Influence of microbial colonization on sperm-mucus interaction in vivo and in vitro*

Two-hundred-and-thirty-three asymptomatic couples with a meanduration of infertility of 5 years were submitted to postcoitaltesting (PT) and to sperm penetration meter test (SPMT) andsimultaneous microbial screening. Cervical swabs and semen specimenswere collected for culture of Mycophma homink, Ureaphma urealyticum,Chlamydia tmhomufis, Neisseria gonorrhoeae, other potentiallypathogenic and commensal aerobic and anaerobic bacteria, herpessimplex virus, vaginal swabs for Trichomonas vaginalis and yeasts.Results of microbial screening were analysed with regard tosperm penetration ability into wives' cervical mucus in vivoand in vitro, but no marked influence was revealed for mostmicmrganisms. Samples of only one of the 233 couples provedto be completely sterile. The findings suggest that in asymptomaticpatients microbial colonization is of minor importance for sperm-mucusinteraction.     

Microbial colonization and sperm--mucus interaction: results in 1000 infertile couples.

After screening a large series of infertile patients (n = 1000 couples), potentially pathogenic microorganisms were identified in genital secretions of the majority of couples. None of the patients displayed signs or symptoms of infection of the lower genital tract. In semen specimens and cervical swabs, mycoplasmas were found in 18 and 12%, potentially pathogenic aerobic bacteria in 50 and 31%, additionally commensal aerobes in 38% and 25%, respectively. The microbial pattern showed great variability with polymicrobial growth most common. The rate of cultures positive for potentially pathogenic anaerobes was markedly influenced by the transport conditions. In endocervical material, Herpes simplex virus (HSV) was identified in 4.5%. Yeasts were found in the posterior vaginal fornix in 10%. Concerning both partners, there were only 10 couples (1%) from whom microorganisms in genital secretions could not be isolated, not including lactobacilli in females. This high prevalence of microbes and the low number of leukocytes indicate colonization rather than infection. When the microbial findings from both partners were analysed with regard to the outcome of sperm-cervical mucus penetration testing in vitro, which was performed simultaneously, no significant correlation was found. Furthermore, microbial prevalence did not differ between couples with 'explained' and 'unexplained' infertility. The results of this study demonstrate that in patients without symptoms of genital tract infection, microbial colonization is of minor importance for sperm--mucus interaction and that extensive microbial screening should be preferentially performed in cases of poor sperm and/or mucus function.     

In-vitro cervical mucus -- sperm penetration tests and outcome of infertility treatments in couples with repeatedly negative post-coital tests

The results of in-vitro cervical mucus—sperm penetrationtests and cross-hostility tests in 178 couples with repeatedlynegative post-coital tests were recorded. Using a protocol ofthree cycles with intra-uterine inseminations (IUI) followedby three cycles with ovulation induction + IUI, the associationbetween the cause of infertility, results of the in-vitro testsand the outcome of infertility treatment was investigated. Wefound that repeatedly negative post-coital tests are a goodindicator of a cervical mucus — sperm penetration problem.The crosshostility test clearly differentiates the abnormalfactor in this interaction, and a good performance of the donors'spermatozoa in the cervical mucus correlates with increasedpregnancy rate. In male factor infertility, failure of the husbands'spermatozoa to penetrate cervical mucus was not indicative ofa deficient fertilization potential in vivo. In these patientsa serious attempt should therefore be made to reverse the infertilityby treatment with IUI or ovulation induction + IUI before attemptingassisted reproduction. Women with polycystic ovaries and repeatedlynegative post-coital tests should be investigated for spermreceptivity of the cervical mucus. Low receptivity of the cervicalmucus may imply that endometrial receptivity and oocyte qualityare also low. Ovulation induction and not IUI alone should thereforebe used as the preferred mode of treatment to improve pregnancyrate.     

Intrauterine insemination

Fifty-six couples with infertility due either to subnormal sperm(n = 40), hostile cervical mucus (n = 5) or idiopathic infertility(n = 11), were treated with intrauterine insemination of washedsperm. A total of 78 treatment cycles were completed. Nine pregnanciesresulted from these insemination cycles, giving an overall pregnancyrate of 8.3% per treat ment cycle. Eight pregnancies occurredIn the andrologic group after 56 treatment cydes. One pregnancywas established in the patient group with idiopathic infertilityafter 15 treatment cycles, while no pregnancy occurred in thepatient group with infertility due to cervical mucus hostility.The mean number of years of infertility in the couples conceivingfollowing treatment was 7 years (range 3–11). The spontaneouspregnancy rate in this patient group is low. The data obtainedin this study suggest that in selected patients intrauterineinsemination will result in an acceptable pregnancy rate. Thereis a need for a randomized prospective study designed to comparethe efficacy of intrauterine insemination with that of alternativetreatment modalities.     

