Cryodamage to plasma membrane integrity in head and tail regions of human sperm

Aim: To investigate the effect of cryopreservation on the plasma membrane integrity in the head and tail regions ofindividual sperm, and the relationship between intact cryopreserved sperm and its motility and zona-free hamster oocytepenetration rate. Methods: The eosin Y exclusion and the hypoosmotic swelling tests were combined to form a sin-gle test (HOS-EY test) to identify the spermatozoa with four types of membrane integrity. Results: After cryop-reservation, there was a marked decline in the percentage of spermatozoa with Type Ⅳ membrane integrity (head mem-brane intact/tail membrane intact), and a significant increase in those with Type Ⅰ (head membrane damaged/tail mem-brane damaged) and Type Ⅲ (head membrane damaged/tail membrane intact) membrane integrity (n = 50, P <0.01). The value of Type Ⅲ integrity had a wide range of variability, whereas Type Ⅱ (head membrane intact/tailmembrane damaged) was uncommon after thawing. A high correlation was observed between the percentage of Type Ⅳintegrity and sperm motility ( n = 50, r = 0.74, P < 0.01 ). However, the values of Type Ⅳ integrity were usuallylower than those of post-thaw motility in most cryopreserved samples. The value of Type Ⅳ integrity did not correlatewith the sperm penetration rate ( n = 25, r = 0.22, P > 0.05). Conclusion: (1) The HOS-EY test has the advan-tage of showing four patterns of membrane integrity in individual spermatozoon; (2) Cryopreservation causes a signifi-cant membrane rupture in the head and tail regions of spermatozoa; Type Ⅲ is the main transitional state of membranecryodamage; (3) Cryodamage to head and tail membrane may occur independently; the presence of an intact tail mem-brane does not necessarily indicate the intacmess of head membrane. (4) Intact membranes are closely related to post-thaw motility, but do not reflect the fertilizing potential.

Evaluation on Sperm Acrosome Integrity of Infertile Men with Varicocele

Objective To determine the sperm acrosome integrity of samples from infertile men with varicocele. Methods Forty-nine infertile men with varicocele were divided into three groups according to the grade of varicocele. Group A （grade Ⅰ）, B （grade Ⅱ）, and C （grade Ⅲ） consisted of 15, 18, and 16 cases, respectively. Besides, 15 semen samples from normospermic donors were used as the control. The acrosome integrity of sperm was examined with fluorescein-labeled Pisum sativum agglutinin. Acrosomal ultrastructure was observed with transmission electron microscopy. Results In three varicocele groups, most samples had high sperm abnormal morphology rates. There were significant differences in acrosome integrity rates between each varicocele group and the control （P〈0.01）. Group C had the lowest acrosome integrity rate among the three groups. Ultrastructural observation showed that acrosome malformations revealed acrosomal membranes defects, swelling, hypoplasia, and dissolution of the matrix. Conclusions Infertile men with varicocele had low level of acrosome integrity. Severe varicocele for infertile men might be associated with severe acrosomal defects. Evaluating sperm acrosome should aid the understanding of the sperm structural state and benefit the treatment for infertile men.

Evaluation on the Morphology and Membrane Integrity of Immotile Human Sperm

Objective To determine the membrane integrity in the head and tail regions of individual spermatozoon, and observe sperm morphology for samples with totally immotile sperm. Methods Ten infertile men with immotile sperm were enrolled into this study （group A）. The membrane integrity in the head and tail regions of individual spermatozoon of immotile sperm was examined by using the combined hypo-osmotic swelling-eosin Y exclusion test （HOS-EY test）. Sperm morphology was observed by light, scanning and transmission electron microscopy. Ten semen samples from normospermic donors were used as the control （group B）. Results The percentage of sperm with intact both head and tail membranes in group A was significantly lower than that in group B （P〈0.01）, whereas the value of sperm with defective head membrane but intact tail membrane in group A was significantly higher than that in group B （P〈0.01）. Abnormal sperm morphology in group A had a high incidence, and immotile sperm with viability and normal morphology could be observed in some cases. Most sperm had multiple ultrastructural defects. Conclussion Some immotile sperm had intact tail membrane but defective head membrane. Immotile sperm with viability and normal morphology could exist in some cases though abnormal sperm were in a great proportion. Carefully evaluating immotile sperm membrane integrity and morphology should benefit the treatment of patients with immotile sperm.

A new method of demodulating the digital frequency modulation signals

A new method of estimating the frequency-known signals from the strong background noise was presented first. Then the new method was used in the demodulation of the digital frequency modulation (FSK) signals. The new demodulation method can complete the demodulation of the FSK signals only with the carrier frequency and without any carrier phase information. The simulation results show that the performance of anti-noise of the new method is better than that of the incoherent demodulation method and the fluctuation of the carrier phase has little effect on the new method. So the new demodulation method has a fine prospect in the practical applications.

