Cairo consensus on the IVF laboratory environment and air quality: report of an expert meeting.

This proceedings report presents the outcomes from an international Expert Meeting to establish a consensus on the recommended technical and operational requirements for air quality within modern assisted reproduction technology (ART) laboratories. Topics considered included design and construction of the facility, as well as its heating, ventilation and air conditioning system; control of particulates, micro-organisms (bacteria, fungi and viruses) and volatile organic compounds (VOCs) within critical areas; safe cleaning practices; operational practices to optimize air quality while minimizing physicochemical risks to gametes and embryos (temperature control versus air flow); and appropriate infection-control practices that minimize exposure to VOC. More than 50 consensus points were established under the general headings of assessing site suitability, basic design criteria for new construction, and laboratory commissioning and ongoing VOC management. These consensus points should be considered as aspirational benchmarks for existing ART laboratories, and as guidelines for the construction of new ART laboratories.

Hepatosplenic T-cell lymphoma of alphabeta lineage in a 16-year-old boy presenting with hemolytic anemia and thrombocytopenia.

The authors report an unusual case of peripheral T-cell lymphoma in a 16-year-old boy who presented initially with jaundice, splenomegaly, anemia, and thrombocytopenia. A lymphoma was found subsequently in the spleen, which was infiltrated extensively in the red pulp by medium-sized, blastic-appearing lymphoma cells. Immunologic characterization of these cells revealed positivity for CD3, CD5, CD45RO, CD56, and T-cell intracellular antigen (TIA), and negativity for CD2, CD3, CD4, CD8, CD57, CD34, and terminal deoxynucleotidyl transferase (TdT). Conventional cytogenetic studies revealed the presence of isochromosome 7q. On follow up, this patient deteriorated rapidly, with evidence of liver and bone marrow involvement. Although the overall clinical and pathologic features of this disease were characteristic of hepatosplenic gammadelta T-cell lymphoma, the T-cell receptor of this tumor showed an immunophenotype of alphabeta not gammadelta lineage. Using the Southern blot technique, the authors demonstrated monoclonal gene rearrangement of the T-cell receptor beta-chain. Thus, they confirmed the existence of hepatosplenic alphabeta T-cell lymphoma. In view of its overall similarity to hepatosplenic gammadelta T-cell lymphoma, this unusual entity probably represents a slight biologic variation of the same disease.

The development of smoothing-independent kinematic measures of capacitating human sperm movement.

The kinematic values which are determined relative to the average path by computer-aided sperm analysis (CASA) instruments are reliant upon the algorithm used for track smoothing, and therefore may differ depending upon the shape and complexity of the trajectories analysed. To overcome this potential source of error in the identification of particular trajectory patterns, kinematic values must be derived so that they are independent of the average path. In this study a number of novel kinematic measures were developed and tested for their ability to differentiate between hyperactivated and non-hyperactivated human spermatozoa. A definition for hyperactivation which relied solely upon smoothing-independent kinematic values was developed. This definition was tested on two groups of trajectories reconstructed at 60 Hz by a CASA instrument. An overall agreement of 99% between the classification of tracks as hyperactivated or non-hyperactivated was observed between the established 60 Hz definition validated for that particular CASA instrument and the new, smoothing-independent definition. Because the new values are only reliant upon (x,y) coordinates for their calculation, and do not require smoothing, it should not be difficult to incorporate them into existing CASA instruments.

Effect of image sampling frequency on established and smoothing-independent kinematic values of capacitating human spermatozoa.

It is known that image sampling frequency affects sperm kinematic values, although no study has considered the relative effect upon hyperactivated and non-hyperactivated spermatozoa. We determined the relative effect of image sampling frequency on the classification of capacitating human spermatozoa, using the established kinematic measures as well as a series of new, smoothing-independent kinematic measures. Spermatozoa were prepared by direct swim-up from semen and sperm movement was recorded using a video system which gave 201 images/s on freeze-frame playback. Trajectories were reconstructed manually and kinematics were determined using the (x, y) co-ordinates of each track point. Lower image sampling frequencies were approximated by considering every second, third, fourth, sixth and eighth track point for each trajectory. Of the 22 kinematic values investigated, only three were not significantly affected by sampling frequency in both hyperactivated and non-hyperactivated spermatozoa. Also, significant differences were observed between hyperactivated and non-hyperactivated tracks at all image sampling frequencies studied for all but four kinematic measures.

