The Split Ejaculate: Assessment of Fertility Potential Using Two in vitro Test Systems

Semen samples (split & whole ejaculates) were obtained from 12 normal men (group A) and 8 oligospermic infertile men with sperm concentrations of less than 20 x 10(6) sperm/ml (group B). All samples were evaluated by standard semen analysis, bovine cervical mucus penetration assay (CMPT), and, in all cases with sufficient sperm, in the human spermatozoa zona-free hamster in vitro penetration assay (SPA). In group A the motile sperm concentration was significantly higher in the ejaculated material of the first two contractions (fraction I or FI) than in the remainder of the ejaculate (fraction II or FII) (p less than 0.02). No significant differences were observed in sperm penetration into zona-free hamster ova or bovine cervical mucus by sperm from FI, FII or the whole ejaculate. Motile sperm concentration was significantly correlated with sperm penetration into bovine cervical mucus (r = 0.65, p less than 0.01), but not into zona-free hamster ova (r = 0.01 NS). In the samples collected by group B, the mean sperm concentration and motile sperm concentration were higher in the first (FI) than in the second (FII) fractions of the split ejaculate or the whole ejaculate (p less than 0.05). No significant differences were found among the FI, FII and the whole ejaculate semen samples for penetration of sperm into bovine cervical mucus. Sperm concentration and motile sperm concentration were significantly correlated with sperm penetration into bovine cervical mucus (r = 0.58, p less than 0.01 and r = 0.57, p less than 0.01, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlation of the bovine cervical mucus penetration test with human sperm characterisitics in 1,406 ejaculates

The bovine cervical mucus penetration test (BCMPT) was performed to determine its usefulness in screening the ability of sperm to successfully penetrate mucus in vitro. Ejaculates were obtained by masturbation from patients attending an infertility clinic. Routine semen analysis was performed using a microcomputerized multiple-exposure photography system. The BCMPT was performed. Overall, the average penetration of the mucus was 38 +/- 0.46 mm. Of the 1,406 ejaculates analyzed, 244 (17%) displayed a negative result (0-20 mm), 291 (21%) a questionable result (21-30 mm), and 871 (62%) a positive result (>30 mm). A highly significant (p < .001) correlation between mucus penetration distance and sperm MD (r = 0.541), MI (r = 0.484), count (r = 0.475), motility (r = 0.448), velocity (r = 0.400) and morphology (r = 0.369) was observed. Overall, the finding of an abnormal semen parameter resulted in a 34 +/- 5% accurate prediction of a negative or questionable BCMPT (<30 mm), while a normal semen parameter resulted in a 90 +/- 4% accurate prediction of a positive BCMPT (>30 mm). Sperm MD showed the strongest positive predictive value (98%), while morphology showed the greatest negative predictive value (50%). Of the 1,406 samples, 25 +/- 2% of the samples with normal semen parameters displayed a negative BCMPT. Conversely, 6 +/- 2% of samples with abnormal parameters showed a positive BCMPT. The BCMPT successfully identifies a significant subpopulation of patients as having an inadequate penetration of mucus with otherwise normal semen characteristics.     

Application of a bovine cervical mucus penetration test

One hundred seventy-eight ejaculates obtained from infertility patients were evaluated by routine semen analysis and by a bovine cervical mucus penetration test (BCMPT). A significant correlation (p less than 0.01) was observed between mucus penetration and both sperm count (r = 0.349) and sperm motility (r = 0.394). One hundred fifty-two of 178 patients (85%) had normal sperm counts (greater than 20 x 10(6)/ml). Of these patients, 68% had good (greater than 30 mm), 26% had questionable (21-30 mm), and 7% had abnormal (less than 20 mm) penetration values. One hundred sixty-one of 178 patients (90%) had normal sperm motilities (greater than 40%). Of these patients, 71% had good, 25% had questionable, and 4% had abnormal penetration values. Conversely, 46% and 18% of patients with abnormal sperm count and motility, respectively, had normal penetration values (greater than 30 mm). A significant relationship (p less than 0.05) was observed between the BCMPT and pregnancy problems not apparent by semen analysis data, and may prove to be a useful adjunct to the use of routine semen analysis in evaluating male fertility.     

