Mechanism Of Morphologically Abnormal Sperm

BACKGROUND Some 10% of couples are subfertile, and in halfof these a male factor is involved. Spermatozoa have abnormal morphologyin about 50% of infertile males. This is perhaps of no surprise since it hasbeen known for some time that only morphologically normal sperm can passthough cervical mucus and the human zona pellucida selectively bindssperm with normal morphology such that men with severe teratozoospermiafrequently have defective sperm–zona pellucida interaction. Otherwise,morphologically abnormal sperm often exhibites a higher risk of aneuploidyrate, with can result in lower cleavage rates, developmental arrest inembryos, abortion although morphologically abnormal sperm can passthrough oocyte.Of all the semen parameters, sperm morphology has consistently beenthe best indicator of male fertility. But, the value of normal spermmorphology is challenged by the large number of classification systems usedto describe what factors constitute a morphologically normal/ abnormalspermatozoon, the various staining procedures employed and the subjectivenaure of the evaluation. Although the cut-off value has been set by WHO,the threshold value was lack of the concordance with clinic which madesome patients accepted some unnecessary treatment.The mechanism of sperm morphology abnormal is unclear, whichresults in no effective methods employed in teratozoospermia. So, it isnecessary to investigate the mechianism of sperm morphology abnormal inorder to provide some theories for treatment of teratozoospermia.OBJECTIVE We compared the influence of different stainingmethods on the results of sperm morphology analysis, so that an techniqueof sperm morphology analysis which is applicable and reliable could beestablished. We studied the relationship between morphology parametersand semen conventional parameters, as well as sperm morphology andsperm function, in order to illuminate the important value of spermmorphology analysis in evaluating male fertile potential. We also study thecorrelation of sperm morphology with accessory sexual gland function,sperm nuclear maturity, endocrine hormone and ROS, in order to initiallyexplored the mechanism of abnormal morphology in sperm. Spermmorphology and subcellular structure in severer asthenozoospermia wasreported so that study the influence of abnormal structure in sperm on spermfunction.PATIENTS 1949 semen samples were collected, involving fertilegroup 295and control group 423,infertile group 463, abnormal semenparameters grouop 682 and teratozoospermia group 86.METHODS ① Semen smears were stained according toWright-Giemisa, Modified Papanicolaou and Coomassie brilliant bluemethod. Then, the influence of these three staining methods on the results ofsperm morphology analysis was compared. ② Semen parameters wereanalysed. The predictive ability of the different semen variables todifferentiate between the fertile of subfertie status of a male was analysedusing ROC curve analysis.③Sperm morphology were analysed, involvingacrosome morphology parameters, TZI and SDI. We investigated therelationship of the above parameters with other semen parameters. We alsoperformed HOST, and induced capacity and acrosome reaction, so that therelationship between sperm morphology and sperm function was studied. ④Seminal activity of neutral alpha-glucosidase, acid phosphatase and fructoseconcentration were measured using spectrophotometry. Sperm nuclearmaturity, serum or seminal hormone and ROS were detected. ⑤Ultrastructure, sperm nuclear maturity and sperm mitochondria functionwere analysed from a severe asthenozoospermic patient.RESULTS ①No significant difference was found in the percentageof morphologically normal sperm between the three staining methods(P>0.05).Comparing with Modified Papanicolaou stain and Coomassiebrilliant blue stain, the rate of Neck/Midpiece defect sperm ,as well as taildefect sperm , significantly decreased after Wright-Giemisa stain(P<0.05).No significant difference was observed in the rate of sperm with I typeacrosome and the rate of sperm with Ⅳ type acrosome between the threestaining methods (P>0.05). However, the rate of sperm with Ⅱ or Ⅲ typeacrosme was higher using Modified Papanicolaou stain than using two otherstains(P<0.05). The significantly positive correlationship was found in therate of normal morphologically sperm (P<0.01, P<0.05 ), as well as in therate of acrosome intactness (P< 0.01 ), among the three staining methods.