Kinematic definition of ram sperm hyperactivation.

Although it is known that ram spermatozoa exhibit hyperactivated motility under capacitating conditions, quantitative analyses of the head and flagellar movement of washed ram spermatozoa have not been published. Motile spermatozoa were recovered from semen by swim-up into HSOF medium, and their movement in 30-microm-deep chambers was videorecorded. Spermatozoa of interest were identified during tape playback (hyperactivated spermatozoa were identified by visual assessment of flagellar movement) and sequential head and tail images were traced onto overhead projector film attached to the video monitor. The flagellar movement characteristics beat angle (FBA), beat envelope (FBE) and curvature ratio (FCR) were determined by first principles, and head centroid kinematics were determined using Cartesian methods. Hyperactivated spermatozoa had significantly higher FBA and FBE and significantly lower FCR values than non-hyperactivated spermatozoa (all P<0.0001). The centroid kinematic values were also found to be significantly different, and kinematic criteria for ram sperm hyperactivation were developed. These criteria were refined by consideration of 60-Hz CASA-derived trajectories, and ram sperm hyperactivation was defined by: VCL > 250.0 microm s(-1) and VSL < or = 100.0 microm s(-1) and LTN < or = 30% and ALHmax > or = 9.0 microm.

Normal fertility in ewes after cervical insemination with frozen-thawed spermatozoa supplemented with seminal plasma.

The effect of seminal plasma (SP) on the motility, capacitation status, penetration through cervical mucus and fertility of frozen-thawed ram spermatozoa was examined. In the presence of SP, motility of frozen-thawed spermatozoa was better (P<0.001) and there were more uncapacitated and less acrosome-reacted cells in comparison with controls (P<0.001). Frozen thawed spermatozoa were also better able to penetrate cervical mucus after addition of SP. Addition of SP increased the percentage of ewes pregnant after insemination of frozen-thawed (39/94, 41.5% v. 51/92, 55.4%; P<0.05) but not fresh spermatozoa (34/55, 61.8% v. 42/58, 72.4% for 0 v. 30% SP in the resuspension medium). Moreover, SP improved pregnancy rates after cervical (14/50; 28% v. 25/49; 51%; P<0.05) but not intrauterine insemination (25/44; 56.8 v. 26/43; 60.5%) with frozen-thawed spermatozoa. In a second experiment, pregnancy rates were 30/45 (66.7%), 9/37 (24.3%) and 24/40 (60.0%) for ewes inseminated with frozen-thawed spermatozoa in the uterus (control), cervix without SP and cervix after supplementation with SP, respectively (P<0.01 for unsupplemented v. supplemented spermatozoa). These experiments demonstrate that impaired function of cryopreserved spermatozoa can be overcome by addition of SP, resulting in normal fertility after cervical AI.

Effect of seminal plasma on capacitation and hyperactivation in human spermatozoa.

While hyperactivated motility is known to be a concomitant of capacitation, and a prerequisite for fertilization, the specific interdependence of capacitation and hyperactivation in human spermatozoa has not been investigated. This study was designed to determine the effect of seminal plasma contamination on the expression of hyperactivated motility and the relationship between hyperactivation and capacitation, since seminal plasma contains decapacitation factor(s). Seminal plasma was obtained by centrifugation of aliquots of liquefied semen layered over 1.5 ml 40.5% Percoll and mixed with human tubal fluid (HTF) medium containing 30 mg/ml human serum albumin (HSA) (HTF) to a final concentration of 5% (v/v) seminal plasma (SP). Motile spermatozoa were isolated from the remainder of the semen by swim-up into either HTF or SP medium. Samples were taken from each treatment immediately post-harvest (0 h) and after 60 min at 37 degrees C (1 h) for hyperactivation and capacitation assessment. The treatments were then divided into two portions, centrifuged and resuspended in either HTF or SP, giving HTF control and SP control treatments and two crossover treatments, 1 h HTF then 1 h SP (H/SP) and 1 h SP then 1 h HTF (SP/H). All tubes were incubated for a further 60 min at 37 degrees C before aliquots were taken for hyperactivation and capacitation assessments. Hyperactivation was estimated using an IVOS v10.6t (Hamilton Thorne Research, Beverly, MA, USA) 60 Hz CASA instrument, and capacitation was estimated using the chlortetracycline (CTC) method. The presence of seminal plasma in the capacitation medium for 60-120 min post-swim-up inhibited the development of hyperactivated motility. This inhibition was reversible, and was not prevented by preincubation for 1 h in HTF medium. There was no difference in the CTC binding patterns between treatments at 2 h, indicating that the capacitation-associated membrane changes were not affected by the presence of a low concentration of seminal plasma. There was no correlation between percentage capacitated and percentage hyperactivated spermatozoa for any treatment. Since the proportions of hyperactivated spermatozoa and capacitated spermatozoa were not related, we conclude that the processes leading to hyperactivation and to the membrane changes associated with capacitation are not tightly interlinked and consider this finding to be due to hyperactivated motility being associated with flagellar movement, while the CTC assay assesses changes in the Ca2+ levels of the sperm head plasma membrane.