ATP content in human semen and sperm quality

Five semen characteristics (count, motility, normal forms, bovine cervical mucus penetration, and ATP) were evaluated in patients attending the infertility clinic. Multiple regression analysis indicated that the only characteristic independently affecting ATP concentration was sperm count, whereas motility and percent normal forms had no additional contribution to ATP level. Penetration into bovine cervical mucus did not depend on ATP levels. Comparison of semen characteristics in the same group between patients who have impregnated their wives and those who have not indicated that with respect to count, motility, morphology, and ATP the only sperm characteristic independently predicting fertility was motility. ATP measurements have limited value in the evaluation of semen quality.     

Human sperm penetration in different media

Sperm penetration into bovine cervical mucus and hen egg white using capillary tube penetration was investigated to verify the suitability of the capillary tube penetration test with hen egg white as a test of human sperm function. Semen samples from 50 consecutive patients were used for penetration tests and spermatozoa of a further 10 semen samples were penetrated into bovine cervical mucus and hen egg white for special motility assessment by computer-assisted motility analysis. Penetration tests revealed the well-known different ability of spermatozoa to penetrate into cervical mucus and a different penetration of spermatozoa into egg white for two nearly equal groups (n = 24 and n = 26, respectively). One group showed penetration comparable with cervical mucus and one group a very fast penetration up to the limit of the scale of measurement. Motility assessment of spermatozoa that penetrated into cervical mucus and egg white revealed significant differences in straight-line velocity, linearity and lateral head displacement. The number of spermatozoa selected actively during the penetration procedure was significantly higher in cervical mucus than in hen egg white. Spermatozoa selected by bovine cervical mucus and hen egg white exhibited a different motility pattern. There was significantly better linearity and less lateral head displacement in egg white than in cervical mucus. Sperm penetration into hen egg white appeared to be influenced by different sources of egg white.     

Physiological integrity of human sperm in the presence of Ureaplasma urealyticum

Several species of Mycoplasma have been isolated from the human genital tract, the most common being M. hominis and Ureaplasma urealyticum. A causal relationship between such infections and sperm dysfunction and infertility has yet to be established. It was the purpose of this study to examine the effects of U. urealyticum infection on the function of sperm as assessed by seminal fluid analysis (SFA), in vitro penetration of bovine cervical mucus (BCMP), and the hamster sperm penetration assay (SPA). No significant differences were noted in the SFA of infected and uninfected samples, either fresh or frozen, fertile or infertile. In addition, no differences were noted in the BCMP or SPA. In sperm from U. urealyticum-infected individuals the basic physiological mechanisms underlying mucus penetration and ovum fertilization seem intact.     

Penetration ability of human spermatozoa into standardized bovine cervical mucus (Penetrak®*) in patients with normal and pathological semen samples**

