The influence of sperm density on the motility characteristics of washed human spermatozoa

To study the effects of sperm density on the results of computer-assisted semen analysis (CASA), 10 washed semen samples were diluted and measured with the CellTrak/S CASA system in a concentration range of 10–180×10⁶ spermatozoa/ ml. All sperm motility parameters were influenced to some extent by sperm density. The motility percentage was influenced significantly in 5 samples ( P< 0.005), the straight line velocity in all samples (P<0.0005 in 7 samples), the curvilinear velocity in 3 samples ( P< 0.005), the linearity in 9 samples (P<0.0005 in 6 samples) and the lateral head displacement in 9 samples (P< 0.005 in 6 samples). In general, the CellTrak/S data are influenced significantly if sperm density exceeds 50 × 10⁶ spermatozoa/ml.
The influence of sperm density on the motility parameters can be explained both by the accuracy of the CASA system and by actual changes in the motility of the spermatozoa. In the light of other published studies, it is concluded that sperm motility measurements with CASA systems should be assessed using 25–50 × 10⁶ spermatozoa/ml, especially in studies concerning lateral head displacement and the linearity, as in sperm hyperactivation studies.     

Kruger strict morphology and post-thaw progressive motility in cryopreserved human spermatozoa

The purpose of this prospective study was to evaluate Kruger strict morphology and conventional semen analysis in predicting cryosurvival and the progressive motility recovery rate of frozen spermatozoa. Our study included 56 semen samples with >10 million spermatozoa per ejaculate. The main outcome measures were conventional semen analysis, strict morphology analysis by the Kruger method, cryosurvival rate and post-thaw sperm motility. A significant reduction in sperm motility after cryopreservation was demonstrated. The freeze-thawing process caused a 66% reduction in rapid progressive motile spermatozoa, a 45% reduction in slow progressive motile spermatozoa and a 2% reduction in nonprogressive motile spermatozoa. The cryosurvival and progressive motility recovery rates were not correlated with parameters of conventional semen analysis, such as sperm concentration, motility, WHO morphology and total motile count, but the progressive motility recovery rate was significantly correlated with the percentage of spermatozoa exhibiting Kruger normal morphology (P = 0.028). The recovery rate of rapidly progressive motility was profoundly decreased compared with slow progressive motility following the frozen-thaw procedure of semen. Kruger strict morphology assessment was a better predictor of the progressive motility recovery rate following the freezing-thaw procedure than parameters of conventional semen analysis.     

Determination of Human Sperm Count and Sperm Motility Using a Laser Beam and the Doppler Effect (LAZYMOT Machine)

For 25 consecutive human semen samples, a comparison was made of sperm count and sperm motility values obtained by routine manual methods and by using a machine that measures these functions by analysing the deflection of an impinging laser beam (Lazymot machine). Sperm counts in undiluted semen were approximately 5 times higher with the laser machine. As sperm counts increased to about 300 million/ml the counts obtained by the two methods converged as the chance of the beam hitting a spermatozoon and not another type of particle increased. In semen diluted 1 + 4 with Baker's solution, the uncorrected laser count agreed well with the sperm count obtained using a haemocytometer. Multiplication of the laser count by 5 did not reach the same count as that measured in the undiluted sample, showing that the dilution had dissolved some of the smaller particles. It was recommended to measure laser percentage motility in undiluted semen but the values obtained bore no relationship to those obtained using a haemocytometer and neither did the values obtained for laser percentage sperm with progressive motility. The mean laser velocity of the total motility was 23-64 micron/sec and for the progressive particles was 48-84 micron/sec, values which were much faster that the acceptably normal values of 8-30 micron/sec found for selected progressively motile spermatozoa timed with a stopwatch. The laser machine detected an increase in counts and the presence of residual motility after cytoplasm had been stripped away from the spermatozoa with a saponin reagent. The laser machine was unable to detect any increase in speed on increasing the temperature to 37 degrees C. It was concluded that the Lazymot machine as presently designed is not useful in the andrological laboratory for routine counting and motility determinations, mainly due to the absence of a size discriminator against the multitude of small particles that are present in human semen.     