Effects of Local Testicular Heating on Bcl-2 and Bax Protein Expression in Spermatogenic Cells in Rats

Objective To investigate the expression of Bcl-2 and Bax in spermatogenic cells induced by local testicular heating in rats Methods Forty adult male SD rats were divided into experimental group and control group at random. According to day 0.5, 1, 3 and 6 after local testicular heating, each group was divided into 4 subgroups： experimental subgroup （n=6） and control subgroup （n=4）. The expression of Bcl-2 and Bax in the spermatogenic cells was detected on day 0.5, 1, 3 and 6 after heat exposure by using immunohistochemistry. Results Compared with control groups, the ratio of positive cells and content of Bcl-2 positive cells significantly decreased in all experimental subgroups （P〈0.01）. The content of Bax positive cells increased in all experimental subgroups （P〈0.01）, the ratio of positive cells which had no significant difference （P〉0.05） except 6 d group decreased （P〈0.01 ）. Redistribution of Bax from a cytoplasmic to perinuclear or nuclear localization could be observed after heating. Conclusions Expression of Bcl-2 would decrease and Bax would increase with redistribution in spermatogenic cells in rats after heating. The change of Bcl-2 and Bax expression in spermatogenic cells would be correlated with the spermatogenic cell apoptosis induced by heating.

Evaluation of Endotoxin in Culture Medium for Human in vitro Fertilization

To determine whether the presence of bacterial endotoxin in the commercial culture media utilized for human in vitro fertilization （IVF）, and evaluate the difference in detecting endotoxin in culture medium between the human sperm motility assay and the 2-cell mouse embryo assay. Methods Thirty-six batches of culture media commonly used in IVF laboratories from 3 manufacturers were determined for thepresence ofendotoxin before using the medium for the assisted reproductive programs （group A）. After being used, 25 specimens among above media were also tested （group B）. The chromogenic limulus amoebocyte lysate （LAL） test was used for quantification the content of endotoxin. In addition, the human sperm motility assay was compared with the 2-cell mouse embryo assay to evaluate the difference in detecting endotoxin in culture medium. Results Endotoxin was not detected in group A. However, 2 samples were positive in group B, Sperm did not show significant change in motility in group A during 24 h of incubation when compared with the control （P〉0. 05）. However, in group A the 2-cell embryo development to blastocyst was suppressed in 3 batches of media. Conclusions Regular screening of each batch of culture medium should be performed if possible although there was no evidence of endotoxin contamination in commercially prepared pre-tested media. Culture environment should be stringently controlled in case the medium is polluted. The sensitivity of the sperm motility assay was lower than that of the mouse embryo assay for detecting low levels of endotoxin or toxic compounds in the medium.

Analysis of DNA Content of Various Types of Spermatogenic Cells in Rat after Testicular Heating with Flow Cytometry

To measure DNA contents of spermatogenic cells and analyze the efficiency of spermatogenesis after testicular heating in rat Methods Eighty adult male Sprague-Dawley rats were randomly divided into experimental group （43 ℃, 30 min） and control group （22 ℃, 30 min）. According to day 0.5, 1, 3, 6, 10, 25, 35 and 50 after local testicular heating, every group was divided into 8 subgroups： experimental subgroups （n=6） and control subgroups （n=4）. DNA contents of various types of germ cells were observed with flow cytometry （FCM） in all groups. Results Compared with control groups, percentages of 4C cell （primary spermatocyte） in 0.5-35 d groups and percentages of 1C cell （spermatid and sperm） in 6-50 d groups significantly decreased in experimental groups （P〈0.05）, and percentages of 2C cell （spermatogonium and second spermatocyte） in 3 -35 d experimental groups increased significantly after heating （P〈0.05）. 4C：2C in all of 8 experimental groups and 1C：2C in 3-35 d experimental groups were down （P〈0. 05）, and in 1 d experimental group 1C：4C was up after heating （P〈0.05）. Conclusions After being heated, the number of spermatocyte firstly decreased, and then that of spermatid and sperm decreased too. Heat influences several stages in spermatogenesis and results in suppression of spermatogenesis. Flow cytometry is an effective method for researching on the change of spermatogenesis and has significance on mechanism about changing of spermatogenic cells induced by heat.