Circulating antisperm antibodies and fertility prognosis: a prospective study

In a prospective study performed on 235 couples with long standinginfertility (median 5 years), circulating antisperm antibodies(ASA) were determined with both a standard tray-agglutinationtest (TAT) and a radloimmunoassay (RIA). Serum levels were correlatedwith results of sperm analysis (SA), postcoital testing (PCT)and in-vitro sperm-cervical mucus penetration testing (SCMIPT),performed with cervical mucus and spermatozoa of patients andin parallel with material of fertile donors. A simultaneousmicrobial screening included Chiamydia trachomatis, mycoplasmas,Herpes simplex virus, gonococci, other potentially pathogenicaerobic and anaerobic bacteria and yeasts. The pregnancy rate12 months after determination of ASA in the serum samples was27% (64/235). Between ASA-positive and -negative male and/orfemale patients (TAT as well as RIA), no significant differencewas found for any of the tested variables of SA, PCT, crossedSCMPT and colonization of genital secretions with microorganisms.Patients who later achieved pregnancy and those who did notdid not differ with regard to serum antibody status (TAT aswell as RIA). The findings suggest that the clinical significanceof circulating ASA determined with the currently available methodsis low. ASA in serum samples of infertile female andlor malepatients do not influence the fertility prognosis.     

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

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

Does treatment with testosterone undecanoate improve the in-vitro fertilizing capacity of spermatozoa inpatients with idiopathic testicular failure?(results of a double blind study)

Seventy-seven couples in whom conventional in-vitro fertilization(IVF)had remained unsuccessful because of low fertilization rateand abnormal sperm characteristics were given either testosteroneundecanoate 120 mg/day, or placebo during 3 months, after whicha new IVF treatment was applied under identical technical conditions.There were no significant changes in sperm characteristics amongthe treated and placebo couples and the fertilization rate showeda similar increase after treatment in both groups. No significantdifference in pregnancies occurred, with 32% pregnancies inthe placebo controls and 17%among couples treated with testosteroneundecanoate. It is concluded that testosterone undecanoate intakedoes not improve sperm characteristics, or the in-vitro fertilizingpotential, or pregnancy rate over those observed in the placebocontrols in cases with primary idiopathic testicular failure.     

Human and bovine cervical mucus penetration as a test of sperm function for in-vitro fertilization

Human and bovine cervical mucus penetration tests (n = 57) wereperformed preceding IVF to test their prognostic value as spermfunction tests for IVF. This evaluation also induded resultsfrom conventional semen analysis and from a computerized spermanalysis system. The bovine cervical mucus penetration testwas shown to be at least as valuable as the human cervical mucuspenetration test in evaluating sperm function. The migrationdistance of the vanguard sperm (P < 0.001) and the spermdensity at a fixed migration distance in the mucus column (P< 0.05) correlated most closely with the IVF results. A clearparallelism with the out come of the ‘swim up’ techniquewas also found. Of the sperm parameters examined, only spermmotility In the ejaculate (P < 0.05) correlated significantlywith the results of IVF. It is concluded that the outcome ofa bovine cervical mucus penetration test depends on the samesperm functions as re quired for IVF. Therefore, this test maybe of predictive value in an IVF programme.     

Blastocyst development and birth after in-vitro maturation of human primary oocytes, intracytoplasmic sperm injection and assisted hatching

Immature oocyte recovery followed by in-vitro oocyte maturationand in-vitro fertilization is a promising new technology forthe treatment of human infertility. The technology is attractiveto potential oocyte donors and infertile couples because ofits reduced treatment intervention. Immature oocytes were recoveredby ultrasoundguided transvaginal follicular aspiration. Oocyteswere matured in vitro for 36–48 h followed by intracytoplasmicsperm injection (ICSI). Embryos were cultured in vitro for 3or 5 days before replacement. Assisted hatching was performedon a day 5 blastocyst stage embryo. Embryo and uterine synchronywere potentially enhanced by luteinization of the dominant follicleat the time of immature oocyte recovery. Mature oocyte and embryoproduction from immature oocyte recovery were similar to theprevious IVF results of the patients. A blastocyst stage embryo,produced as a result of in-vitro maturation, ICSI, in-vitroculture and assisted hatching, resulted in the birth of a healthybaby girl at 39 weeks of gestation.     