Pentoxifylline stimulates various sperm motion parameters and cervical mucus penetrability in patients with asthenozoospermia

Pentoxifylline (PTX) was incubated in vitro with human spermatozoa to examine its effects on sperm motility characteristics and bovine cervical mucus penetrability (BCMP). Sperm motion parameters were assessed by computer-assisted motion analysis (CASA) using HTM-IVOS and BCMP was evaluated using the Penetrak kit. In vitro incubation with PTX (1  mg  ml⁻¹; 3.6  mm, 30  min) did not significantly change percentage motility, average path velocity (VAP), straight-line velocity (VSL) or beat cross frequency (BCF) of spermatozoa from normozoospermic or asthenozoospermic samples. However, it significantly increased curvilinear velocity (VCL), amplitude of lateral head displacement (ALH) and hyperactivated motility (HA), and significantly decreased linearity (LIN) of spermatozoa from both samples. Pentoxifylline was found to increase BCMP scores for spermatozoa from asthenozoospermic samples, but did not affect scores for spermatozoa from normozoospermic samples. Bovine cervical mucus penetrability (BCMP) was found to be positively and significantly correlated with the percentage motility of both non-PTX-treated and PTX-treated spermatozoa for asthenozoospermic samples. These results demonstrated that PTX enhanced several motion sperm parameters as well as BCMP in asthenozoospermic samples and suggest a potential use of the methylxanthine in infertile patients with motility defects undergoing artificial insemination.     

Effect of recombinant β-defensin 1 protein on human sperm motility and viability

The reduction of sperm motility and subsequently reduced ability to undergo capacitation and acrosome reaction are considered as common causes of male infertility. The β-defensin family is a group of well-known secretory proteins with antimicrobial activity that contribute to the process of “sperm maturation” during the passage of spermatozoa in the epididymis when spermatozoa attain its motility. One member of this family is “β-defensin 1” which is present in seminal plasma and spermatozoa. The aim of this study was the incubation of human processed spermatozoa with recombinant β-defensin 1 (500 ng/ml) for 1, 2 and 3 hr at 37°C under 5% CO₂ atmosphere and assessment of sperm viability and motility in 59 semen samples. The analysis of semen samples such as sperm concentration, motility, viability, morphology and semen volume was performed according to the World Health Organization (2010; World health organization laboratory manual for the examination and processing of human semen (p. 287). Geneva, Switzerland: World Health Organization) criteria. The result of the current study shows that the incubation of spermatozoa with recombinant β-defensin significantly maintained percentage of sperm viability and motility compared to processed spermatozoa incubate in the absence of β-defensin in the studied time intervals (p < .05). Therefore, we concluded that recombinant β-defensin 1 protein as an agent with antimicrobial activity can maintain sperm viability and motility in in vitro condition.     

Pentoxifylline increases sperm penetration into zona-free hamster oocytes without increasing the acrosome reaction

Summary. Several drugs have been used to stimulate human sperm motility, including 3-deoxy-adenosine, caffeine, and pentoxifylline. Pentoxifylline is an inhibitor of the phosphodiesterase and may stimulate sperm motility by increasing the intracellular levels of cAMP. In this study we have evaluated the effect of pentoxifylline in the outcome of the sperm penetration assay into zona-free hamster oocytes. Twenty-seven semen samples, obtained for diagnostic purposes, were used. After the motile sperm were selected by the swim-up technique, the samples were divided into two aliquots. One aliquot was incubated with 1 mg ml⁻¹ of pentoxifylline at 37 °C, 5% CO₂ for 30 min. The control aliquot was incubated with culture medium. The samples were then washed and resuspended in fresh, pentoxifylline-free medium, at a sperm concentration of 10 × 10⁶ cells ml⁻¹. One hundred microlitres of each sperm suspension was then deposited under oil and 30–40 zona-free hamster oocytes were added. After 6 h of gamete coincubation, the percentage of penetrated oocytes and the number of decondensed sperm heads were evaluated. The percentage of acrosome-reacted sperm was evaluated using the Pisum sativum lectin. The percentage of zona-free hamster oocytes penetrated was increased after pentoxifylline-treatment. The percentage of acrosome reacted sperm and the number of decondensed sperm heads per egg were not different between the control and the pentoxifylline-treated groups. The results suggest that the beneficial effect of pentoxifylline upon the sperm cells is not mediated by stimulation of the acrosome reaction.     