Expression of Core Domain of Porcine Zona Pellucida 3β in E.coli

To obtain the recombinant core domain of porcine zone pellucida 3β （cZP3β） for the further research on its functions Methods The nucleotide sequence region from 44 to 306 codons of pZP3β entire eDNA was obtained by PCR and then was cloned into pET-3c vector. After being identified, recon was transformed into E.coli BL21 （DE3） pLysS and then induced by IPTG. Results The recombinant cZP3β was expressed in E. coli up to 15% of total cellular proteins, and was made sure by Western blot analysis. Conclusion The research on expression of core domain of pZP3β could benefit to further investigation of its immunogenicity and the development of antigen preparation.

Effects of Mifepristone (RU486) on Development and Ultrastructure of Mouse Blastocysts

Objective To investigate effects of mifepristone （MP） on the development and ultrastructure of mouse blastocysts in vivo Methods Sixty female mice were equally divided into 4 groups： control group （group A）, 1.9 mg/kg MP group （group B）, 5.6 mg/kg MP group （group C）, and 16.8 mg/kg MP group （group D）. The female mice of 4 groups undertook a superovulation method. The development of obtained blastocysts was evaluated, and ultrastructural changes of the blastocysts were observed by transmission electron microscope. Results In comparison with group A, the development rate of blastocysts in group B showed no difference （P〉0.05）, while the development rate of blastocysts in both group C and group D was significantly lower （P〈0.05）. With doses of 5.6 mg/kg and 16.8 mg/kg, the blastocysts showed granular appearance of the cytoplasm, irregular cell borders, enlarged perivitelline space and degeneration. Ultrastructure of the blastocysts in group B was similar to group A, except a little number of fat droplets in the cytoplasm. In group C, the microvilli on apical surface was decreased in number or even disappeared, mitochondria were under developed, a lot of filamentous crystals were found in the cytoplasm. Cellular junctions were defected. In group D, the blastocyst cells were irregular in shape, mitochondria were frequently vacuolated, the nucleolus was enlarged, nuclear membrane was ruptured, and chromatin was slack. Conclusion MP could damage to the ultrastructure of mouse blastocyst, and was responsible for the inhibition of blastocyst development. The inhibitory effect of MP would be in a dose-dependent fashion.

Evaluation of Sperm Parameters of Infertile Men with Retrograde Ejaculation

Objective To investigate sperm parameters of infertile men with retrograde ejaculation. Methods Twelve infertile men with retrograde ejaculation （group A） were enrolled into this study. Sperm samples were obtained from the postejaculation urine. After sperm recovery and washing procedure, sperm parameters were assessed. Twelve semen samples from normospermic donors were Used as the control （group B）. Results In all retrograde cases, motile sperm with forward movement were observed in the medium. Motility of group A was significantly lower than that of group B （P〈0.01）. In group A, sperm motility ranged from 11% to 56%, sperm with intact both head and tail membranes was 42.2 ±12.3%, sperm count ranged （13-85）×^ 106/ml, and the sperm survival time was highly shortened. Sperm with normal morphology and intact acrosome were observed in retrograde specimens. Conclusion Sperm parameters recovered from retrograde specimens were highly variable between subjects. The toxicity of urine caused deleterious to sperm functions. Motile sperm could be collected by sperm recovery procedure. Sperm parameters could meet the requirement for the use of assisted reproductive techniques for treating infertile men with retrograde ejaculation.

Expression of Nonfusion Extracellular Porcine Zona Pellucida Protein 3β in E.coli

Objeetive To obtain the recombinant non fusion extracellular porcine zona pellucida protein 3β （pZP3β） in E.coli Methods By modificated the transition initiation region （TIR） in primers, synthetic nucleotide was gained by PCR. Such gene was cloned into pET-3c vector and transformed into E.coli BL21（DE3）pLysS. Results The recombi.ant nonfusion extracelhtlar pZP3β was expressed in E.coli to 10% of total cellular proteins, and identified by the Western blot method. Conclusion Modification of nucleotide without changing amino acid sequences is an effective means to increase non fusion expression rate of recombinant proteins, such as pZP3β in E. coll.

Effects of Cryopreservation on the Ultrastructure of Human Testicular Sperm

Objective To investigate effects of cryopreservation on changes of the ultrastructure of human testicular sperm and evaluate the efficacy of cryopreserving testicular tissue as a source of sperm for assisted reproduction. Methods Testicular biopsy tissues were obtained from infertile patients （n=12） with obstructive azoospermia and cryopreserved. Testicular sperm motility was observed after in vitro culture procedure. The ultrastructure of testicular sperm （n=6） was examined by transmission electron microscope. Results After cryopreservation, 10 biopsy tissues frozen revealed motile sperm, and 2 samples showed non-motile sperm. Some testicular sperm in frozen-thawed group had normal morphology in fine structures. Sperm head in frozen-thawed tissue showed a proportion of nuclei with more electron-dense granules of chromatin. In a few frozen-thawed sperm heads, formation of vesicles and degeneration were observed. The frozen-thawed testicular sperm frequently showed swollen or/and ruptured of the plasma membrane and acrosome membranes. Conclusion Cryopreservation of testicular tissue is simple and efficacious for testicular sperm extraction. And the freezing-thawing procedure of testicular tissue causes damage to ultrastructural morphology of human testicular sperm.

