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.     

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.     

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.     

Comparison of the penetration ability of human spermatozoa into bovine cervical mucus and zona-free hamster eggs

In vitro bovine cervical mucus (BCM) penetration tests, sperm penetration assays (SPA) using zona-free hamster eggs, and routine semen analyses were performed on a total of 136 freshly collected semen samples from men who were seen at an infertility clinic. The correlations between bovine cervical mucus penetration and other semen parameters were the percent motile spermatozoa (r = 0.48), progressive motility grade (r = 0.44), sperm count (X 10(6)/ml) (r = 0.47), the percent normal morphology (r = 0.32) and the percent eggs penetrated (r = 0.46) (P less than 0.0001 for each correlation coefficient). When known fertile (n = 32) and infertile (n = 18) groups were tested, positive mucus penetration was associated 75% correctly and positive egg penetration was associated 90% correctly to clinical status. The mucus test had no false-negative results and the SPA had no false-positive results in these groups. It appears, then, that the mucus test and sperm penetration assay, although contributing different elements of data to an infertility evaluation, are both useful adjuncts to a semen analysis.     

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.     

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,     

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)     

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.     

Bovine cervical mucus penetration by human spermatozoa: lack of association with conception

Penetration of bovine cervical mucus (BCMP) by human spermatozoa has been reported to be a test that may aid in assessment of male fertility. We determined the predictive value of BCMP for conception in 127 infertile couples. Fresh semen and swim-up specimens were tested with the Penetrak assay. Out of 127 patients 16 (13%) achieved pregnancy with 6 months to 2 years of follow-up. In the 62 men with sperm counts of greater than 20 million/ml and motility of greater than 60%, 55 (89%) had adequate BCMP while adequate penetration was found in only eight of 24 (33%) with both sperm count and motility below these values (p less than 0.001). A normal BCMP did not predict pregnancy, and a reduced BCMP had no prognostic value in designating couples not likely to conceive. The comparison of penetration values for semen following swim-up suggests that this preparation may improve the correlation of BCMP with fertility. These results indicate that the semen parameters of sperm density and percent motility predicted spermatozoal performance in the BCMP assay, and that the assessment of BCMP did not predict the likelihood of pregnancy.     

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.     

Human sperm velocity and postinsemination cervical mucus test in the evaluation of the infertile couple

Homologous artificial insemination (AIH) followed by postinsemination cervical mucus test (PIT) was performed in 100 couples in a prospective study to evaluate suspected cervical or male factor infertility. A portion of the ejaculate was submitted for standard semen analysis as well as turbidimetric analysis of sperm velocity. In a review of 333 cases evaluated for infertility, the average sperm velocity in a normal semen analysis was 96.5 mu/sec, for those with normal postcoital test (PCT) 96.6 mu/sec, and in those that established a pregnancy 91.6 mu/sec. These are significantly higher than the values obtained for abnormal semen analysis and abnormal PCT (64.6 and 63.6 mu/sec, respectively; p less than 0.001). Patients with normal PIT (WHO criteria for normal PCT consisting of greater than or equal to 7 motile sperm/hpf) had sperm velocity of 87 mu/sec compared to velocities of 46 mu/sec for abnormal PIT (p less than 0.001). Four of the five patients with abnormal PIT (in spite of normal semen analysis and normal cervical mucus) had sperm velocities less than 75 mu/sec. Likewise, all 8 patients who had normal PIT in spite of abnormal semen analysis had sperm velocities greater than 75 mu/sec, even though the sperm motility was below normal in 5 of them. Sperm velocity is a more sensitive indicator of sperm function when compared to standard semen analysis results.     

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.     

Stability of basic semen measures and abnormal morphology within individuals

Semen from 15 healthy volunteers was assessed for basic semen measures every 2 weeks over a 6-month period to determine the relative stability of these factors. The parameters were: sperm count, semen volume, sperm motility, and normal morphology, along with the type of abnormal morphologic forms. Basic semen measures were generally more stable than the morphologic forms. Using three samples, seven semen factors (sperm count, normal morphology, semen volume, sperm motility, microcephaly, tapering and round tail) were found to be stable and contributed to the identification of the fifteen individuals with a classification accuracy of 75.0%. While individuals do have characteristic patterns of spermatogenesis, the biologic basis for these findings is as yet undetermined.     

