Study On Serum Hormone And Sperm Morphology By SEM And Sperm Y-chromosome Microdeletions Of Patients With Idiopathic Oligospermic

Introduction: According to WHO report, Human reproduction capability is poor thanbefore. However, sperm density and motility were being decreased from 120×10~6/mlto 26×10~6/ml during 50 years. Recent work by National study indicates the reason thatabout 50％couples with infertility is male reason. Intracytoplasmic sperm injection(ICSI) is a recent major advance in the treatment of such couples. ICSI offersidiopathic oligospermia couples the possibility of fathering own genetic children.However, what is a useful marker of spennatogenesis in serum7 How is spermmorphology construction by scanning electron microscope (SEM) and by spermmorphology Quick Staining kit? The reason of gene idiopathic oligozoospermia is theaim of present study.Objective:To investigate the correlation of serum hormone and sperm morphology construction by scanning electron microscope (SEM) and the Y-chromosome microdeletions with idio-pathic oligozoospermia patient.Materials and methods:A total of 60 consecutive men who presented with clinical and laboratory dataindicating idiopathic oligospermia. Mean male age was 32.1±2.8(rang 29～36)(group1), The control group included 60 men having normal seminal parameters was31.0±2.2(rang 28～35)(groug 2).1. Patients and seminal analyses: Semen samples were produced by masturbationafter 2～7 days of sexual abstinence and collected into sterile containers. Thesperm density was detected by computer-aided sperm analysis(CASA) at least two seminal analyses which were carried out as described in the World HealthOrganization Manual(WHO, 1999). Each patient underwent physicalexamination. The sperm density in semen was detected by routine semen analysis.Group 1 sperm density was less than 5×10~6/ml and more than 0. Group 2 spermdensity was higher than 20×10~6/ml.2. Hormone Analyses: Hormones were measured using commercially available kits.testosterone (T), free testosterone (FT), sex hormone-binding globulin (SHBG)and inhibin B(IHN-B). Blood and semen samples were collected from two groups.Serum was collected two times at 8:00and 10:00 am, and mixed serum amply.Serum concentration was measured by using specific an enzyme linkedimmunosorbent assay (ELISA) with two monoclonal antibodies in serum ofpatients with oligospermia.3. Semen processing: Semen samples were obtained by masturbation after 2～7 daysof sexual abstinence. After liquefaction at incubator, semen parameters wereassessed as outlined by the WHO criteria. Sample was examed by spermmorphology Quick Staining (Diff-Quik) Kit. Semen analyses were performed toseparate motile spermatozoa from non-sperm cells, The ejaculate was layeredover the gradient and centrifuged at 2000r/min for 20min. After centrifugation ,thesupernatant was removed, and sperm resuspended in 2ml freshmedium(Sperm-washing medium, COOK). Sperm washing according to assistedReproduction Technology (ART). The semen was divided into two parts equally.One part of semen was fixed in 2.5％(w/v) glutaraldehyde solution in a sodiumacetone series, dried in a critical point drier using carbon dioxide, mounted on thespecimen holder, coated with gold, and examined under a scanning electronmicroscope (SEM), detecting the sperm deformity index (SDI).The another part ofsemen was performed and checked by PCR amplification of selected regions ofthe Y-chromosome. The male-specific region of the Y-chromosome (MSY) STSprimers amplify both anonymous sequences of the chromosome or genes. At the same time lymphocytes Y- chromosomal microdeletions of blood was detected.The PCR amplification genomic DNA for clinical diagnosis requires strictcompliance with excellently laboratory practice and basic principles of qualitycontrol.Results:1. Inhibin B significantly lower in group 1 compared with group 2. Serum inhibin Bconcentrations were significantly higher with group 2(mean values25.07±19.49pg/ml and 189.06±47.06 pg/ml, P＜0.01). In contrast, no differenceswere detected between these two groups with respect to serum T(mean values437.27±109.1 ng/dl and 436.66±97.52 ng/dl),FT(mean values 17.03±5.94 pg/mland 18.63±4.32 pg/ml),SHBG(mean values 186.21±68.74 pg/ml and184.24±67.68 pg/ml) or testicular size respectively(P＞0.05).2. The study of sperm morphology construction by SEM detecting SDI, there wassignificant differences between two groups(1.644±0.55 vs 0.864±0.23, P＜0.05). SDIof sperm of Idiopathic oligozoospermia had more deformity than that of normalgroup. The study of sperm morphology by SEM is better way than other method.3. Multi-PCR and PCR-RFLR were used to analyze the sperm deletion of DAZ genecopies in the AZFc region of Y-chromosome. Chromosoma(quantity andconstruction were detected by G-band in the two groups). In the severeoligozoospermic patients and fertile males, the rates of AZFc deletion was 15％, noother type Y-chromosomal microdeletions was found, but no deletion was detectedin the normal control group. There is significant differences in the semenY-chromosomal microdeletions between two groups. The rates of oligospermiaAZFc microdeletion (15％)is higher than lymphocytes.Conclusion:1. Measuremcnt of serum IHN-B concentration could reflect the function of tesis,which is useful for early diagosis and treatment of oligospermia. Inhibin Bmeasurement is a useful non-invasive predictor of spermatogenesis, and thus, all infertile males should have serum inhibin B concentrations determined in additionto other measurement.2. SDI of oligospennia is significant higher than fertile group by SEM. SEM may bea better method to exam sperm of infertile males.3. There is a high frequency of sperm Y-chromosomal microdeletions in patientswith oligosperia than that of serum lymphocytes Y-chromosomal microdeletions,which suggests that Y-cbormosomal microdeletions might be important genoticcauses sperm density abnormal and rnale spermatogensesis failure.