Summary: Semen samples from 89 men were analyzed and evaluated with a standardized bovine cervical mucus penetration test (Penetrak®). 29 patients had a normozoospermia, 22 patients a teratozoospermia and 38 patients an oligozoospermia of various degree. The most important parameters of semen analysis were compared with sperm mucus penetration. The sperm mucus penetration test correlated best with progressive motility (r = 0.6758), followed by total motility (r = 0.6302), sperm count (r = 0.6190), and % normal spermatozoa (r = 0.5493) (p < 0.01 for each correlation coefficient). In the group of patients with normozoospermia 6 cases had been detected with insufficient sperm mucus penetration and in both groups with subfertility 15 cases with adequate sperm mucus penetration. 8 semen samples with normal sperm count and normal sperm motility were used for IVF. Only 6 patients with normal sperm penetration (> 30 mm/ 90′) the spermatozoa fertilized the ova, but not from two patients, who had a sperm mucus penetration of less than 30 mm/90′. The study demonstrated that standardized bovine mucus penetration test detects disturbances of sperm motility which may not be discovered by conventional semen analysis. Zusammenfassung: Penetrationsfähigkeit menschlicher Spermatozoen in standardisierten Rinderzervikalschleim (Penetrak®) bei Patienten mit normalem und pathologischem Spermiogramm Die vorliegende Studie umfaßt 89 Manner, bei denen, neben den klassischen Spermiogramm-Parametern, die Penetration der Spermatozoen in Rinderzervikalschleim mit Hilfe eines standardisierten Testes (Penetrak®) geprüft wurde. 29 Patienten hatten eine Normozoospermie, 22 Patienten eine Teratozoospermie und 38 Patienten eine Oligozoospermie unterschiedlichen Schweregrades. Bei Vergleich der wichtigsten Spermiogrammparameter mit der Penetrationstiefe der Spermatozoen in den Rinderzervikalschleim ergab sich, daß die Penetrationstiefe der Spermatozoen am besten mit der Progressivmotilität korreliert (r = 0.6758), gefolgt von der Gesamtmotilität (r = 0.6302) der Spermatozoendichte (r = 0.6190) und dem prozentualen Anteil normal geformter Spermatozoen (r = 0.5493) (p < 0.01). In der Gruppe der Patienten mit einer Normozoospermie konnten 6 Fälle erfaßt werden, die einen unzureichenden Spermatozoenpenetrationstest aufwiesen, dem gegenüber in den beiden Gruppen mit einer Subfertilität 15 Patienten mit normalem Spermatozoenpenetrationstest. Von 8 Patienten mit normaler Spermatozoendichte, normaler Spermatozoenmotilität und weitgehend normaler Spermatozoenmorphologie wurde das Ejakulat für IVF benutzt. Zur Fertilisation der Eier kam es jedoch nur bei den Patienten, insgesamt 6, die auch einen normalen Spermatozoenpenetrationstest hatten (> 30 mm/90′), nicht jedoch bei zwei Männern, bei denen der Spermatozoenpenetrationstest unter 30 mm/90′ lag. Aufgrund der vorliegenden Ergebnisse kann festgestellt werden, daß mit Hilfe des standardisierten Rinderzervikalschleim-Penetrationstestes offensichtlich Störungen der Spermatozoenmotilität aufgedeckt werden können, die im konventionellen Spermiogramm nicht erfaßt werden.     

Effects of kallikrein on sperm motility, capillary tube test, and pregnancy rate in an AIH program

From 1984 to 1986, 172 couples were enrolled in an artificial insemination homologous (AIH) program, because of negative postcoital test, fair PT, positive PT with unexplained infertility, and impotentia coeundi. Patients were randomly assigned to the following procedures: AIH with native semen (N, n = 68), washed sperm (V, n = 50), semen mixed with kallikrein (K, 5 IE/ml semen, n = 45), and timed intercourse. The overall pregnancy rate (PR) was 22% per couple (13% inseminations, 9% spontaneous). PR from insemination (spontaneous) was 13% (8), 13% (13), 23% (0) in PT negative, fair, and positive patients, respectively, and 18% (9), 13% (7), and 11% (9) in AIH groups N, V, and K. Among in vitro studies with K added semen, an improvement of sperm motility was demonstrated in half of the specimens. In the capillary tube test (CTT), with wives' cervical mucus, a deterioration was more frequently seen than an improvement after 2 h, independent of the early effect of K in semen, K induced changes of CTT were less pronounced when donors' cervical mucus was used. The results of the in vitro studies offer an explanation for the low PR in the AIH program with K.     

Effects of cryopreservation on penetration ability of human spermatozoa into bovine cervical mucus

Cryopreserved sperm exhibit lower fertilizing capacity in comparison to fresh sperm, partly due to effects of glycerol as the common cryoprotectant medium. Since standard semen analysis is not a good predictive method to assess sperm fertilizing capacity, functional tests like cervical mucus penetration may provide more useful information. A total of 24 semen samples were examined before and after cryopreservation for sperm parameters as well as number and motility of penetrated sperm into bovine cervical mucus (BCM) as an alternative for human cervical mucus. Freezing and thawing procedures have negative effects on sperm penetration into cervical mucus. No significant relation was noticed between sperm motility percentage or its penetration into BCM before and after cryopreservation, which denotes the variability in resistance of sperm to damaging effects of freezing.     

