Effects of chelating agents on the rheological property of cervical mucus

As an ongoing effort to elucidate the mechanisms involved in the calcium-dependent fertility regulation process, the viscoelastic properties of the mucus obtained from lamb cervix and human semen, as well as their water and total protein contents after exposure to EDTA, a chelating agent, or Nonoxynol-9 (N-9), a spermicidal agent, were examined. The viscosity was measured using a Cone Plate Digital Viscometer, while the water and total protein contents were determined by the lyophilization process and the Lowry method, respectively. The significant changes in the rheological properties of mucus, such as its viscosity and the water content, upon exposure to EDTA were demonstrated. The viscosity of cervical mucus and human semen were significantly increased by EDTA treatment (as compared to the controls): lamb cervical mucus (2.9 ± 0.3 vs. 2.2 ± 0.3 cps) and human semen (5.0 ± 0.3 vs. 4.3 ± 0.3 cps), respectively. The hydration rate was decreased by EDTA treatment as compared with the control (93.6 ± 0.7 vs. 96.8 ± 0.8%). Among tested samples, the reduction in the percentage of sperm penetration through the cervical mucus was the highest in the mucus containing EDTA, which had the lowest water content (93.6 ± 0.7%), indicating that there is a positive relationship between the hydration rate of the cervical mucus and its ability to permit the penetration of spermatozoa. This result indicates that spermicidal activity exerted by high concentrations of EDTA is in part due to its effect on the rheological properties of cervical mucus or semen.     

Synergistic effect of gramicidin and EDTA in inhibiting sperm motility and cervical mucus penetration in vitro

Gramicidin, a linear polypeptide with antiviral and antimicrobial properties, was compared in vitro with a commonly used spermicidal detergent—nonoxynol-9 (N9). The inhibition of sperm functions was evaluated by computer-assisted semen analysis (CASA) for sperm motility, in cervical mucus penetration assay, and by colorimetric tetrazolium salt and lactate dehydrogenase release assays routinely employed for testing the toxicity of drugs. The effective 100% inhibitory concentration (IC₁₀₀) of gramicidin in a 2-min sperm immobilization assay by CASA was equal to 4 μg/ml, whereas IC₁₀₀ of N9 was equal to 200 μg/ml. The presence of 0.1% of chelating agent, EDTA, reduced IC₁₀₀ of gramicidin to 10 ng/ml, while less than a twofold enhancement in N9 activity was observed upon combination with EDTA. Likewise, the gramicidin/EDTA combination was 100,000 times more potent than N9/EDTA in the sperm penetration assay. Quantitative toxicity tests confirmed that gramicidin is a potent spermostatic rather than spermicidal agent. Further development of a gramicidin/EDTA formulation is warranted as a nontoxic topical contraceptive with activity against viral and microbial sexually transmitted diseases (STDs).     

Analysis of pre-ovulatory changes in cervical mucus hydration and sperm penetrability