Minimum sperm trajectory length for reliable determination of the fractal dimension.

The aim of this study was to determine the minimum track length required for the reliable calculation of the fractal dimension of trajectories of capacitating human spermatozoa. Manually reconstructed trajectories classified previously as hyperactivated or non-hyperactivated were re-analyzed. The trajectories were reconstructed at 60 Hz, and each comprised 61 points (corresponding to 1 s movement). The trajectories were 'split' to give the equivalent of 2 x 31 point, 3 x 21 point, 4 x 16 point and 6 x 11 point track segments and the fractal dimension determined for each. The fractal dimensions of each track segment within each trajectory were compared using paired t-tests. No significant difference was observed between the fractal dimensions of track segments of equal length, irrespective of the motility pattern. However, significant differences in fractal dimension values were observed when segments of different lengths were compared (P<0.01). For the non-hyperactivated tracks, the fractal dimension was consistently below the hyperactivation threshold level for only the 31 point segments. The hyperactivated tracks consistently had fractal dimension values above the threshold level when segments of 16 points or longer were analysed. Therefore, the minimum track length for the determination of reliable fractal dimension values was 31 points, corresponding to an image sampling time of 0.5 s at 60 Hz, although it could be used as a screening method for the identification of hyperactivated motility for track segments of greater than 11 points (corresponding to 0.17 s movement).

Quantitative observations of flagellar motility of capacitating human spermatozoa.

For technical reasons sperm head movement is assessed in kinematic analysis, while flagellar movement is the determining factor of head movement, not vice versa. It follows then that the development of new kinematic values to describe the movement of capacitating human spermatozoa should include the analysis of their flagellar movement. The aim of this study was to establish quantitative differences between flagellar movement patterns of hyperactivated and non-hyperactivated spermatozoa which could then be used in the evaluation of new centroid-based kinematic values. Spermatozoa were prepared by swim-up from semen into culture medium supplemented with 30 mg/ml human serum albumin. Sperm movement was recorded in 50 microm-deep chambers using a 200 Hz video system. Sperm movement was classified based on flagellar movement, with 24 non-hyperactivated and 26 hyperactivated spermatozoa included in the study. Flagellar analysis was performed using both a semi-automated analysis system (SIAM FLAG; 30 images at 200 Hz) and manual methods (100 Hz). Hyperactivated spermatozoa had significantly larger flagellar beat angles (> or = 87 degrees) and significantly lower flagellar beat frequencies (< or = 29.4 Hz) than non-hyperactivated human spermatozoa. In addition, the flagellar wave amplitude was significantly greater and the bend diameter significantly smaller for hyperactivated spermatozoa in the proximal region of the flagellum (up to 20 microm from the head-midpiece junction). The velocity of the hyperactivated wave was low in this region, although it was significantly slower than the non-hyperactivated wave in all regions of the sperm tail.

A critical review of the physiological importance and analysis of sperm movement in mammals.

The identification of human sperm hyperactivated motility has potential importance in sperm function tests, as well as in quality control assays and in reproductive toxicology investigations. However, relatively little is known about this phenomenon and the variety of definitions used for hyperactivation has led to a great deal of confusion as to its occurrence and physiological relevance. This presentation is a critical review of a number of aspects of hyperactivated motility, including its identification and potential role(s) in mammalian fertilization. The initial sections of the review consider the mechanisms involved in the development and maintenance of mammalian sperm motility, and the structural and functional changes in spermatozoa which occur during transport through the female reproductive tract. The methods available for the quantification of aspects of sperm movement are also discussed, with an historical overview of sperm movement analysis.