②Difference between fertile and subfertile groups were most significant fornormal morphologically sperm, concentration, motility, grade A, motilesperm rate, viability, HOST scores and pH (P<0.05 ). No difference wasobserved in both groups for semen volume, WBC (P>0.05). ROC curveanalysis showed no significant difference of the area under the ROC curvewas found between sperm morphology and all other semen parameters. Thebest discriminating parameter between the two groups was spermmorphology evaluated according to WHO criteria at a cut-off point of 14%normal spermatozoa. The other cut-off values were at 72.03×106/mL forconcentration, 28.08% for motility, 53% for viability, 47% for HOST scores,and 7.1for pH .③Comparing with abnormal semen parameters group, therate of intact acrosome was increased in normal semen parameters group(P<0.05). The rate of intact acrosome was positively correlated with thepercentage of morphologically normal sperm and sperm viability(P<0.05,P<0.01), and negatively correlated with the semenleukocytospermia concentration (P<0.05).Comparing with control group,TZI and SDI value significantly increased in infertile group (P<0.05). Thesignificantly positive relationship between TZI and SDI with WBC wasobserved (P<0.05). However, no important relationship was obtainedbetween TZI and SDI with sexual abstinence time, concentration, motility,HOST scores, and viability (P> 0.05).④ HOST scores of 0% control groupand of 41.5% in abnormal semen parameters group were in 0-50%,significant difference was found between these two group(P<0.05). HOSTscores of 19.5% control group and of 19.2% of abnormal semen parametersgroup were in 50-60%, no difference was found between these two group(P> 0.05). HOST scores of 80.5% control group and of 39.5% abnormalsemen parameters group were in 60-100%, significant difference was foundbetween these two group(P<0.05).No significant relationship of HOSTscores was found with the rate of morphologically normal sperm, headdefects sperm, Neck/Midpiece defects sperm and tail defects sperm(P>0.05). Comparing with sperm motion patameters before capacitation, GradeA, VSL,VAP,LIN,WOB were significantly decreased after capacitation(P<0.05), but Grade B and BCF significantly increased(P<0.05). Nodifference was found in other sperm motion parameters between before andafter capacitation(P> 0.05), as well as in the rate of morphologicallynormal sperm(P> 0.05). After capacitation, the rate of morphologicallynormal sperm positively correlated with VCL ,VSL,VAP(P<0.05),However, no relationship was found between the rate of morphologicallynormal sperm and other motion parameters(P>0.05). Comparing withmorphologically normal sperm group, the rate of sperm with acrosomereaction significantly increased in Small head sperm group, as well as inTapered head, Pyriform head, Round head, Irregularity sperm group,Borderline morphology sperm group, Other abnormal sperm group(P<0.05 ) . ⑤ Comparing with control group, fructose concentration wassignificantly higher,however, activity of neutral alpha-glucosidase and acidphosphatase were significantly lower in group with abnormal semenparameters(P<0.05). Inversely correlation was found between activity ofneutral alpha-glucosidase and the percentage of other defect sperm(p<0.05),as well as between activity of acid phosphotase and the percentage ofhead defect sperm(p<0.05).⑥Comparing with control group, the rate ofsperm with AO positivity , CMA3 positivity, AB positivity significantlyincreased(P<0.05). Significantly negtive correlation was obtained betweenAO positivity , CMA3 positivity, AB positivity with the percentage ofmorphologically normal sperm(P<0.01). Also a significantly positivecorrelation between head defects, Neck/Midpiece defects, and tail defectswith AO positivity(P<0.05,P<0.01). CMA3 positivity significantlycorrelated with head defects ( P < 0.01 ) . The significantly positivecorrelationship was found among AO staining, CMA3 staining, and ABstaining(P<0.01). Meanwhile, SDS test significantly correlated withSDS-EDTA test(P<0.01). The results of logistic regression analysisshowed that only AB staining independently related with morphologicallynormal sperm rate(B=0.122, OR=1.129,95.0% C.I. = 1.041-1.225,P=0.004). After swimming up, AO positivity, CMA3 positivity, ABpositivity, and the percentage of stable sperm after SDS-EDTA in swim-upfraction