Changes in cervical mucus occur during the proliferative phase of the menstrual cycle and are known to correlate with receptivity to sperm and to the endocrine milieu. Prior studies, however, have often lacked biological incisiveness and technical objectivity and precision. This study analyzed daily changes in mucus water content (hydration) prior to the LH surge (LH+0) in normal women, in relation to daily levels of serum LH, FSH, estradiol and progesterone, and to daily tests of sperm penetration of the mucus. Cervical mucus was studied for 12 cycles in 10 ovulating women. Three to ten mucus specimens were collected per cycle, over the days LH-8 to LH+0. Each specimen was subjected to measurement of both water content (hydration) and penetration by spermatozoa from fresh specimens of normal human semen. For the latter, a new microscale assay was developed and applied, which was amenable to very small volumes of mucus. The new technique determines objective measures of both the numbers of penetrating sperm (motile and non-motile) and the distance penetrated by the forwardmost vanguard sperm. In these experiments, variations in semen quality were controlled by performing a companion penetration assay in an artificial 1.5% polyacrylamide gel.The patterns of change in mucus hydration varied quantitatively among women, with preovulatory baseline levels ranging from 93.8-96.5%. All normal cycles (as defined by endocrine profiles) displayed a significant increase in hydration over a one-day period occurring 3-4 days before the LH peak. The magnitude of this shift varied among women between 2 and 3% (absolute hydration), a distinction well within the precision of the hydration assay. This quantum increase in hydration was more pronounced than the corresponding increase in serum estradiol on the same day. The change in mucus hydration, and the associated increase in sperm penetrability, were more consistent among cycles than the changes in reproductive hormones.There was a strong but non-linear correlation between mucus hydration and sperm penetrability. Once the value of hydration rose above approximately 97.5%, there was a substantial increase in penetrability. This 'cut-off point' in sperm penetrability was in the middle of the range of hydration values (across women) which preceded the quantum jump in hydration - which, itself, preceded the surge of L.H. Hydration began to increase approximately 2 days before measurable increases in sperm penetration of the mucus in vitro.These results demonstrate that mucus hydration may be a valuable marker of the approach to ovulation and delineation of the fertile period. They also provide new methods for assessing sperm penetration into both large periovulatory and very small samples of collected mucus.     

Effects of the levonorgestrel-releasing intrauterine system on cervical mucus quality and sperm penetrability

BackgroundIn levonorgestrel intrauterine system (LNG-IUS) users, the prevention of sperm penetration through cervical mucus has not been demonstrated.Study DesignSubjects were enrolled in an investigator-blinded study to compare quality and sperm penetrability of mid-cycle cervical mucus between LNG-IUS users and hormone-free controls. Cervical mucus was microscopically examined using World Health Organization (WHO) cervical mucus analysis (CMA). CMA score ≥10 of 15 points indicated cervical mucus favoring sperm penetration. Mucus was incubated with sperm using the WHO simplified slide test (SST) and Kremer sperm cervical mucus penetration test (SCMPT).ResultsData from 14 LNG-IUS users and 16 controls showed 14% of LNG-IUS users had CMA score ≥10% vs. 69% of controls (p=.004). SST showed no sperm penetration for LNG-IUS users, significantly less than controls (0% vs. 64.3%, p<.001). SCMPT demonstrated no sperm mucus penetration for LNG-IUS users at 2 and 6 h (0% vs. 85% in controls with 2-h score ≥6, p<.001; 6 h 0% vs. 79% in controls, p<.001).ConclusionsMid-cycle cervical mucus of LNG-IUS users is poor quality and prevents endocervical sperm transport in vitro.     

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.     

CORRELATION OF THE BOVINE CERVICAL MUCUS PENETRATION TEST WITH HUMAN SPERM CHARACTERISTICS IN 1406 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 1406 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 (&gt;30 mm). A highly significant (p&lt;.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 (&lt;30 mm), while a normal semen parameter resulted in a 90+/-4% accurate prediction of a positive BCMPT (&gt;30 mm). Sperm MD showed the strongest positive predictive value (98%), while morphology showed the greatest negative predictive value (50%). Of the 1406 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.     

男性生育力的综合评价

选择１２０例身体健康、生育力正常的青壮年男性及１２０例临床诊断为不育症患者的精液标本行精液常规分析,精子穿透牛宫颈粘液实验及精子穿透去透明带金黄地鼠卵实验。对所得精子功能多项指标采用两类指标组合指标法确定９５％男性生育力多元医学参考值范围,提供对男性生育力进行综合评价的简便方法。该法用于判断男性生育力的敏感性为９０％,特异性为９３．７％,准确度为９３．２％。     