Fractal analysis of capacitating human spermatozoa.

While head centroid-derived kinematic values have been determined for the trajectories of hyperactivated human spermatozoa, the definitions are not robust with respect to image sampling frequency and track analysis methods. The determination of the fractal dimension of a trajectory has been suggested as an alternative descriptive parameter for hyperactivated motility. Here, we have investigated two methods for the determination of the fractal dimension of a trajectory. A simple but useful equation was found to be: D = log(n)/[log (n + log (d/L)], where the n is the number of intervals in the trajectory, d is the planar extent of the curve and L is the length of the trajectory. This equation was not influenced by scaling of the trajectory. A fractal dimension (D) >=1.30 was found to define hyperactivated trajectories, and D <= 1.20 defined non-hyperactivated trajetories, reconstructed at both 30 and 60 Hz. However, when circling tracks were studied, all had D > 1.30, even though they were classified as non-hyperactivated by curvilinear velocity and/or amplitude of lateral head displacement values. An analysis of a series of non-ideal track segments suggested a relationship between a track's linearity and its fractal dimension. It was determined by a linear regression analysis that the fractal dimension of a trajectory was inversely proportional to its linearity (r = -0.77, P < 0.001). Although the fractal dimension of a trajectory is a good indicator of its regularity (describing its space-filling properties), it should not be used as the sole criterion for the classification of a trajectory as hyperactivated.

Variable kinematics of capacitating human spermatozoa.

Human spermatozoa were prepared by swim-up from semen into in-vitro fertilization (IVF) culture medium, and their movement recorded by videomicrography using NTSC video to give 60 images/s on freeze-frame playback. Trajectories in which spermatozoa appeared to switch from one type of motility pattern to another (from non-hyperactivated to transitional or star-spin hyperactivated), were reconstructed manually and the movement characteristics determined for short consecutive sequences along each track. The spermatozoa were able to switch between all of these motility patterns, apparently at random. These observations illustrated that hyperactivated motility is a reversible state in human spermatozoa, with phase changes from hyperactivated to non-hyperactivated motility patterns along trajectories. It was not necessary for spermatozoa to pass through the transitional hyperactivated motility phase when switching from non-hyperactivated to star-spin hyperactivated motility or vice versa.

Calcium-activated proteases in the bovine parathyroid gland: potential role in degradation of parathyroid hormone to peptide fragments.

Our studies suggest that protein kinase C is involved in low calcium (Ca2+)-stimulated secretion of parathyroid hormone (PTH) but not directly in high Ca(2+)-stimulated intracellular degradation of PTH to secreted carboxyl-terminal fragments (C-PTH), an important component of Ca(2+)-regulated PTH secretion. The present study was undertaken to determine the presence of calcium-activated proteases, 84 kDa (micro)-calpain and 80 kDa (milli)-calpain, in the bovine parathyroid, and whether they could degrade PTH to C-terminal fragments. Immunocytochemistry of bovine parathyroid tissue using antibodies raised against bovine heart micro- and milli-calpain detected both isoforms of calpain. Western blotting of total bovine parathyroid cell protein prepared from primary cell cultures confirmed the presence of both isoforms of calpain, demonstrated by specific milli- and micro-calpain bands. Purified bovine PTH (bPTH) was incubated in vitro with human erythrocyte micro-calpain and the cleavage products were separated by reverse-phase HPLC. Eluant fractions were assayed with an RIA with equimolar sensitivity to C-PTH and bPTH, and peak areas integrated. Micro-calpain produced a C-PTH peak from bPTH which co-eluted with the major C-PTH secreted by parathyroid cells in culture. C-PTH production by micro-calpain, expressed as per cent area under the curve, increased from 0% in the absence of either micro-calpain or Ca2+, to 71.5% when a 5:1 molar ratio of bPTH to calpain was used. Amino acid sequencing and analysis of the immunoreactive PTH cleavage products indicated the presence of two fragments of bPTH in the C-PTH peak, bPTH47-48 and bPTH69-84. In summary, both isoforms of calpain are present in the bovine parathyroid and calpains may play a role in the Ca(2+)-dependent degradation of PTH to secreted C-terminal fragments.

Kinematics of capacitating human spermatozoa analysed at 60 Hz.