The hypo-osmotic swelling test and the sperm mucus penetration test in determining fertilization of the human oocyte

The usefulness of the hypo-osmotic swelling (HOS) test and thesperm mucus penetration (SMP) test as sperm function tests forin-vitro fertilization was analysed in 56 couples. Using logisticregression analysis only the SMP test was independently relatedto fertilization (P = 0.004), no false negative results wereobtained, i.e. no fertilization if sperm from the ejaculatefailed to penetrate mucus. The HOS test was of no predictivevalue. The results justify a further examination of the SMPtest in other IVF centres.     

Accuracy of sperm-cervical mucus penetration tests in evaluating sperm motility in semen: a systematic quantitative review

BACKGROUND: Our objective was to determine the accuracy of in-vitro sperm penetration into cervical mucus or substitutes in evaluating sperm motility in semen. METHODS: This was a systematic quantitative review of test accuracy studies. The Cochrane library (2000:4), Medline (1966-2001), Embase (1988-2001) and SciSearch (1981-2001) were searched, in addition to manual searches of conference papers and bibliographies of known primary and review articles. Primary studies measuring in-vitro sperm penetration into cervical mucus, or substitutes (i.e. sperm-mucus penetration test, SMPT) and comparing results with sperm motility in semen were included. RESULTS: There were 18 primary diagnostic studies published in 17 papers, involving a total of 2580 samples. Fourteen primary diagnostic tests used vanguard distance as diagnostic criteria (SMPT(vd)) and the pooled likelihood ratio (LR) for positive (LR+) and negative (LR-) tests were 2.29 (1.82-2.87) and 0.52 (0.44-0.63) respectively. Four studies used diagnostic criteria based directly or indirectly on swim-up sperm count per high power field (SMPT(sc)) instead. Their pooled LR+ and LR- were 5.24 (3.36-8.18) and 0.15 (0.06-0.39) respectively. CONCLUSIONS: SMPT(vd) has a low accuracy in the evaluation of sperm motility in semen. However, SMPT(sc) was found to be more accurate. This method of using sperm concentration, instead of vanguard distance, as diagnostic criteria of in-vitro SMPT has potential as a useful laboratory-based sperm function test.     

Comparative study of the effect of human cervical mucus and a cervical mucus substitute, Healonid, on capacitation and the acrosome reaction of human spermatozoa in vitro

The present study was designed to investigate the effect ofhuman cervical mucus on capacitation and the acrosome reactionof human spermatozoa and compare its effect to that of a cervicalmucus substitute, sodium hyaluronate (Healonid). Spermatozoafrom donors of proven fertility were isolated from semen usingcervical mucus, Healonid or a direct swim-up (acting as thecontrol). Sperm capacitation and the acrosome reaction weremonitored by the chlortetracycline assay. In the mucus-treatedgroup, there was a significantly higher percentage of capacitatedspermatozoa, but a low incidence of spontaneous and A23187-inducedacrosome reactions compared to the control. The use of Healonidduring sperm isolation mimicked the effect of mucus relativelysuccessfully. Since mucus and Healonid show very little chemicalsimilarity, this finding would imply that cervical mucus exertsa physical effect during its interaction with spermatozoa, althougha chemical effect cannot be completely dismissed. In conclusion,this study confirms early reports describing the ability ofcervical mucus to capacitate spermatozoa but at the same timeconserve sperm function. The finding that Healonid exerts analmost identical effect on spermatozoa would lend support toits use as a cervical mucus substitute during in-vitro fertilityassessments and research studies.     

Chlamydial serology in 1303 asymptomatic subfertile couples

The clinical significance of antichlamydial antibodies (Chlam Ab) was determined in a total of 1303 subfertile couples consulting for infertility investigation and treatment. Median age of the women was 30 (range 22-44) years and of the men 33 (range 21-53) years. The median duration of infertility was 4 (range 1-21) years. All patients were asymptomatic for genital tract infection. A comprehensive infertility investigation included examination of the endocrine, cervical, and tubal factor, and semen analysis, antisperm antibody (ASA) testing, sperm-mucus interaction testing in vitro using a standardized protocol, and post-coital testing (PCT). Screening for Chlam IgG Ab was performed in serum of both partners, obtained at the same time. Simultaneous microbial cultures in genital secretions of both partners included a broad spectrum of potentially pathogenic bacteria. Elevated titres of Chlam IgG Ab as seromarker for previous infection were found in 20.8% of all women, and in 12.6% of men. Chlam Ab were significantly more frequent in partners of seropositive patients (in 51.8% of women with a Chlam Ab positive partner, compared to 15.8% of the other women). Microbial screening outcome was not significantly related to results of chlamydial serology in both partners. In women, elevated titres of Chlam Ab were significantly associated with a tubal factor, but were not related to reduced quality of the endocervical mucus (CM), including the in-vitro penetrability of the CM (using partners' or donors' spermatozoa). In males, Chlam Ab were not significantly related to the outcome of semen analysis, including screening for ASA (IgG and/or IgA) in semen, and several parameters of sperm functional capacity. After exclusion of couples with tubal disease, subsequent male fertility did not significantly differ in males with or without Chlam Ab. The results suggest that during basic infertility investigation, positive chlamydial serology as an easy screening procedure indicates a higher risk for a tubal infertility factor. However, in asymptomatic patients, Chlam IgG Ab in serum are not associated with a cervical factor or with the male factor, using several determinants for evaluation of semen quality including subsequent fertilizing capacity.      