Longevity of Gynol II and Ortho Creme in the Prentif cervical cap

A review of the literature yielded no scientific basis for current recommendations on the length of time spermicide-filled cervical caps can be worn while continuing to protect against pregnancy. Ten cervical cap users provided 99 samples of cap contents after a variety of insertion periods using Gynol II, Ortho Creme and vehicle controls. Testing for spermicidal effect was performed in the laboratory using quality semen from two donors and employing the Multiple Exposure Photography system of semen analysis. Criteria for determining spermicide effectiveness were developed. Results suggest that 3 to 4 days is the reasonable limit for cap use with Gynol II and 5 days with Ortho Creme, contrasted with 1 day limits for Gynol II and 2 day limits for Ortho Creme using existing criteria. Limited experience with samples exposed to semen in vivo suggest that spermicidal potency is not lost through repeated intercourse. Recommendations for validating these limits with clinical studies are made.     

In vitro characteristics of fresh and frozen-thawed ram spermatozoa after sex sorting and re-freezing

The in vitro function of sex-sorted, frozen-thawed ram spermatozoa derived from fresh or frozen semen was investigated. Sorted, frozen-thawed spermatozoa had higher (P < 0.05) motility, viability, acrosome integrity and mitochondrial activity than non-sorted, frozen-thawed controls immediately following thawing and after incubation at 37 degrees C for 3 and 6 h. Similarly, frozen-thawed, sorted, re-frozen-thawed spermatozoa outperformed (P < 0.05) non-sorted controls upon thawing (mitochondrial activity) and following a 3-h incubation (motility, viability/acrosome integrity and mitochondrial activity), but there were no differences after incubation for 6 h (P > 0.05). Velocity characteristics (computer assisted sperm assessment 0-6 h post-thaw) of sorted spermatozoa derived from either fresh or frozen semen remained inferior (P < 0.05) to non-sorted spermatozoa, as did their ability to penetrate artificial cervical mucus after thawing. Direct comparison of cryopreserved spermatozoa derived from either fresh or frozen semen revealed that frozen-thawed, sorted, re-frozen-thawed spermatozoa had comparable (P > 0.05) motility, viability/acrosome integrity, mitochondrial activity, average path velocity and oviducal binding capacity immediately post-thaw, but reduced (P < 0.05) quality after 3 and 6 h of incubation. These findings indicate that, under the tested in vitro conditions, sex-sorted spermatozoa derived from fresh semen are superior in some respects to those derived from frozen semen. Further, that the use of either technique, while reducing velocity characteristics and cervical mucus penetration, results in comparable, if not enhanced motility, membrane and mitochondrial function in the post-thaw population of spermatozoa when compared with non-sorted, frozen-thawed controls.     

Properties of a new acid-buffering bioadhesive vaginal formulation (ACIDFORM)

Vaginal prophylactic methodology may prevent heterosexual transmission of the HIV and other sexually transmitted disease-causing organisms as well as unplanned pregnancies. A new delivery system (ACIDFORM) was designed with acid-buffering, bioadhesive, and viscosity-retaining properties to (1) maintain the acidic vaginal milieu (the low pH inactivates many pathogens and spermatozoa), (2) form a protective layer over the vaginal/cervical epithelium (minimizing contact with pathogenic organisms), and (3) provide long-term vaginal retention. A Phase I clinical study with ACIDFORM provided initial information about its safety and showed the formation of a layer over the vaginal/cervical epithelium [1; Amaral et al., Contraception 1999;60:361-6]. To study the properties of the gel (without active ingredient) in more detail, ACIDFORM's acid-buffering, bioadhesive, viscosity-retaining, and spermicidal properties were compared in vitro to marketed formulations, and its long-term stability was assessed. ACIDFORM, either when titrated with NaOH or when mixed directly with semen, is highly acid buffering and much more effective than Aci-Jel, a commercial acid-buffering vaginal product. ACIDFORM adheres well to two model membranes (excised sheep vagina and cellophane) and is more bioadhesive than Conceptrol, Advantage S, Replens, Aci-Jel, and K-Y jelly. On dilution, ACIDFORM also retains its viscosity better than these marketed products. ACIDFORM is spermicidal and is stable for at least 2 years. These results suggest that ACIDFORM has advantages over presently marketed vaginal delivery systems. The gel may either be useful by itself as an antimicrobial contraceptive product or as a formulation vehicle for an active ingredient with antimicrobial and/or contraceptive properties.     