were lower than those in pellet(P<0.05). ⑦Comparing withcontrol group, serum T significantly increased in teratozoospermic group(P<0.05). Significantly negative correlation was found between serum T andthe rate of morphologically normal sperm(P<0.05).No relationship wasfound of T and FSH between semen plasma and serum(P> 0.05). Nodifference was observed between control group and teratozoospermic groupin the lever of seminal T and FSH(P> 0.05). No relationship of the lever ofseminal T and FSH was found with the rate of morphologically normalsperm(P> 0.05). The percentage of stable sperm after SDS-EDTApositively correlated with the lever of seminal T(P<0.05). No relationshipwas found of seminal T and FSH with AO positive, CMA3 positivity, ABpositivity, and the percentage of stable sperm after SDS(P> 0.05).⑧ Nodefference was found in WBC concentration between control group andteratozoospermic group(P> 0.05), as well as Basal ROS, FMLP-inducedROS, PMA-induced ROS, ROS score(P> 0.05). Comparing with controlgroup, the percentage of tail defects sperm significantly increased interatozoospermic group(P<0.05). No correlation was found between BasalROS, FMLP-induced ROS, PMA-induced ROS, ROS score with the rate ofmorphologically normal sperm(P> 0.05).Significantly negative correlationwas obtained between Basal ROS, FMLP-induced ROS, PMA-induced ROS,ROS score with the percentage of stable sperm after SDS(P<0.05,P<0.01).However, no correlation was found between Basal ROS, FMLP-inducedROS, PMA-induced ROS, ROS score with AO positivity, CMA3 positivity,AB positivity, and the percentage of stable sperm after SDS-EDTA(P>0.05). ⑨ Activity of neutral alpha-glucosidase in semen plasma was 13.92mU /ejaculate which was lower than threshold. However, activity of acidphosphatase and fructose concentration were normal. The sperm withoutmitochondria was 40%. After sperm nucleus maturity test, AO positive rate ,CMA3 positive rate, AB positive rate respectively was 22.5%, 21%, 29%.Under transmission electron microscopy, most sperm ultrastructure wereabnormal.CONCLUSIONS ①We can consider the three staining methods,Modified Papanicolaou stain, Wright-Giemisa stain, and Coomassie brilliantblue stain, as reliable and applicable staining methods to detect thepercentage of human morphologically normal sperm and the rate ofacrosome intactness. Among the three staining methods, ModifiedPapanicolaou is better to be used in examing the percentage of humanmorphologically normal sperm, and Wright-Giemisa stain is more suitablefor detecting the rate of acrosome intactness.② Sperm morphology could beconsidered as an important means of identifying male infertility.③ To someextent, male fertility propotion could be established through acrosomemorphology analysis. Morphologically normal acrosome could result inlower viability. Morphologically abnormal acrosome and sperm in higherextent could result from higher WBC concentration in semen. ④Nosignificant influence of morphologyically abnormal in sperm was found onsperm tail membrane function. Even though no morphologically differenceduring capacitation, sperm with abnormal morphology could not performhyperactivation and acrosome reaction. ⑤ Seminal neutral alpha-glucosidase,acid phosphatase and fructose could not be associated with spermmorphology, which could imply no influence of abnormal function inepididymis and accessory sex gland on sperm morphology.⑥ It could be animportant reason for sperm morphology abnormal that the replacement ofhistones by protamines abnormally occurred which could result in absenceof protamine, sperm DNA damagement, and degrade of spermfunction.Sperm recovered after swim-up could possess higher nuclearmaturity. ⑦ Increased serum T could result in sperm morphology abnormal.Blood and seminal plasma T, FSH levels were not correlated. Seminalplasma T could not associated with sperm morphology. However, decreasedseminal T could result in high sperm chromatin stability which could notmake the sperm chromatin decondense to form male pronucleus when spermpassed into oocyte.⑧ Increased lever of ROS could not influence spermmorphology, but sperm chromatin stability, resulting in sperm beingsubjective to decondense after ejaculation. ⑨ Abnornal sperm ultrastructure,especially abnormal mitochodria and flagellae, could be important risk ofsperm motility. Impaired flagellar structure could be associated withrespiratory tract infections.