The motility of demembranated human spermatozoa is inhibited by free calcium ion activities of 500 nmol/L or more

A number of studies have demonstrated that high calcium ion activities inhibit sperm motility, but little is known about the effect of different calcium activities close to the physiological range. Therefore, we investigated whether raising calcium activities within the submicromolar range would inhibit the motility of demembranated human spermatozoa. Spermatozoa were demembranated with Triton X-100 and motility was measured objectively by computer assisted semen analysis. Motility, reactivated by 1 mol adenosine 5'-triphosphate (AlphaTauP)/L, was short lived, with maximum activity only sustained for about 1 min. Reactivated motility was not affected by 50 micromol cAMP/L. The amplitude of lateral head displacement was significantly greater at room temperature than at 37 degrees C, but there were no significant differences between the percentage of sperm motile or their velocity at the two temperatures. The calcium buffer 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) at 1 mmol/L was included in the demembranation-reactivation medium, and free calcium ion activities were calibrated using the fluorescent calcium probe Fura-2. Calcium ion activities of > or =500 nmol/L significantly inhibited the percentage of demembranated-reactivated spermatozoa that were motile, and the velocity and lateral head displacement of these cells. The range of intracellular calcium activities in spermatozoa from 24 cryopreserved ejaculates was 110-534 nmol/L; roughly twice the value in fresh spermatozoa. Therefore, calcium ion activities in the range observed in cryopreserved spermatozoa can inhibit the activity of demembranated human spermatozoa.     

Maintenance of human sperm motility and prevention of oxidative damage through co-culture incubation

Summary. Co-culture incubation is one of the important techniques used in basic and clinical research of assisted reproduction. In this study, sperm samples from 40 healthy donors were prepared for co-culture incubation with Vero cells which had been derived from the kidney fibroblasts of the African green monkey, Cercopithecus aethiops. We found that the motility characteristics of ejaculated human sperm co-cultured with Vero cells were largely maintained and the percentage of hyperactivated sperm in the co-culture group was not affected. While the sperm of the control group completely lost the motility at 12 h incubation at 37°C in 5% CO₂, the sperm co-cultured with Vero cells still maintained 74% of the original motility. Lipid peroxidation and accumulation of 8–hydroxy-2'-deoxyguanosine in spermatozoa were also reduced by the co-culture incubation, which strongly indicates that intercellular interactions may play some role in the maintenance of sperm functions. We conclude that the oxidative damage in vitro of the sperm can be reduced by the co-culture system and thereby maintains the function of sperm from oxidative damage.     

The relationship between ejaculated ram sperm mitochondrial protein synthesis and motility

Summary. D-chloramphenicol, at concentrations of 20 and 40 μg/ml, inhibited over 80% of the newly synthesized mitochondrial proteins expressed by incorporation of ³H-amino acid mixture. At concentrations of 20 and 40 μgml⁻¹, D-chloramphenicol enhanced the collective motility of washed ram spermatozoa. The collective motility measured by the multichannel Reflectospermiograph system, significantly enhanced the motility wave frequencies and amplitudes by 24–17% and 65–32%, respectively. Furthermore, the longevity of the collective motility was prolonged by 12–19%. In about 20% of the cases, when the original sperm motility was low, it was found that 40 μg ml⁻¹ D-chloramphenicol has maximum stimulation effect on sperm motility in inverse fashion. Since the mitochondria are located adjacent to the motility initiation area, it can be speculated that the mitochondrial protein(s) directly inhibiting the axonemal-ATPase activity or indirectly blocking sperm metabolite, are essential for maintaining sperm motility.     

Effect of Two Methylxanthine Derivatives and Four Prostaglandins on the Motility of Spermatozoa from Volunteers and Oligozoospermic Patients

Caffeine has a dose dependant effect in vitro on the motility of human spermatozoa obtained from volunteers. In doses of 6 mM and 60 mM it delayed the fall in motility with time of spermatozoa when added within an hour of ejaculation. 6 mM caffeine had similar effects on the motility of spermatozoa obtained from oligozoospermic patients. However, 6 mM caffeine was ineffective in modifying the motility of previously cryopre-served spermatozoa. In vitro theophylline 6 mM was effective in delaying the fall in motility with time of human spermatozoa obtained from volunteers and oligozoospermic patients within one h of ejaculation. In the doses used (prostaglandin E₁, 25 μg/ml, E₂, 25 μg/ml, F_1α. 5 μg/ml and F_2α, 5 μg/ml) none of the prostaglandins delayed the fall in in vitro motility with time of human spermatozoa obtained from volunteers or oligozoospermic patients, in fact prostaglandins E₁, E₂, and F_2α caused an inhibition of the motility of spermatozoa obtained from volunteers.     

