 is smaller than the amplitude of the variation of the intensity of the blinking pattern in the second detection channel 2, with the frequency of the intensity variation in the first detection channel 1 being twice that of the frequency of the intensity variation in the second detection channel 2. Additionally, in FIG. 2B the image of the sperm head 5 in both detection channels 1 , 2 varies with respect to its geometrical shape over the cycle of movement of the spermatozoon between a roundish and an elliptical shape.

A rotating spermatozoon (FIG.2A) may be identified by a relatively strong blinking, i.e. variance of brightness, in both detection channels 1 , 2, wherein the frequency and the intensity variation in both detection channels 1 , 2 is the same, wherein the amplitude of the intensity variation in both detection channels 1 , 2 is substantially the same, featuring a fixed phase shift (different from zero).

Finally, FIGS.3A-3C illustrate the determination of the kinematics of a spermatozoon using two detection channels 1 , 2 which are both based on radially asymmetric illumination. In the exemplary scenario illustrated in FIGS.3A-3C, the first illumination light field 6 and the second light field 8 may correspond to mutually exclusive parts of a radially symmetric light field pattern. For example, the first illumination light field 6 may correspond to a right sided illumination of the sperm sample and the second light field 8 may correspond to a left sided illumination of the sperm sample. This exemplary configuration of the light fields 6, 8 is also illustrated in FIG.3A. In particular, an illumination pattern in which the first illumination light field 6 and the second illumination light field 8 are viewed together may be point symmetric. Alternatively, both light fields 6, 8 may correspond to different segments of an annularly shaped illumination light pattern. Except for the different second light field 8, the scenarios depicted in FIGS.3A-3C correspond to those depicted in FIGS.2A- 2C with regard to the meaning of the elements shown therein and the order of analyzed motion patterns of the spermatozoon. That is, the results shown in FIG.3A are based on a rotating spermatozoon, whereas the results shown in FIGs.3B and 3C are based on a spermatozoon with a disturbed rotation in the form of sideways rocking and vertical rocking. Therefore, the same elements are been labelled with the same reference numbers and will not be described in detail again.

Since the second illumination light field 8 used in the scenario depicted in FIGS.3A- 3C is the same as the second light field 8 used in the scenario depicted in FIGS.2A- 2C, the statements made above with regard thereto apply here as well. In the examples depicted in FIGS.3A-3C, the first illumination light field 6 corresponds to a so to speak conjugate version of the second illumination light field 8 and leads to images in the first detection channel 6 of the sperm head 5 which are similar to the images of the sperm head 5 obtained in the second channel 2. As may be seen by comparing the second series of pictograms 4 between the first detection channel 1 and the second detection channel 2 in FIG. 3A and 3B, in the exemplary case of mutually excluding one sided illumination patterns, the temporal order is reversed. In both cases the intensity variation shows a phase shift between channel 1 and 2. Flowever, if the first illumination light field 6 and the second illumination light 7 viewed together do not form a symmetric illumination pattern, this will not be the case. In general, when each of the two illumination light fields 6, 8 provides an asymmetric, one-sided illumination of the sperm sample, the measured intensity correlates strongly with the orientation/angle of the sperm head 5 as compared to symmetric illuminations. A vertically rocking sperm (FIG.3C) generates changes in intensity in both detection channels 1 , 2. In particular, the variation of the intensity in the form of a blinking sperm head 5 is synchronized in both detection channels with no phase shift between both channels. In addition, the image of the sperm head 5 substantially maintains its overall geometrical shape; only its scaling varies over time.

FIG.4A illustrated the experimental setup used for the analysis of longitudinal rolling of human sperm by dark-field microscopy.

FIG.4B shows single frames of a single spermatozoon obtained at different times (t = 0, 48, 96, and 136 ms) via dark-field microscopy. The scale bar represents a dimension of 25 pm.