Comparison of cervical mucus of 24/4 vs. 21/7 combined oral contraceptives

BackgroundFew studies have examined the action of combined oral contraceptives (COCs) on cervical mucus. We hypothesized that midcycle cervical mucus of women taking COCs is of poor quality when compared to their own midcycle mucus prior to initiating COCs. We sought to compare the effect upon quality and sperm penetration of the cervical mucus on the last hormone-free day with a 24/4 regimen to a 21/7 regimen.MethodsThis is an open-label, investigator-blinded, randomized, controlled, crossover equivalency study. All subjects received, in random order, 2 months of a 21/7 regimen and 2 months of a 24/4 regimen, each containing 20 mcg ethinyl estradiol and 1 mg norethindrone acetate. Analysis of cervical mucus quality (CMQ) and sperm penetration took place midcycle and on the last day of the hormone-free interval during the second month of each COC treatment.ResultsFrom April 2010 to November 2011, 18 subjects completed all study visits. Mean midcycle CMQ was poor (mean CMQ=1) and did not differ between 24/4 and 21/7 regimens (p=.92). On the last day of the pill-free interval, the quality and sperm penetration were poor with both regimens.ConclusionThis study indicates that thickening of cervical mucus is a major mechanism of contraceptive action of COCs and that both 21/7 and 24/4 regimens result in poor quality and impenetrable mucus on the last day of the pill-free interval.     

Spermicidal activity of dermaseptins

ObjectiveThe present study was undertaken to elucidate the spermicidal efficacy of two synthetic antimicrobial peptides, dermaseptin (DS1 and DS4).MethodsTwenty samples of fresh semen were obtained from patients aged between 23 and 35 years. The ability of DS to kill sperm was evaluated by the Sander–Cramer test under in vitro conditions.ResultsThe data showed that sperm motility was inhibited with various concentrations of DS at different intervals ranging from 2 to 240 min. The effective 100% inhibitory concentration (EC₁₀₀) of DS4 in 2 min during the sperm immobilization assay was 100 μg/mL whereas the sperm immobilization of EC₁₀₀ of DS1 was 200 μg/mL. The presence of 0.1% chelating agent ethylenediaminetetraacetic acid (EDTA) reduced the EC₁₀₀ of DS4 to 10 μg/mL whereas less than a two-time enhancement in DS1 activity was observed upon combination with EDTA. The action of DSs on sperm motility was observed to be dose dependent. Supplementation with pentoxifylline and that with calcium are known to enhance the motility of sperm but they did not prevent the spermicidal action of DSs.ConclusionThis present study indicates that DS is an effective agent to kill sperm. In view of this fact, it is suggested that DS4 has antibacterial, antiviral, antifungal and potentially spermicidal activities and could be a potent vaginal contraceptive.     

Sperm immobilizing effect of triterpene saponins from Acacia auriculiformis

A mixture of two partially triterpenoid saponins (Tg), isolated from Acacia auriculiformis was tested for sperm immobilizing activity by using in vitro system. The lowest concentration (ED) required for an obvious immobilization of human sperm by using a modified Sander-Cramer test was found to be 0.35 mg/ml in physiological saline. The ability of the compound as a sperm immobilizing agent was compared with that of Triton X-100 and found to be more potent. Cervical mucus penetration test was also performed and the ED successfully prevented sperm entry in human cervical mucus. Supra-vital staining with eosin-nigrosin indicated death of the treated sperm. Electron microscopic study of Tg-treated sperm showed plasma membrane disintegration and dissolution of acrosomal cap which is presumably the cause for the spermicidal effect of the saponins. No permanent lesion was observed after application of 1.25 mg/ml Tg solution in physiological saline to the eye of rabbits for ten consecutive days.     