Fertilization and early embryology: Evidence of sperm entry into assumed unfertilized human oocytes after sub-zonal sperm microinjection

Sub-zonal sperm microinjection (SUZI) as a treatment for malefactor infertility can facilitate fertilization, however, inmany cases oocytes remain unfertilized even though the spermis placed in close contact with the oolemma. In order to improveour understanding of gamete interaction in cases of failed fertilization,we have analysed the failed fertilized oocytes from both SUZIand conventional in-vitro fertilization. The fluorochrome Hoechst33342 (which binds specifically to DNA) was used to check forthe possible presence of paternal chromatin in the unfertilizedoocytes. A significantly higher (P < 0.01) number of microinjectedoocytes showed signs of fertilization 2–3 days after spermmicroinjection compared to normally inseminated oocytes, 30/175(17.1%) and 2/79 (2.5%) respectively. In addition, four outof eight couples returning for a second treatment by SUZI displayedanomalies in fertilization in both cycles. The semen characteristicsof patients with or without anomalies in fertilization was notdifferent. The irregularities observed in the fertilizationprocess infer that certain male factor patients have intrinsicsperm anomalies lying at the sperm membrane and/or chromatinlevel that could lead to anomalies in the appearance of thepronuclei.     

Sperm counts in enzymatically liquefied cervical mucus: quantitative validation using donor cervical mucus

The post-coital test evaluates the penetration of spermatozoainto cervical mucus; it relies on subjective meaurements andtherefore lacks precision. Enzymatic liquefaction of cervicalmucus allows sperm concentration to be measured in post-coitaltest samples, but the reliability of such measurements is notknown. Donor cervical mucus was used as a model to test theaccuracy and sensitivity of sperm quantification in liquefiedcervical mucus. Donor cervical mucus was dissolved by enzymatictreatments in the presence of known numbers of spermatozoaand the recovery of sperm cells was assessed after liquefactionof the samples. Enzymatic treatment of cervical mucus with acombination of bromelin and glycosidases resulted in reliableand fast liquefaction of the samples. The accuracy of spermconcentration measurements was 89±10% (mean±SD,n=50), and the sensitivity limits were 1x106 and 0.2x106 spermatozoa/mlfor quantitative concentration measurement and qualitative spermdetection respectively. This study demonstrates that liquefactionof cervical mucus by combined protease and glycosidases allowsaccurate and sensitive determination of sperm concentrationin the sample. Therefore we believe that valuable data can beobtained for sperm concentration and total sperm counts in post-coitaltests, that should help to improve the reliability of the post-coitaltest.     

A self-migration method for preparation of sperm for in-vitro fertilization

Human sperm samples (n = 211) were prepared for in-vitro fertilization(IVF) and embryo transfer by a self-migration procedure in Earle'smedium containing highly purified hyaluronic acid (Hya) (MW3 000 000) included to increase the viscosity of the medium.The method resulted in the recovery of a significantly higherpercentage of motile spermatozoa compared with the traditionalcentrifugation method, 87.5 ± 0.9% versus 76.1 ±1.3% (P < 0.001). When comparing media with and without Hyain the selfmigration method for preparation of normal spermsamples, the media containing Hya resulted in the recovery ofa significantly higher percentage of motile spermatozoa, 89.0± 0.8% versus 73.8 ± 2.0% (P < 0.001). In agroup of 80 consecutive couples entering our IVF programme,sperm samples from 44 of the men were allocated at random forthe self migration method in medium containing Hya and spermsamples from 36 men for preparation by centrifugation and swim-up.Significantly more pregnancies were achieved in the group preparedin medium containing Hya. It is concluded that self-migrationof sperm in a medium containing Hya is simple and rapid, andresults in a high recovery of motile spermatozoa which can beused for in-vitro insemination of human oocytes with favourableresults.     