FIG.4C shows results obtained from dark-field imaging of a sperm population. In the upper half of the figure, single frames, obtained at t = 185, 363, 540, and 718 ms are shown. The four sperm heads selected for analysis are marked with numbers (1-4). The scale bar represents a dimension of 25 pm. In the lower half of the figure, the temporal variation in the brightness (blinking) of the selected sperm heads is shown in four diagrams, each diagram corresponding to each of the four selected spermatozoa, as marked in the single frames. The vertical lines in the diagrams correspond to the time-points of the single frames shown in the upper half of the figure.

FIG.4D shows a diagram which illustrates an exemplary distribution of rotation frequencies among freely swimming sperm in a representative sample (n = 164).

FIG.4E depicts the experimental setup for a laser-based optical tweezer.

FIG.4F shows four bright-field images obtained at t = 0, 55, 155, 698 and 185 ms of an optically trapped sperm cell using the setup depicted in FIG.4E.

FIG.4G shows a diagram which illustrates a representative time course of the rotation frequency of trapped sperm. Each sperm cell is represented by a different plot symbol (each set of plot symbols being labelled with a number between 1 and 4). Error bars indicate the accuracy of the Fast Fourier analysis used to determine the frequency. FIG.4H shows a diagram which illustrates an exemplary distribution of rotation frequencies in trapped sperm (n = 32).

FIG.4I shows an image series of a sperm cell trapped parallel to the optical axis. The images have been obtained at t = 0, 95, 165, and 205 ms. The bar indicates the 360° rotation of the tip of the head.

EXAMPLES

Materials and methods

Sperm preparation and buffer conditions

The studies were performed in accordance with the standards set by the Declaration of Helsinki. Samples of human semen were obtained from healthy volunteers and DIS patients with their prior written consent, under approval of the institutional ethical committees of the medical association Westfalen-Lippe and the medical faculty of the University of Muenster (4INie). Ejaculates were allowed to liquefy at 37 °C for 30-60 minutes. Motile sperm were purified 316 by a ‘swim-up’ procedure: Liquefied semen (0.5-1 ml) was layered in a 50 ml falcon tube below 4 ml of human tubal fluid (HTF) medium, containing (in mM): 97.8 NaCI, 4.69 KCI, 0.2 MgS04, 0.37 KH2P04, 2.04 CaCI2, 0.33 Na-pyruvate, 21.4 lactic acid, 2.78 glucose, and 21 HEPES, pH 7.35 (adjusted with NaOH). Motile sperm were allowed to swim up into the HTF layer for 60-90 min at 37 °C. After swim-up, sperm were washed twice (700 x g, 20 min) with HTF and the sperm concentration was adjusted. To study the motility of non- capacitated sperm and the action of bicarbonate, the medium was supplemented with human serum albumin (HSA, Scientific Irvine, USA; 3 mg/ml) only immediately prior to the experiment; the HSA was required to prevent attaching of sperm to the surface of the recording chambers. Otherwise, sperm were capacitated for 3 h at 37 °C in HTF++ medium, containing (in mM): 72.8 NaCI, 4.69 KCI, 0.2 MgS04, 0.37 KH2P04, 2.04 CaCI2, 0.33 Na-pyruvate, 21.4 lactic acid, 2.78 glucose, 25 NaHC03, 21 HEPES, pH 7.35 (adjusted with NaOH), and supplemented with 3 mg/ml HSA.