Spermicidal activity of dermaseptins

OBJECTIVE: The present study was undertaken to elucidate the spermicidal efficacy of two synthetic antimicrobial peptides, dermaseptin (DS1 and DS4). METHODS: Twenty samples of fresh semen were obtained from patients aged between 23 and 35 years. The ability of DS to kill sperm was evaluated by the Sander-Cramer test under in vitro conditions. RESULTS: The data showed that sperm motility was inhibited with various concentrations of DS at different intervals ranging from 2 to 240 min. The effective 100% inhibitory concentration (EC(100)) of DS4 in 2 min during the sperm immobilization assay was 100 microg/mL whereas the sperm immobilization of EC(100) of DS1 was 200 microg/mL. The presence of 0.1% chelating agent ethylenediaminetetraacetic acid (EDTA) reduced the EC(100) of DS4 to 10 microg/mL whereas less than a two-time enhancement in DS1 activity was observed upon combination with EDTA. The action of DSs on sperm motility was observed to be dose dependent. Supplementation with pentoxifylline and that with calcium are known to enhance the motility of sperm but they did not prevent the spermicidal action of DSs. CONCLUSION: This present study indicates that DS is an effective agent to kill sperm. In view of this fact, it is suggested that DS4 has antibacterial, antiviral, antifungal and potentially spermicidal activities and could be a potent vaginal contraceptive.     

Treatment with Norplant subdermal implants inhibits sperm penetration through cervical mucus in vitro

Attempts were made to collect several samples of cervical mucus in each of thirty-three cycles of women using NORPLANT subdermal implants, in seven cycles from NORPLANT-2 rod users, and in 33 control cycles. The attempts to collect a mucus sample were successful on 20 of 77 and 7 of 14 occasions in NORPLANT capsule and rod users, respectively, due to the scanty amount and stickiness found in the majority. All 59 attempts in control subjects were successful. The distance travelled by the fastest sperm and by the bulk of spermatozoa through the cervical mucus in vitro was assessed after 10 min of incubation with a normal semen sample. The best score for each cycle was considered for the comparison between treated and control cases. Sperm penetration was greatly impaired in mucus samples of implant users with penetration by the fastest sperm exceeding 10 mm in only two instances and exceeding 20 mm in none. In 30 of 33 control samples, the fastest sperm travelled 21 mm or more and in 28, the bulk of spermatozoa travelled further than 10 mm. Unsuccessful attempts to collect mucus and poor sperm migration were observed in NORPLANT implant users even when circulating estradiol levels were comparable to those seen in the late follicular phase of the normal menstrual cycle. These results suggest that the principal mechanism by which NORPLANT implants prevent pregnancy is by interference of sperm migration through cervical mucus.     

IMPROVEMENT IN THE CERVICAL MUCUS PENETRATION TEST BY USING STANDARD SPERM CONTROL

The objective of the present experiments was to establish an acceptable standard for the cervical mucus penetration test (CMPT) by determining the minimal progressive motile spermatozoa concentration (PMSC) that will yield the highest score for proven fertile donor sperm specimens. For this purpose, fresh and frozen-thawed samples were used. Semen was obtained from 29 fertile donors and different PMSC (8, 10, and 14 10⁶/mL) were prepared for each sample. The same mucus specimen was used for testing each sperm sample in the three different dilutions. No difference in the scoring of the CMPT between the fresh vs. frozen-thawed groups was found. When PMSC of 14 10⁶/mL was used, almost all specimens scored the highest rank. The present study revealed that only semen samples with a minimal PMSC of 14 10⁶/mL cells can be used in the CMPT. The information that the freeze-thaw process does not affect the CMPT results supports the concept of cryopreservation of pooled fertile donor specimens in aliquots with adequate concentration of progressive motile spermatozoa for later use as a CMPT control.     