Semen Quality in Infertile Men with a History of Unilateral Cryptorchidism

Objective To evaluate semen quality in infertile men with a history of unilateral cryptorchidism Methods Semen samples were obtained from 47 infertile men with a history of unilateral cryptorchidism. Semen analysis and membrane integrity including both eosin Y stain and hyo-osmotic swelling tests were performed using standard procedures. All men were divided into three groups according to their age at surgery of orchidopexy as follows： group A, 2-7 years （n=23）, group B, 8-13 years （n=14） and group C, 14-17years （n=10）. Results There was wide range for sperm count and motility for all the subjects studied. Majority of men had high percentage of abnormal sperm morphology. Among the three groups of men, group A and B had significantly higher sperm count, motility and membrane integrity than that of group C. Group C also had the highest frequency of oligoasthenoteraozoospermia （50%） than group A （17%） and group B （35%）. Conclusion Semen quality of men with a history of unilateral cryptorchidism appears to be related to their age of performing orchidopexy. Surgery at early age of childhood may improve spermatogenesis which results in better semen quality in adulthood.

A retrospective study on sperm cryopreservation in 1034 patients diagnosed with cancer in southern China

Sperm cryopreservation is an effective fertility preservation method for cancer patients before anticancer treatments. However, thereare little data on fertility preservation in large cohorts of patients with cancer in southern China. This retrospective cross-sectionalstudy aimed to assess the fertility preservation status of 1034 newly diagnosed male patients with cancer in the Human SpermBank of Guangdong Province in southern China (Guangzhou, China). Of these, 302 patients had reproductive system tumors,mostly testicular cancers (99.0%), and 732 had other tumors, including lymphoma (33.1%), gastrointestinal cancer (16.3%),nasopharyngeal carcinoma (15.7%), leukemia (7.7%), sarcoma (3.6%), and others (23.6%). Patients with reproductive systemtumors had lower sperm concentration and prefreezing and post-thawing progressive motility than those with non-reproductive systemtumors (all P < 0.001). Differences in sperm concentration, progressive motility, and normal morphology rate were observed betweenpatients with and without anticancer surgery before sperm cryopreservation (all P < 0.05). As of April 30, 2022, 63 patients usedtheir cryopreserved sperm for assisted reproductive technology treatments and 39 pregnancies were achieved. This study providesvaluable data on the fertility preservation status in newly diagnosed cancer patients in southern China, demonstrating that patientswith reproductive system tumors had poor sperm quality for their pretreatment fertility preservation.

Comparison of Clinical Efficacy of Three Methods for Cervical Ripening followed by Surgical Evacuation in Early Missed Abortion

Objective To compare clinical efficacy of three cervical preparations for surgical evacuation in first trimester missed abortion. Methods Two hundred and ten women with early missed abortion were equally randomized into three groups. Three cervical preparations, Dilapan-dilator method （group A, n= 70）, mifepristone method （group B, n= 70）, and misoprostol method （group C, n= 70）, were used 24 h before curettage for terminating missed abortion, respectively. Clinical outcomes and complications for these three methods were compared. Results No differences were found in rates of successful treatment among the three groups （P〉O.05）. The incidence of fitting number 8 Hegar dilator was slightly high in group A, but it was not different compared with that in group B or group C （P〉0.05）. Bleeding in previous 24 h in group C was significantly greater than that in group A or group B （P〈0.01）. The cases of blood loss （〉50 ml） during evacuation in group B were more than those in group A （P〉0.05）. Women in group B had less abdominal pain than those in group A or group C （P〈0.01）. Side effects in group C were more than those in group A on nausea （P〈0.01）, vomiting （P〈0.05）, and diarrhea （P〈0.01）. Group B had the highest acceptability among the three groups （P〈0.05）. Conclusion Three methods have the similar clinical efficacies in cervical ripening for surgical treatment of early missed abortion. Based on individual characteristics, different approach would be chosen to avoid some adverse events and improve the clinical application of Dilapan-dilator.