Sperm chromatin heterogeneity as an infertility factor

Semen samples from husbands with a history of unexplained infertility (n = 33), of women with habitual abortion (n = 36), or normal fertile donors (n = 20) were subjected to conventional semen analysis (SA), Acridine orange test (AOT), and zona-free hamster egg penetration test (HEPT). The three tests operate independently. The most discriminatory test was AOT (p = 0.0001) followed by HEPT (p = 0.019). The frequency of sperm chromatin heterogeneity as detected by AOT red fluorescence was highest in habitual abortion (39.4%), followed by unexplained infertility (16.4%), and, last, donors (9.4%). However the percentage of penetration was highest in habitual abortion (50.7%), followed by donors (43.1%), and least in unexplained infertility (33.9%). Conventional semen parameters (sperm density, motility, abnormality, and vitality) were the least to discriminate between the three groups. The presence of abnormal sperm chromatin may lead to infertility as a result of early pregnancy loss.     

Relationship between the sperm penetration assay and other tests of sperm function

Semen from 88 men of infertile couples and 33 fertile donors differed in seminal fluid analysis (sperm density and motility) (SFA) as well as in the penetration of hamster ova (SPA) and bovine cervical mucus (MPT). In the fertile group, significantly more subjects had adequate SFA, SPA, or MPT results than in the infertile group. When the two groups were subdivided into those with normal or those with abnormal SFA, no differences were noted in SPA, MPT, or postcoital test (PCT) scores. The SFA parameter most consistently reflected in the results of the SPA, MPT, and PCT was sperm density. This was most evident when the SFA was poor. The worst prognosticator of fertility was the SFA, with 30% of the fertile donors having an abnormal SFA. The worst prognosticator of infertility was the MPT, with 79% of the patients penetrating in the fertile range. The SPA was a significantly better predictor than either the SFA or MPT. SPA and MPT results were positively correlated only in the overall infertile group. The SPA, MPT, and PCT measure sperm qualities distinct from those revealed by the SFA, and from each other, and in combination provide the best assessment of fertility.     

Relationship between hypoosmotic swelling test, semen analysis, and zona-free hamster ovum test

The hypoosmotic swelling test is a simple test for measuring the functional competence of the human sperm membrane. Fifty-four patients with idiopathic infertility were assessed by hypoosmotic swelling test and the results were compared with those of routine semen analysis and zona-free hamster ovum human sperm penetration test (ZSPT). Semen samples with abnormal semen parameters had lower percentage of swollen sperm in comparison with those with normal semen parameters. A positive correlation was observed between sperm concentration and sperm swelling (r = .50, p less than .05). A strong positive correlation was observed between the percentage of sperm motility and the percentage of swollen sperm (r = 0.60, p less than .01), and between motile sperm concentration and sperm swelling (r = .62, p less than .01). On the other hand, sperm swelling correlated only weakly with the percentage of sperm penetration. The results indicate that the hypoosmotic swelling test appears to evaluate different functional qualities of sperm than ZSPT.     

Multivariate discriminate analysis of the relationship between the hypo-osmotic swelling test and the in-vitro fertilizing capacity of human sperm

Multivariate discriminant analysis was used to evaluate the usefulness of routine semen parameters and the hypo-osmotic swelling test (HOST) as predictors of the in-vitro fertilizing capacity of human sperm as assessed by the zona-free hamster egg penetration assay (HEPA). Eighty-eight semen samples from untreated patients attending an infertility clinic were analysed. Semen samples were classified into the following three groups before statistical analysis: group 1--positive sperm penetration (greater than or equal to 10%, n = 39); group 2--borderline penetration rates for HEPA (greater than 0% but less than 10%, n = 39) and group 3--negative sperm penetration (0%, n = 10). The percentage of sperm with normal morphology and sperm count were found to be significant in discriminating between semen samples exhibiting different in-vitro fertilizing capacity. These two discriminating variables in combination gave an overall correct classification rate of 45.5%. The multivariate discriminant analysis was also performed after excluding the data of group 2 semen samples (n = 39), which exhibited borderline sperm penetration rates. As a result, three discriminating variables including semen volume, sperm count and the percentage of sperm with normal morphology were selected. These three variables in combination could accurately predict whether a semen sample would exhibit positive sperm penetration (group 1) or negative sperm penetration (group 3) with an overall accuracy of 75.5%. The percentage of swollen sperm after hypo-osmotic treatment was not related to the HEPA result, as determined by linear correlation and multiple regression analyses, and did not give additional information about the in-vitro fertilizing capacity of sperm as evaluated by multivariate discriminant analysis.     