Effect of temperature and pH on contraceptive gel viscosity

The rheological properties of four commercially available spermicidal gels (two polyacrylic acid derivatives and two carboxymethylcellulose based) and their dilutions with a vaginal fluid simulant (pH 4.2) and a semen simulant (pH 7.7) were measured at 25 degrees C and 37 degrees C over a biologically relevant range of shear rates. All four gels were shear thinning with temperature-dependent rheological properties. The two types of gels responded differently to dilution. The rheological properties of the polyacrylic acid derivative gels were strongly dependent on the type of diluent used. Their viscosities after dilution with the semen simulant were 100 times greater than after comparable dilutions with the vaginal fluid simulant, this effect being due primarily to the higher pH. The cellulose gels did not exhibit such an effect. These results suggest that the polyacrylic acid and cellulose gels interact differently with the vaginal environment in vivo. Such differences could lead to differences in the extent and durability of epithelial coating.     

Human Sperm Velocity and Postinsemination Cervical Mucus Test in the Evaluation of the Infertile Couple

Homologous artificial insemination (A1H) 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 μ/sec, for those with normal postcoital test (PCT) 96.6 μ/sec, and in those that established a pregnancy 91.6 μ/sec. These are significantly higher than the values obtained for abnormal semen analysis and abnormal PCT (64.6 and 63.6 μ/sec, respectively; p < 0.001).

Patients with normal PIT (WHO criteria for normal PCT consisting of ≥ 7 motile sperm/hpf) had sperm velocity of 87 μ/sec compared to velocities of 46 μ/sec for abnormal PIT (p < 0.001). Four of the five patients with abnormal PIT (in spite of normal semen analysis and normal cervical mucus) had sperm velocities < 75 μ/sec. Likewise, all 8 patients who had normal PIT in spite of abnormal semen analysis had sperm velocities > 75 μ/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.     

Efficacy of nonhormonal vaginal contraceptives from a hydrogel delivery system

This investigation describes the synthesis of a biodegradable hydrogel composed of a core surrounded by four concentric sheaths containing dextran, copolymers of polylactide and epsilon-caprolactone. The hydrogel was impregnated with iron (II) d-gluconate dihydrate, which causes complete spermiostasis due to lipid peroxidation, ascorbic acid to increase the viscosity of the cervical mucus and mixtures of polyamino and polycarboxylic acids to sustain vaginal pH close to 4.5. The combined effects of the agents in the daily eluates of the hydrogel were efficacious up to 16 days, within 30 s, as shown by sperm penetration tests. For in vivo studies, rabbits were chosen as the experimental model because they are easy to handle and the female is always in estrus. The anterior vagina of estrous female rabbits was instilled with the hydrogel, and then inseminated with the semen from a fertile male. Postinsemination flush from the female rabbits showed that all of the sperm were dead. These observations demonstrate the potential for the development of a biocompatible, nonhormonal, intravaginal contraceptive device.     

Effects of norgestimate in combination with ethinyl estradiol on cervical mucus

The antifertility effect of norgestimate (Ng) 0.125 mg in combination with ethinyl estradiol (EE) 0.035 mg on cervical mucus of 5 apparently normal and ovulatory volunteer women was studied for 2 cycles: a control cycle followed by a study cycle in which the medication was given. Throughout both cycles, basal body temperature, cervical mucus properties, sperm penetration in cervical mucus and karyopyknotic index of vaginal cells were evaluated. Disappearance of cyclicity of these parameters in the study cycle as well as monophasic basal body temperature were suggestive of inhibition of ovulation caused by the combination pill. Deterioration of cervical mucus properties throughout the study cycle and inhibition of sperm penetration may be a reflection of anovulation. However, a direct antifertility effect of Ng on cervical mucus cannot be excluded.