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.     

Characteristics of Anti-Spermatozoal Antibodies Responsible for the Shaking Phenomenon with Special Regard to Immunoglobulin Class and Antigen-Reactive Sites

The interaction between spermatozoa and cervical mucus in the presence of antispermatozoal antibodies was studied in the SCMC-test and in the Miller-Kurzrok test.
A high correlation was found between: 1. The "shaking phenomenon" in the SCMC-test; 2. sperm penetration into cervical mucus; 3. the sperm agglutination titre in seminal plasma and in cervical mucus.
The authors performed investigations which indicate that antispermatozoal IgA in semen and in cervical mucus is probably responsible for the "shaking phenomenon" in the SCMC-test and for the reduced penetration of spermatozoa into cervical mucus. Experiments with the Miller-Kurzrok test make it probable that antispermatozoal antibodies in semen, which are responsible for the "shaking phenomenon", are directed against the surface antigens of the sperm tails. Antispermatozoal antibodies in cervical mucus, which are responsible for the "shaking phenomenon", are probably directed against the surface antigens of the sperm acrosome.     

Effect of rapid dilution of semen on sperm-bound autoantibodies

The effect of rapid dilution of autoantibody-positive semen on sperm-bound antibodies was studied in 12 male patients (0-100% IgA and 10-100% IgG). The direct immunobead test (IBT) and the sperm cervical mucus contact (SCMC) test were used to detect sperm-bound antibodies on spermatozoa before and after rapid dilution and swim up in HAM F-10 + 10% human blood serum. All patients tested had normal semen parameters. Sperm-bound antibodies detected after swim-up of semen samples ejaculated into 15 ml HAM F-10 + 10% serum (diluted) did not differ significantly from undiluted samples. Most values were lower (IBT and SCMC) but were not statistically significant. Sperm motility, forward progression, and morphology were statistically improved. Pregnancies resulting from washed semen (diluted) in combination with artificial insemination (AIH) or gamete intrafallopian tube transfer (GIFT) took place in spite of antibodies still present on the sperm membrane and can probably be attributed to improved semen quality, minimizing of ovum-sperm distance, and cervical mucus elimination.     

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.     

Male infertility due to asthenozoospermia and flagellar anomaly: detection in routine semen analysis

Major monomorphous teratozoospermia, due to sperm tail structural anomalies, were detected in 42 out of 4231 infertile patients during routine semen analysis. The flagella were very short or absent in 16 cases, shortened with thickness irregularity in 18 cases, and of normal length with diameter anomalies in eight cases. These syndromes were always associated with poor forward motility, while mobility and penetration into human cervical mucus were sometimes impaired only partially. With good sperm smears and an experienced observer, the diagnosis could be made without the need for electron microscopic analysis. All the affected patients were sterile and several syndromes could have been transmitted genetically.     

精索静脉曲张患者精液质量及其不育患者手术前后精液变化的观察

目的观察精索静脉曲张（VC）患者的精液质量和精子形态学改变，以及VC不育患者手术前后精液的变化。方法121例VC患者精液按WHO标准常规分析并对精子形态学进行评价，23例健康男性精液检查结果作为对照。并对21例VC不育患者术前及术后的精液进行检测分析。结果121例VC患者的精子密度、（A＋B）级活动力精子（％）、活率、有效精子数、活动精子数、活力指数以及正常形态精子比例较对照组明显降低（P〈0．01）；畸形精子中小头、锥形头和无定形头精子数较对照组增多（P〈0．01）。21例VC不育患者手术后精子质量和精子形态学较术前明显改善。结论VC可以引起精液质量下降导致不育，精子形态学分析是判定VC患者精子受损的一个敏感指标，手术能有效地改善精液质量。     

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.