Catalase improves motility,vitality and DNA integrity of cryopreserved human spermatozoa

Cryopreservation of human spermatozoa offers a pre‐therapeutic possibility of preserving progenity in patients with testicular tumours. We aimed to investigate effects of cryopreservation and addition of catalase on sperm motility, vitality and DNA integrity in fresh and swim‐up spermatozoa. Semen samples were collected from 50 fertile men. Each sample was divided into two parts. First part was subdivided into two equal aliquots: both cryopreserved with and without catalase. The second part was subdivided into two equal aliquots: both processed by swim up and then cryopreserved with or without catalase. Semen analyses, sperm vitality and sperm DNA integrity were performed. Sperm concentration showed significant decrease while percentage of progressive motility, sperm vitality and % of DNA damage showed significant increase in processed and cryopreserved processed samples when compared with fresh and cryopreserved fresh samples. There was no significant difference in sperm concentration while there was significant increase in % of progressive motility and sperm vitality and % of DNA damage showed significant decrease in samples with catalase when compared with samples without catalase (either fresh or processed). Catalase supplementation (fresh and processed) during cryopreservation results in better post‐thawing percentage of progressive motility and percentage of sperm vitality and improved DNA integrity.     

Adenosine triphosphate (ATP) in human spermatozoa

The seminal adenosine triphosphate (ATP) content was determined by bioluminescence after treatment with trichloroacetic acid (TCA) in 81 semen samples 1.5 h after ejaculation obtained from men attending our fertility clinic, and selected to contain either 20% or less spermatozoa with good progressive motility (n = 22), or 60% or more spermatozoa with good progressive motility (n = 59) (Study I), and in 18 semen samples from fertile men 30 min and 3.5 h after ejaculation (Study II). The latter samples were divided into 2 equally large groups according to sperm motility. In Study I the mean sperm ATP concentration was significantly higher in the semen samples with bad motility (0.63 nmol per living spermatozoa x 10(-6)) than in semen samples with good motility (0.39 nmol per living spermatozoa x 10(-6); P less than 0.01). In Study II the ATP concentration per living spermatozoa was also lower in the group with the best motility in comparison with the spermatozoa with lower motility (P less than 0.01), both 30 min and 3.5 h after ejaculation. During the 3-5 h incubation the sperm ATP concentration decreased by 21% (P less than 0.01) in the former group of samples but remained unchanged in the latter group. The results indicate that, in semen samples with highly motile spermatozoa, the consumption of ATP is higher than in semen samples with impaired sperm motility. It is therefore essential that the time between ejaculation and ATP measurement is as short as possible to obtain comparable results. Repeated ATP measurements in combination with an analysis of the number of living spermatozoa, may provide further information on the fertilizing capacity of spermatozoa.     

Cyclic AMP-dependent protein kinases in semen from men with subnormal sperm motility or morphology

Photoaffinity labelling by 8-N₃-/³²P/cAMP of human sperm homogenates with normal pathology and normal progressive motility of the gametes (N) revealed the presence of 2 specific cAMP-binding activities with MW 47 000 (R-I) and 52 000 (R-II). Similar amounts of R-I and R-II were found in sperm homogenates obtained from semen samples with either reduced progressive motility (A50: 30–50% progressive motility and A30: < 30% progressive motility) or abnormal morphology of the gametes (T: 5–30% normal heads). In contrast, the seminal plasma of the respective semen samples of N, A50, A30 and T incorporated the 8-azido cAMP photolabel in similar quantities into 2 additional cAMP-binding proteins with MW 42 000 and 37 000. Quantitative analysis of the photoaffinity labelling of the seminal plasma obtained from normal (N) or subnormal semen (A50, A30, T) showed that cAMP was predominantly bound to the 37 000 protein. Furthermore, the cAMP-dependent protein kinase and cAMP-binding activities of sperm homogenates were quantitatively comparable within the 4 groups. These results provide the first evidence that neither quantitative nor qualitative differences in the molecular and/or enzymatic properties of cAMP-dependent protein kinases can be detected between normal human sperm (N) and those with either reduced progressive motility (A50, A30) or abnormal morphology (T).     

