Study On The Correlation Between Sperm Morphology And Outcomes Of In Vitro Fertilization

Objectives:1. Study on the role of sperm morphology parameters in the use of IVF or ICSIfor assistant reproductive technology, choose the reasonable methods to improve thefertilization rate and avoid failure of fertilization.2. Establish the Cut off value of sperm morphology parameters on IVFpregnancy according to this group; study the effects of sperm morphology parameterson IVF outcomes.3. Study on the mechanisms of abnormal reproductive of patients with specialabnormal sperm morphology, provide the basis of the clinical diagnosis and treatmentof such patients.Study subjects:1. From May,2011to October,2013,265infertile couples visiting thereproductive medical center of First Hospital of Jilin University were included, all ofthe women’s fallopian tubes are blocked.2. Select3special cases with abnormal sperm morphology:95%acephalicsperm(Case1),98%acephalic sperm(Case2),100%globozoospermia (Case3);Control group:20%acephalic sperm,15%acephalic sperm.Methods:1. Use computer-assisted semen analysis system for semen analysis. Spermmorphology smear was prepared by Diff-Quik staining method. The morphology wasanalyzed according to the criteria of sperm morphology analysis of the5th edition ofWHO Laboratory Manual for the Examination and Processing of Human Semen.Calculate the percentage of normal and abnormal sperm morphology. Computationalacrosome index and acrosome integrity were analyzed on the same smear according tothe Menkveld method. 2. Sperm cells were embedded in epoxy resin using Epon812group. Lightmicroscopy after positioning ultrathin sections was observed under transmissionelectron microscope spermatozoa ultrastructure; Parafin-embed testicularseminiferous tubules, prepare parafin sections, detect HE staining of spermatogenesispathological state; Use sperm chromatin diffusion test combined fluorescence in situhybridization analysis of sperm nuclear DNA integrity of sperm and spermaneuploidy of chromosomes. Analyze500cases of sperm DNA damage andchromosome number18/X/Y case under a fluorescence microscope, the spermnuclear DNA damage index was calculated by the rate of sperm aneuploidy.3. Use SPSS17.0statistical software for analysis. T-test was used to analyzemeasurement data; Chi-square test was used for analyzing count data. MedCalcsoftware was used for ROC curve analysis. P <0.05indicates significant difference.Results:1. Role of sperm morphology parameters(1) Depending on the manner in vitro fertilization, IVF group is divided into197cases and68cases rescue ICSI group. Comparison of sperm morphologyparameters between the two groups showed that the percentage of morphologicallynormal sperm IVF group, acrosome index, acrosome integrity and rescue ICSI groupwere significantly different (P<0.05). There was no significant difference betweenICSI group and r-ICSI group in the abnormal sperm index(P>0.05).(2) Use ROC curve to analyze sample of265cases of various parameters onsperm morphology evaluation capacity fertilization methods and reference values.The results show acrosome integrity maximum area under the ROC curve was0.717,The difference between the areas under the curve of other parameters was notsignificant. We did specificity and sensitivity analysis for all sperm morphologyparameters, in which the best was acrosome index. The percentage of normal spermmorphologically, acrosome index and acrosome integrity cutoff of select method were1.44%,15.35%,28.57%, respectively.(3) According to fertilization in different ways, the binary logistic regressionanalysis with the insemination of sperm morphological parameters were correlated. The results showed that acrosome integrity and insemination with independentcorrelation has significant difference(OR=1.038, P<0.05).2. Affect of sperm morphology parameters on IVF outcomes(1) Using ROC curve analyze197cases of various parameters of spermmorphology on evaluation capacity and the reference value for IVF pregnancy or notoutcome. The results showed acrosome integrity largest area under the ROC curvewas0.553, but the difference between other parameters area under the curve was notstatistically significant. The percentage of normal sperm morphology, spermdeformity index, acrosome index, acrosome integrity cutoffs in IVF pregnancyoutcome evaluation were3.92%,1.37,26.34%and30.92%.