Efficacy of zona-free hamster egg sperm penetration assay as a predictor of in vitro fertilization

The efficacy of the zona-free hamster egg sperm penetration assay (SPA) as a predictor of in vitro fertilization (IVF) is studied. Indications for IVF were tubal factors in 35 couples and male factors in 24 couples. The diagnostic characteristics of SPA in reference to IVF for the whole group (n = 59) were reasonably reliable. Sensitivity was 74%, specificity was 84%, and diagnostic accuracy was 81%. However in patients with male factors, SPA was less reliable in terms of sensitivity (70%), specificity (57%), and diagnostic accuracy (63%), than in patients with tubal factors where the indicators were 80%, 97%, and 94%, respectively. Semen from patients with male infertility shared notable variations in terms of sperm density and motility which may account for the low SPA predictivity. SPA seems to be a useful screening test for prediction of IVF outcome. However other semen variables should be considered before interpretation, especially in male infertility where the diagnostic accuracy is low.     

Filtering capacity of bovine cervical mucus towards abnormal forms of human-ejaculated spermatozoa

The filtration capacity of bovine cervical mucus (BCM), fresh bovine cervical mucus (FBCM) or frozen and thawed bovine mucus (BCMF) was studied, using human-ejaculated spermatozoa in comparison with the properties of human cervical mucus (HCM).
Thirty semen specimens of good quality were used in cervical mucus (CM) penetrations, using capillary tubes containing CM of 30 HCM, 77 FBCM and 77 BCMF samples. Spermiocytograms were carried out on semen and cervical mucus at 1st and 3rd centimeter of the capillary tube after one hour of incubation. Smears were stained by Papanicolau and the patterns of seven forms of sperm cells (normal, tapering, macrocephals, microcephals, pinhead, neck pathology and amorphous cells) were counted on a total of 100 cells in each slide.
A clear, highly significant ( P < 0.0001) selection of cells penetrating the CM has been shown, preventing certain abnormal forms from penetrating the CM (macrocephals, neck pathology and amorphous cells) and enabling good penetration of normal sperm forms and moderate penetration of tapering, microcephals and pinhead cells. On the average, a specimen with normal sperm forms of 72% showed a spermiocytogram of 90% normal forms at the 3rd centimeter of migration. The same patterns of filtration were recorded in all the three sources of CM. It is suggested that BCM be used as a filter towards abnormal sperm forms in a highly teratozoospermic ejaculate in order to improve its quality for artificial inseminations.     

The use of hens'' egg white as a substitute for human cervical mucus in assessing human infertility

Semen samples from 179 patients with longstanding infertility were allowed to penetrate capillaries filled with a fraction of fresh hens' egg white (HEW) as a substitute for cervical mucus (CM) for the in-vitro sperm-cervical mucus penetration test (SCPMT). Results were correlated with those obtained from human CM (HCM) used in parallel on the same semen sample, compared to results of sperm analysis and post-coital testing (PCT) and analysed for their prognostic value for a subsequent pregnancy in a prospective study. The overall pregnancy rate after 6 months was 26.3% (47/179). Whereas routine semen analysis (semen volume, pH, sperm count, progressive motility, morphology and fructose concentration) did not differentiate between patients who achieved pregnancy and those who did not, significant differences were found for the penetration distance (P less than 0.02) and the motility grade of sperm in HEW (P less than 0.02). Although the mean sperm velocity in HEW was reduced compared to HCM, a close positive correlation was found between the sperm density and the penetration distance (P less than 0.001). The percentage of samples with good HEW penetration increased significantly in parallel with better results of post-coital testing (P less than 0.001). The results indicate that, when fresh human CM is not available, HEW can be used as a medium for the SCPMT as a valuable adjunct to semen analysis which provides information about sperm functional capacity.     