Rolling analysis in populations

The longitudinal rolling of sperm was recorded in glass chambers (depth of ~ 100 pm) under an inverted microscope (IX73, Olympus, Germany), equipped with condenser (IX2-LWUCD, Olympus, Germany) with a custom-made dark-field filter, a 10x objective (UPLFLN10X2PH1 , Olympus, Germany), and additional 1.6x magnification lenses (16x final magnification). The samples were illuminated with a red light-emitting diode (LED; M660D2, Thorlabs, Germany) and a custom-made power supply. Over 4-10 minutes, short movies (~ 725 ms) of sperm in several fields of view in the observation chamber were recorded at 124 Hz with a high-speed sCMOS camera (Zyla, 4.2 plus, Andor, UK). Longitudinal rolling (i.e. longitudinal axis rotation) was assessed with a custom-made program written in the ImageJ macro language (Rasband, 1997-2016). In brief, moving sperm heads were tracked and rotation was indicated by an oscillating change in the brightness of the head. The rotation frequency was computed from the average temporal distance between two intensity peaks. The sperm head is approximately plane-symmetrical with planes intersecting the length axis and, therefore, lights up twice per 360° rotation. Thus, the rotation frequency (FR _O is given by FR _0† = Feiin _k x 0.5. Sperm that displayed less than three relative maxima within the observation time were excluded from the analysis, yielding a cut-off for FR _0† of about 1.5 Hz; immotile sperm were excluded from the analysis. Immediately prior to the experiment, sperm were diluted 1:9 into HTF ⁺⁺ for control experiments and to study the effect of bicarbonate on sperm prepared in HTF, in HTF (supplemented with HSA) for experiments under non capacitating conditions, or HTF ^0Ca to study the action of Ca ²⁺ .

Optical trapping of sperm

The trapping of sperm cells was achieved with an optical tweezer (Ashkin, A., Dziedzic, J. M., Bjorkholm, J. E. und Chu, S. (1986): Observation of a single-beam gradient force optical trap for dielectric particles. Optics Letters 11 , 288-290), using a continuous-wave (cw) diode laser (Lumics LU0975M500, Germany) at a wavelength of 976 nm. The laser beam was expanded by a telescope setup of two lenses to a diameter of 1.7 mm (full width at half maximum) and directed into a 10Ox oil immersion objective (MO, Plan-Apochromatic 100x/1.40 Oil DIC M27, Zeiss, Germany) with a numerical aperture (NA) of 1.4, allowing for tight focusing of the laser beam onto the head of a sperm swimming inside an observation capillary. To assess the rotation frequency of trapped sperm, the laser light reflected by the sperm head into the objective was directed onto a photomultiplier tube (PMT, H10721 -20, Hamamatsu, Japan) by a beam splitter (reflectivity approx. 4%). A long-pass filter in front of the PMT blocked both the bright-field illumination and ambient light. The PMT signal was sampled with a frequency of 2 kHz, every 10 points were averaged. The rotation frequency was determined by a fast Fourier transformation in a moving time window of 1.5 seconds. In parallel to the quantification of the back-reflected laser light, we recorded the trapped sperm with a bright-field microscope. A blue LED, equipped with a 450 nm short-pass filter and a collimator lens, served as a light source. The bright-field image was reflected by a dichroic mirror, projected by a lens onto the chip of a charge-coupled device (CCD) camera (UI-378 3140CP-M-GL, IDS, Germany) and recorded with a frame rate of 200 Hz. To measure the rotation frequency of a sperm at different conditions, we trapped sperm inside a microfluidic capillary (dimensions (height x width): 0.4 x 0.33 pm; p-Slide III 3in1 , Ibidi, Germany) with three separate inlets to establish a continuous, parallel laminar flow of three solutions (1 . control stream with sperm, 2. barrier stream, 3. stimulus stream) with a flow speed of 65 pm/s. The barrier stream was supplemented with fluorescein (1 pM). Fluorescein was excited with the blue LED; fluorescence light was collected through the microscope objective and reflected by two dichroic mirrors through a long-pass filter onto a second PMT (H10721 -210, 386 Hamamatsu, Japan). Using a custom- built mechanical scanning table, the microfluidic capillary was moved in the horizontal plane orthogonal to the flow direction, dragging a trapped sperm within 6.8 s from the control stream through the barrier stream into the stimulus stream. The fluorescein fluorescence, recorded synchronously to the back-reflected laser light and the bright- field images, provided a readout of the position of the trapped sperm inside the flow profile. The stimulus stream consisted of one of the following buffers: HTF ⁺⁺ for control experiments and to study the effect of bicarbonate on sperm prepared under bicarbonate-free conditions, HTF (supplemented with HSA) for control experiments under non-capacitating conditions, or HTF ^0Ca to study the action of Ca ²⁺ . For paired- plot analysis, the change in frequency was determined after it reached a stable value.