Hyperactivation is a concomitant of eutherian sperm capacitation, characterized by the development of high amplitude flagellar waves with a corresponding increase in velocity. In humans, kinematic values have been derived which describe the movement characteristics of spermatozoa analysed at 30 images/s. However, these values are frame rate-dependent, and modern computer-aided sperm analysis (CASA) instruments used for studying sperm movement now use 60 images/s. This study used first-principles manual track analysis to derive the range of movement characteristics which describe hyperactivated motility of human spermatozoa at 60 images/s. Standard terminology for centroid-derived movement characteristics, as recommended by the World Health Organization, was used. US-standard (NTSC) video recordings of capacitating human sperm populations were replayed using a non-interlaced freeze-frame video cassette recorder, and individual tracks reconstructed on acetate overlays. Tracks were classified as either forward progressive or hyperactived based upon flagellar beating patterns, then reconstructed manually at x3540 and analysed using both manual methods and basic geometric calculations from (x, y) coordinates (Cartesian methods) similar to those used by CASA instruments. In all, 40 hyperactivated and 40 forward progressive tracks were studied. A set of Boolean arguments defining hyperactivated motility was derived, and there was generally good agreement between the limits derived by manual and Cartesian methods. The limits for the definition of hyperactivated motility of human spermatozoa at 60 Hz derived by Cartesian methods were: curvilinear velocity > or = to 180 micrograms/s and linearity < or = to 45% and wobble < 50% and amplitude of lateral head displacement ALHmean > 6.0 micrograms or ALHmax > 10.0 micrograms.(ABSTRACT TRUNCATED AT 250 WORDS)

Workshop report: clinical CASA--the quest for consensus.

The goals of the Workshop were to establish the current state-of-the-art in the clinical use of computer-aided sperm analysis (CASA), to identify areas of contention or confusion in the use of CASA technology, and to reach consensus on these matters to facilitate the wider use, and usefulness, of these instruments in clinical and research applications. CASA assessments of sperm morphology were not considered as they were discussed in a separate Workshop (Advanced Techniques in Sperm Preparation and Imaging) on analytical techniques. Four topics were considered: (a) CASA and semen analysis; (b) the diagnostic value of sperm kinematics in semen; (c) human sperm hyperactivation; and (d) CASA and fertility prediction. In all, 17 specific consensus points were identified.

Methods of sperm preparation for assisted reproduction.

Because seminal plasma contains factors which inhibit the fertilising ability of spermatozoa, it is essential that spermatozoa be separated from it quickly and efficiently. Four basic approaches for this exist: (1) simple dilution and washing; (2) sperm migration (either direct from liquefied semen, from a suspension of washed spermatozoa or from a washed sperm pellet); (3) selective washing procedures (mainly Percoll and Nycodenz density gradients); and (4) adherence methods for the elimination of debris and dead spermatozoa (glass wool, glass beads and Sephadex columns). While the success of a sperm preparation method is often assessed by its yield of motile spermatozoa, it is also vital that sperm preparations for clinical use should be free of any microbiological contaminants present in semen. Other relevant considerations in choosing a method include its technical complexity as well as its material, apparatus and time costs. Any possible exposure of spermatozoa during preparation to deleterious influences that may cause iatrogenic sperm dysfunction must obviously be avoided at all costs. Consequently, methods involving centrifugal washing steps prior to the selection of motile spermatozoa should be discontinued. Direct swim-up from semen remains the simplest way to obtain populations of highly motile spermatozoa and, dependent upon the absolute yield required, can be a very rapid procedure with normal semen samples. Abnormal samples, especially those with increased viscosity, may benefit from a prior filtration on a glass bead or Sephadex column. The initial motile sperm preparation should be washed once (perhaps twice) to minimise seminal plasma contamination of the final preparation. Several rapid, simple discontinuous Percoll gradients giving excellent yields are available.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation and inhibition of protein kinase C in cultured bovine parathyroid cells: effect on the release of C-terminal fragments of parathyroid hormone.