Bovine mucus penetration test and routine semen analysis of fresh and cryopreserved human spermatozoa

Semen samples from 18 fertile men and 18 infertile men were evaluated both fresh and after freezing and thawing using traditional semen parameter, the hypoosmotic swelling test (HOS-test) and the bovine cervical mucus penetration test (BMP-test). Major differences in the semen analysis between the fertile and infertile men were observed for motility, morphology (head and tail defects), the bovine mucus penetration test and the hypoosmotic swelling test. Cryopreservation resulted in a decrease regarding motility, the BMP-test and the HOS-test. Percentagewise the losses due to cryopreservation were higher in the infertile group than in the fertile group. The results of this study show that the bovine mucus penetration test as well as the HOS-test are valuable additions to the routine semen analysis of fresh and cryopreserved spermatozoa.     

Morphological factors influencing the penetration of human sperm into cervical mucus in vitro

The efficiency of cervical mucus in filtering out single, multiple and associated abnormalities of human spermatozoa was determined. Twenty semen samples which gave a normal in vitro cervical mucus penetration test (CMPT) were analysed before and after migration using a detailed classification system (13 categories). The % of normal forms was significantly increased in cervical mucus (59.5 vs 33.2%), whereas the % of sperm with single, multiple or associated abnormalities of the midpiece or of the flagellum were found to decrease significantly in cervical mucus. Sperm with single or multiple abnormalities confined to the head migrated similarly to normal forms. The decrease in amorphous and elongated tapering sperm was explained by their more frequent association with other defects of the midpiece and/or of the flagellum.     

Presence of Sperm Agglutinating Antibodies in Infertile Men and Inhibition of in vitro Sperm Penetration into Cervical Mucus

The relation between presence of antispermatozoal antibodies in infertile men and the inhibition of the in vitro sperm penetration into cervical mucus (CM) was studied with the sperm cervical mucus contact (SCMC) test. The tests were performed with semen from infertile men and from semen donors. The CM used permitted good penetration of normal spermatozoa. The so called "shaking phenomenon", the result of a specific interaction of spermatozoa and CM, was expressed in the shaking percentage (S%).
The S% did not change beyond the experimental error within 30 min after mixing semen and CM. The S% was 30 at the most in 194 out of 198 SCMC tests with normal donor semen and normal pre-ovulatory CM. Significant negative correlations ( P < 0.005) were found between the readings of the sperm penetration meter (SPM) test on one hand and the S%, the sperm agglutination titer in the serum and the sperm agglutination titer in the seminal plasma (SP) on the other hand. Significant positive correlations ( P < 0.005) were found between the S% and the sperm agglutination titer in the SP. The sperm agglutination titer in serum and in SP correlated significantly better ( P < 0.02) with the S% in the SCMC test than with the readings of the SPM test.
It was concluded that: 1. A high S% is highly specific for the presence of antispermatozoal antibodies in infertile men, 2. The SCMC test is more suited than the SPM test for studying the effect of antispermatozoal antibodies on the penetration and migration of spermatozoa into CM,     

Reappraisal of the hypo-osmotic swelling test to improve assessment of seminal fertility status

The hypo-osmotic swelling (HOS) test has been proposed as a useful assay for evaluation of the functional competence of the human sperm membranes. To assess this further, the HOS-test was evaluated in 187 semen samples collected from fertile men and from male patients consulting for infertility. These samples were classified as normal, oligo-, astheno- or oligoasthenozoospermic on the basis of their standard semen variables. The percentage of total sperm tail swelling and of sperm exhibiting different tail swelling patterns was recorded. In the fertile men and in the group of patients with normal semen variables, significantly more (P less than 0.001) HOS-reactive sperm were observed after hypo-osmotic treatment in comparison with those groups exhibiting abnormal semen parameters. Swelling of the sperm in a hypo-osmotic medium was highly correlated with both progressive motility (r = 0.62, P less than 0.001) and sperm viability (r = 0.65, P less than 0.001). A weak positive correlation was also observed between sperm swelling and sperm morphological features (r = 0.31, P less than 0.005) and between sperm swelling and sperm concentration (r = 0.31, P less than 0.005). No significant correlation was observed between sperm swelling and in-vitro sperm fertilizing capacity as assessed by the zona-free hamster oocyte penetration assay. However the majority of the semen samples (87.3%) showing a normal penetration rate (greater than or equal to 10%) also exhibited a 60% (or higher) reaction in the HOS-test.(ABSTRACT TRUNCATED AT 250 WORDS)