A new CentriSwim procedure to increase the recovery of motile spermatozoa

A prospectively controlled in vitro study was performed to compare sperm concentration, sperm motility and progressive sperm motility recovered following the standard swim-up procedure and a new CentriSwim procedure. The CentriSwim procedure involves creating a centrifugal force to counteract the force of gravity during sperm swim-up procedure. Two aliquots of semen from 12 normozoospermic ejaculates and 12 laboratory-induced oligoasthenozoospermic specimens were diluted, centrifuged, and 1.0 ml of media layered over the sperm pellet. One aliquant was processed by standard swim-up technique. The other aliquant was processed by CentriSwim procedure involving centrifugation at 200 rpm on a 2-cm radius upward-directing arm, at an angle of 60 degrees for 10 min, creating roughly 0.8 g centrifugal force at room temperature (22-24 degrees C) to counteract the force of gravity. The numbers of spermatozoa recovered from the upper 0.5 ml of the medium following CentriSwim from the normozoospermic ejaculates and laboratory-induced oligoasthenozoospermic specimens were significantly higher than following standard swim-up procedure. No statistical differences in the recovery of percentage sperm motility and progressive sperm motility between the two techniques were observed. In conclusion, the CentriSwim procedure yields higher numbers of motile spermatozoa than the standard swim-up technique.     

Comparison of measurements of human sperm motility characteristics by the automated CellSoft system and time-exposure photomicrography

Human sperm motility characteristics in 28 semen samples with sperm concentrations less than 40 x 10(6) ml-1, as determined by the World Health Organization manual analysis (WHO, 1987), were measured by the automated CellSoft semen analyser (Cryo Resources Ltd, New York, NY, USA) using different system parameter settings (Mortimer & Mortimer, 1988a). The results were compared with those obtained by time-exposure photomicrographic (TEP) analysis. It was found that the settings of the minimum video frame rate and the threshold velocity used to distinguish motile from immotile sperm by the automated CellSoft system had a significant influence on measurements of percentage motility but not on linear velocity. At the five different parameter settings used in the present study, the automated CellSoft system gave significantly lower mean values for percentage motility in comparison with the WHO manual and TEP analyses. Measurements for linear velocity between the automated CellSoft system and TEP analyses were found not to be significantly different in these defined semen samples.     

Predictive value of CASA parameters in IUI with frozen donor sperm

The objective of this study was to determine if characteristics of sperm motion determined by computer-aided semen analysis (CASA) after thawing and preparation on discontinuous gradient could predict pregnancy outcome after intrauterine insemination (IUI) from frozen donor sperm. A retrospective analysis of 100 non-selected women undergoing 171 consecutive donor insemination cycles was conducted between January 2006 and April 2007. Semen samples from all donors were analysed after thawing and density gradient preparation. Women who became pregnant and those who did not were comparable in terms of age, ovarian stimulation regimen and indication of IUI with donor semen. Pregnancy rate per cycle was 21.8%, and pregnancy occurred after 2.5 IUI cycles on average. Motility parameters of sperm measured by CASA (VAP, VCL, VSL, LIN, STR, and ALH) and total spermatozoa concentration after preparation on discontinuous gradient showed no difference in both groups. Progressive and total motile spermatozoa concentration, as well as progressive and total motile percentages was significantly higher in pregnancy group. The receiver operating characteristic (ROC) curve analysis showed that total motile percentage >17% and motile concentration >0.9 × 10⁶/mL best predicted pregnancy. In a multivariate analysis, only total motility percentage was able to predict pregnancy. Sperm motility parameters of frozen-thawed prepared donor sperm obtained by CASA do not seem to predict pregnancy in IUI cycles. Total motile and progressive percentages and concentrations remain the best prognostic elements for pregnancy in IUI with donor semen.     

Semen analysis with regard to and functional aspects sperm number,sperm morphology

The new World Health Organization （WHO） Manual for Semen Analysis contains several improvements. One is that the 20 million spermatozoa per mL paradigm has been ousted in favour of proper calculations of lower reference limits for semen from men, whose partners had a time-to-pregnancy of 12 months or less. The recommendation to grade the progressive motility as described in the third and fourth editions of the WHO manual was not evidence-based, and WHO was therefore motivated to abandon it. However, the new recommendation is not evidence-based either, and it is difficult to understand the rational for the new assessment. It may have been a compromise to avoid returning to the rather robust system recommended in the first edition （1980）. The unconditional recommendation of the ＇Tygerberg strict criteria＇ is not evidence-based, and seems to be the result of an unfortunate bias in the composition of the Committee in favour of individuals known to support the ＇strict criteria＇ method. This recommendation will have negative effects on the develop- ment ofandrology as a scientific field. Given the importance of the WHO manual, it is unfortunate that the recommenda- tions for such important variables, as motility and morphology, lack evidence-based support.     