The response of the parathyroid gland to low Ca2+ may be mediated in part by protein kinase C (PKC). We assessed the effect of two PKC activators, SC-9 and SC-10, and one PKC inhibitor, H-7, on Ca(2+)-regulated PTH release and degradation in primary cultures of bovine parathyroid cells. Both SC-9 and SC-10 stimulated PTH release, compared to high Ca2+ alone, in parathyroid cells incubated in high Ca2+, with maximal PTH release of at least twofold occurring at a concentration of either activator of 10 nM (p less than 0.05). We have previously shown that another PKC activator, PMA, not only enhances PTH release in the presence of high Ca2+ but suppresses low Ca(2+)-stimulated PTH secretion. In the present study, neither SC-9 nor SC-10 caused a comparable suppression of PTH release at low Ca2+. However, the PKC inhibitor, H-7 (1 microM), blocked low Ca(2+)-stimulated (compared to the low Ca2+ control) PTH secretion by approximately 50% (p less than 0.01) and did not affect high Ca2+ suppression of PTH secretion. H-7 (1 microM) was able to oppose the stimulation of PTH release by the PKC activators SC-9, SC-10, and PMA at high Ca2+ and negated the PTH release-inhibiting effect of PMA at low Ca2+. Culture medium from these experiments was subjected to reversed-phase HPLC and the eluted fractions analyzed by RIA for the presence of intact and C-terminal fragments of PTH.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of phorbol myristate acetate on the intracellular degradation of bovine parathyroid hormone.

The suppression of PTH release by high extracellular calcium (Ca2+) has been associated with secretion of biologically inactive carboxyl-terminal fragments of PTH (C-PTH), while relatively more intact PTH is released under low extracellular Ca2+ conditions. In the presence of high extracellular Ca2+, phorbol myristate acetate (PMA) has been shown to stimulate PTH release to levels observed at low Ca2+, suggesting that protein kinase-C (PKC) is involved in the regulation of PTH secretion. We have examined the effect of PMA on PTH secretion and the release of PTH fragments at high and low calcium concentrations. Primary cultures of bovine parathyroid cells were incubated for 90 min in 0.5 mM (low) or 2.0 mM (high) Ca2+ with or without 1.6 microM PMA. Reverse phase HPLC using an 18-60% gradient of acetonitrile in 0.1% trifluoroacetic acid was performed on the medium from these incubations, and the eluant fractions were analyzed with a carboxyl (C)-terminal-specific PTH RIA. Medium from cultures exposed to low Ca2+ exhibited two large peaks of PTH immunoreactivity, coeluting with intact PTH-(1-84) and a synthetic human C-PTH-(39-84). PMA treatment at low Ca2+ resulted in the secretion of a greatly reduced amount of intact PTH, suggesting that PKC may increase the production of PTH fragment. At high extracellular Ca2+ PMA caused an increase in total immunoreactive PTH release similar to that seen at low Ca2+. However, on HPLC analysis, proportionally more PTH eluted in the position of the C-PTH fragment than was seen with low Ca2+ stimulation of PTH secretion. It, therefore, appears that the degradation of PTH to C-PTH may be linked to activation of PKC and can be separated from the Ca2+ regulation of PTH release occurring at the cell membrane.

A simplified approach to sperm-cervical mucus interaction testing using a hyaluronate migration test.

Fifty-one comparisons were made of human sperm migration into capillary tubes containing either human cervical mucus ('Kremer test') or a synthetic mucus substitute consisting of a 5 mg/ml solution of sodium hyaluronate (average mol. wt 2 x 10(6)) in a phosphate-buffered medium. The results of these two tests were highly significantly correlated and dependent upon the same sperm characteristics reflecting sperm progressive ability (including the specific movement characteristic of lateral head displacement amplitude), morphological normality and cellular vitality as well as the concentration of these more functional cells in the semen. The result of the hyaluronate migration test, in conjunction with the mucus quality measures of Insler score and pH, allowed a 92.2% correct prediction of the Kremer test outcome (90.9% of normal tests and 93.1% of abnormal tests). In this data set, these values also corresponded to the sensitivity and specificity of the analysis, respectively. From these studies, we propose the hyaluronate migration test as a useful adjunct to routine semen analysis, sperm movement analysis and the more traditional in-vitro tests of sperm-cervical mucus interaction in the diagnostic investigation of infertile couples. It effectively assesses the mucus-penetrating potential of a semen sample without the need for relatively large quantities of midcycle cervical mucus; it will therefore augment (as an internal control), although not necessarily replace, the homologous Kremer test and reduce the quantity of both patient and donor mucus needed for comprehensive crossed-hostility format testing of sperm-mucus interaction.