(2) According to the parameters of sperm morphology on IVF pregnancyoutcome predicted intercept point value, the semen samples were group as normalsperm morphology, sperm deformity index, acrosome index and acrosome integritystudy sperm morphology parameters on the outcome of IVF effects. The results showthe percentage of morphologically normal sperm, the acrosome index and acrosomeintegrity of clinical pregnancy group were significantly different (P<0.05). Acrosomeindex between groups were significantly different of2PN fertilization rate(P<0.05).3. To investigate the mechanisms of abnormal reproductive of patients withspecial abnormal sperm morphologyCase1: Normal liquefaction time, moderate viscosity, volume1.5ml, pH7.9, theconcentration of83.55×106/ml,(a+b) levels of4.19%. Diff-Quik staining showed95%of the headless sperm. Chlamydia, Mycoplasma, Neisseria gonorrhoeae, TORCH,seminal plasma biochemistry, hormones, peripheral chromosome, AZF gene testswere normal. Sperm chromosome aneuploidy rate is0.6%. Sperm nuclear DNAdamage index was84.4%.Woman gets10eggs at the date of oocyte retrieval.4M Ⅱ oocytes, After ICSIget42PN fertilized all cleavage. Transplant two embryos of7Ⅱ grade quality. Nopregnancy after transplantation.Case2: Liquefaction normal, moderate viscosity, volume3.1ml, pH7.5, theconcentration of26.14×106/ml,13%(a+b) stage, the survival rate of60%. Diff-Quik staining showed98percent of headless sperm. UU culture was positive. PRL levelwas high. AsAb, Chlamydia trachomatis, peripheral chromosome, AZF gene testswere normal. Testicular biopsy smears:20%of spermatogonia,20of primaryspermatocytes,2%secondary spermatocytes, accounting for25%of the sperm cells,support cells accounted for30%,3%of sperm, per high-power field visible1-2sperm.Sperm chromosome aneuploidy rate was0.8%. Sperm nuclear DNA damage indexwas95%.Ultrastructure: specimen head, neck and middle, the tail can be seen in variousdegrees of structural abnormalities: the entire film only to see most of the sperm tail;poor acrosome development, deletion, acrosome membrane structure is not complete;nuclear vacuoles; mitochondrial size uneven derangement; portion of the sperm tailwire wrapped multiple axes, and the axis wire structure is incomplete.Testicular pathology: Limiting membrane can be seen in most of theseminiferous tubules sclerosis, cellular components disappear, only can see. Limitingmembrane was thickenned obviously with no sperm production. Some seminiferoustubules were relatively normal. Leydig fibrosis, inflammatory cells were foundoozing.Both spouses were disagreed using assisted reproductive progesterone.Case3: Liquefaction time>60mins, slightly thick, amount1.2ml, pH7.8,concentration of35.99×106/ml,(a+b) levels of0.00%, the survival rate of38%.Diff-Quik staining showed100%round sperm. Hormones, peripheral chromosome,AZF tests were normal. Sperm chromosome aneuploidy rate was0.2%.Woman gets21eggs at the date of oocyte retrieval,20MⅡ oocytes. After ICSIobtaining two2PN cleavages, embryo transplant a non-premium grade5Ⅱ.14d aftertransplantation, blood HCG418U/L,34d after transplantation see intrauterine singlelive births, seen in germ and beating heart tube. A healthy baby boy was born in June,2013.Control group:20%acephalic sperm:Sperm chromosome aneuploidy rate was0.4%. Sperm nuclear DNA damage index was34%.15%acephalic sperm:Spermchromosome aneuploidy rate was0.2%. Sperm nuclear DNA damage index was27%. Conclusions:1. Sperm normal morphologically, acrosome index and acrosome integrity can beused as an important basis in select fertilization methods. Acrosome morphologyparameters were better than the percentage of normal sperm morphology.2. Percentage of normal sperm morphology, acrosome index, acrosome integritycan predict IVF pregnancy outcomes.3. Abnormal ultrastructure of sperm and high sperm DNA damage may be thecause of abnormal reproductive of patients with abnormal sperm morphologyabnormalities,and even such patients can get successfully assisted reproductive afterICSI,but their long-term safety of future generations should be a long-term follow-upstudy.