Effect of pentoxifylline on motility and membrane integrity of cryopreserved human spermatozoa

The purpose of this study was to examine the effects of pentoxifylline used before and after semen cryopreservation-thawing on sperm motility and membrane integrity. Twenty-four semen samples were split into four equal aliquots. Aliquots were incubated at 37 degrees C for 30 min, followed by cryopreservation with TEST-yolk freezing medium using slow programmable freezing protocol. After 2 weeks the sperm samples were thawed, washed twice in Quinn's Sperm Washing Medium (modified HTF with 5.0 mg/mL Human Albumin) and incubated at 37 degrees C for 30 min. Aliquots were treated by adding 3 mmol/L pentoxifylline to: (1) fresh sperm samples during incubation period prior to cryopreservation, (2) sperm samples as a supplement to the cryoprotectant prior to cryopreservation, and (3) thawed sperm samples during incubation period. One aliquot received no treatment (control group). The addition of 3 mmol/L pentoxifylline to fresh semen during incubation period prior to cryopreservation significantly decreased progressive and total motility compared with controls. However, the addition of 3 mmol/L pentoxifylline to cryopreserved semen after thawing significantly increased progressive and total motility compared with controls. After post-thaw, no differences in motion characteristics between sperm samples treated by adding 3 mmol/L pentoxifylline as a supplement to the cryoprotectant and control groups were observed. Post-thaw hypoosmotic swelling (HOS) test scores did not improve with the addition of pentoxifylline compared with the control group. It is concluded that pentoxifylline enhanced post-thaw motility of cryopreserved human spermatozoa when added after thawing. No improvement was found by freezing sperm with pentoxifylline.     

Investigations on the influence of bradykinin upon the motility of ram spermatozoa

The influence of bradykinin, a component of the kallikrein-kinin system, on the motility of ram spermatozoa was examined in vitro (photometric motility evaluation, penetration test in cervical mucus of sheep, estimation of the percentage of forward-moving spermatozoa in the thermal resistance test). Whereas the motility was found to be influenced by bradykinin the acrosomal status remained undisturbed by it. With fresh as well as with frozen semen, the drug mainly increased the motility of samples with a low initial motility. With frozen semen, the penetration test revealed greater motility rises at 22 degrees C than at 38 degrees C. The importance of the results in relation to the angiotensin-converting enzyme (kininase) as well as the motility evaluation capabilities of the methods used are discussed. There was, however, no positive drug effect after adding the drug in the course of cryopreservation prior to freezing semen in straws although bradykinin solutions frozen in liquid nitrogen maintained their effectiveness.     

Semen parameters in Norwegian fertile men

The World Health Organization (WHO) provides guidelines for assessing the various semen variables. A set of reference ranges is given in the WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction, but several studies indicate that the values should be revised. Furthermore, semen parameters obtained at different laboratories are not directly comparable even if the same methods are used. Thus, it is recommended that each laboratory establish its own reference ranges. In this study, semen from 99 men who had recently achieved a pregnancy were analyzed to establish reference ranges for semen variables. The reference values were based on the group with time to pregnancy (TTP) 12 cycles or less (92%) and abstinence time from 2 to 7 days. The 5th and 10th percentiles for sperm concentration were 10.6 and 16.9 x 10(6)/mL, respectively, and 33% (5th percentile) and 43% (10th percentile) for spermatozoa with progressive motility. These values were below the WHO lower limit. The percentages of ideal spermatozoa (percentage with normal morphology according to WHO strict criteria) were 3 (5th percentile) and 4 (10th percentile). Thirty-nine percent reported that their partners became pregnant during the first cycle after they had stopped using contraception. The semen parameters in this group were compared with the others. Overall, the semen parameters were more favorable in the group with TTP = 1 cycle than in the group with TTP > 1. Sperm concentration, progressive motility, and percentage of ideal spermatozoa according to WHO strict criteria were significantly different in the 2 groups. However, when analyzed by multiple logistic regression, only "total numbers of sperm with progressive motility" remained in the model (P = .002). This is in accordance with previous studies indicating that a combination of semen characteristics provides a better predictor of male fertility potential than the single parameters. In conclusion, new reference ranges for semen variables deviating from the WHO values are established for our laboratory.