Changes in the sialylglycoconjugate distribution on the human sperm surface during in-vitro capacitation: partial purification of a 20 kDa sialylglycoprotein of capacitated spermatozoa

Changes in the distribution of sialylglycoconjugates on thesurface of uncapacitated and in-vitro capacitated human spermatozoawere studied by means of two sialic acid specific lectins [Maackiaamurensis agglutinin and Sambucus nigra agglutinin). On theuncapacitated sperm surface, sialylglycoconjugates were foundbe localized from the post-acrosomal region of the sperm headto the tail middle piece, whereas after invitro capacitationthese molecules were only found in a small area of the post-acrosomalregion. The surface of capacitated human spermatozoa was alsoinvestigated by specifically radiolabelling its terminal sialicacid residues. A 20 kDa glycoprotein, which was partially purifiedby anion-exchange chromatography, was the main component ofthe sialylglycoconjugate pattern after in-vitro capacitation.     

Fertilization and early embryology: Behaviour of spermatozoa in human oocytes displaying no or one pronucleus after intracytoplasmic sperm injection

The behaviour of sperm cells after intracytoplasmic sperm injection(ICSI) was investigated by analysing 192 unfertilized and 37one-pronuclear (1PN) oocytes following ICSI. Eighty-two unfertilizedoocytes were directly fixed whereas 110 were first parthenogeneticallyactivated by puromycin. In contrast to the findings in unfertilizedoocytes after in-vitro fertilization, most unfertilized oocytesafter ICSI (n = 76) contained evidence of the presence of spermatozoain the cytoplasm. Few oocytes (n = 6) contained prematurelycondensed sperm chromosomes (PCC), whereas the majority containedeither intact sperm heads (n = 31) or swollen sperm nuclei (n= 39) along with metaphase II chromosomes of the oocyte. Followingactivation by puromycin, swollen sperm nuclei and PCC were nolonger observed, whereas unchanged sperm heads persisted in12 oocytes displaying a single pronucleus. A non-decondensedsperm nucleus along with decondensed maternal chromatin werealso discovered in 32 out of 37 oocytes displaying a singlepronucleus after ICSI. The findings in unfertilized and 1PNoocytes after ICSI indicate that successful sperm injection,even followed by oocyte activation, is not sufficient to guaranteenormal fertilization. It seems that partial sperm membrane damageprior to injection is also required to ensure normal sperm decondensation.     

Infertility: Dummy embryo transfer using methylene blue dye

The aim of this prospective work was to evaluate different cathetersand techniques used for embryo transfer. Studies were performedon 105 IVF patients before the start of treatment cycles. Eachpatient was used as her own control to study the expulsion ofmethylene blue (MB) dye after dummy embryo transfer. Group A(n = 35) underwent the test twice, before and after aspirationof the cervical mucus. Group B (n=30) underwent the test twicewith and without the presence of two air bubbles in the embryotransfer catheter. Group C (n = 40) underwent the test twiceusing two different catheters, the Wallace and the Craft catheters.The results showed that the dye was extruded at the externalos in 57% of the cases when the cervical mucus was not aspiratedcompared to 23% when the mucus was aspirated (P = 0.01). Thedye was extruded in 33% of the cases with air bubbles in thecatheter as compared to 27% when no air was present (P >0.05). When the Wallace catheter was used expulsion occuredin 25.5% compared to 77.5% when the Craft catheter was used.We concluded that using soft catheters and complete aspirationof cervical mucus significantly reduced the expulsion of thedye. The presence of air bubbles did not affect rate of expulsionof the dye.     

Possibilities and limitations of techniques of assisted reproduction for the treatment of male infertility

Since relatively few spermatozoa are needed for oocyte fertilization during gamete intra-Fallopian transfer (GIFT) or in-vitro fertilization (IVF), these methods have been applied in couples with infertility due to male causes. Forty-six couples with male factor infertility were enrolled in this study and results were compared with those attained in 48 couples treated with the same techniques for other than male causes. Overall, GIFT resulted in 26% ongoing pregnancies. GIFT seems to be particularly successful when the sperm concentration is 20 x 10(6)/ml or more, but sperm motility and/or morphology are poor. Nine pregnancies occurred out of 26 GIFT cycles in 18 cases selected on this basis. The ongoing pregnancy rate after IVF was 16% per patient. The latter treatment should be attempted in male immune infertility and in cases with a low sperm concentration, with or without abnormal sperm motility and/or morphology. In these circumstances, five pregnancies were attained out of 28 cycles in 14 cases. For similar sperm concentrations, the conception rate per cycle attained with techniques of assisted reproduction was more than twice that attained with conventional treatment of male infertility.