Circulating Antisperm Antibody (ASA) in Women is not Associated with Missed Abortions at the First-trimester Pregnancy

Objective To determine the role of the circulating antisperm antibody （ASA） in the pathogenesis of missed abortion at the first-trimester pregnancy. Methods Sixty-two patients with a history of missed abortion at the first-trimester pregnancy were enrolled into this study. Indirect immunobead test （IBT） was used to measure the circulating ASA levels. Fifty healthy women with the first-trimester pregnancy set as the control. Results No case had the positive level of ASA according to the World Health Organization criteria （50% or more of the motile sperm with immunobead binding）. Only 1 case in patient group and 1 case in the control had 10%-20% of the motile spermatozoa with ASA-IgG bead binding. In both patient and control groups, ASA-IgA was found to be completely negative binding. Conclusion The circulating ASA is not associated with the pathogenesis of missed abortions at the first-trimester pregnancy.

Evaluation of Adiponectin, Resistin, IL-6, TNF-α in Obese and Non-obese Women with Polycystic Ovary Syndrome

Objective To evaluate possible clinical effects of adiponectin, resistin, 1L-6, and TNF-a in obese and non-obese patients with polycystic ovary syndrome （PCOS）. Methods Seventy selected PCOS patients were recruited for this study, and were divided into two groups based on their body mass index （BM1）： 35 obese （BM1 25 kg/m2, group A）, 35 non-obese （BMI〈25 kg/me, group B）. In addition, 35 healthy non-obese women （BMI〈25 kg/m2） were enrolled as the control （group C）. Serum levels of FSH, LH, T, glucose, insulin, adiponectin, resistin, 1L-6, TNF-a. were detected, and compared the differences of them among three groups. Results Blood glucose levels among three groups had no difference （P〉0.05）. Blood insulin level was significantly greater in group A than in group B or group C （P〈0.05）, and a significant difference existed between group B and group C （P〈0. 05）. The ratio of glucose/insulin was significantly higher in group A than in group B or group C （P〈0.05）, and the ratio in group B was significantly higher than that in group C （P〈0.05）. Adiponectin level was significantly lower and resistin level was significantly higher in group A than in group B or group C （P〈0.05）. IL-6 level in group C was significantly lower than that in group A or group B （P〈0.05）, and significant difference was found between group A and group B （P〈0.05）. TNF-a level was a slight high in group B, whereas there was no statistical difference among three groups （P〉0.05）. Conclusion Disturbances of some metabolic and inflammatory adipokines could involve the pathogenesis of PCOS in both obese and non-obese women. Low-grade chronic inflammation might have negative effects on the development of PCOS in non- obese women.

Expression of Survivin Gene among Human Normal Endometrium,Atypical Hyperplasia of Endometrium and Endometrial Carcinoma

Objective To investigate the expression of survivin gene among human normal endometrium,atypical hyperplasia of endometrium,and endometrial carcinoma.Methods Tissue samples of human normal endometrium during proliferative phase （NE,n=20）,atypical hyperplasia of endometrium （AHE,n=11）,and endometrial carcinoma （EC,n=9） were collected.Besides,Paraffin embedded sections of NE （n=20）,AHE （n=20）,and EC （n=20） were used.The expression of survivin gene was determined by immunohistochemistry and the real-time reverse transcription polymerase chain reaction.Results Survivin immunostaining appeared in the cytoplasm of endometrial epithelial cells.Both survivin staining and mRNA had higher levels in AHE or EC than those in NE （P〈0.01）.No difference was found on survivin staining and mRNA between AHE and EC （P〉0.05）.Conclusion High expression of survivin gene in human endometrium is associated with the risk of atypical hyperplasia progressing to endometrial carcinoma.The high level of survivin expression is useful as a predictive indicator for endometrial carcinoma.

Status of reproductive health knowledge for junior high school students in Guangzhou city

Objective To understand the status of reproductive health knowledge for junior high school students in Guangzhou city and provide the base data for reproductive health education. Methods A total of l 210 students including 657 boys and 553 girls aged 12 ~15 years were surveyed with anonymous self-administrated questionnaire. The students were from a middle school in suburb of Guangzhou city. The questionnaire contained basic concepts of reproduction, love, sex, contraceptives, abortion and sexually transmitted diseases （STDs）. Analysis consisted of descriptive statistics and Chi square tests for differences between boys and girls. Results The students mostly had experienced menarche （94%） or first spermatorrhea （83.6%）. About 58. 4% of students just knew little about process of puberty, fertilization, and contraception. Far more boys than girls considered that the effect of abortion on physical injury was harmless （30.4% and 9.6%, P〈O.O01）. More than 60% of respon- dents knew the route of transmission of STDs well but were poor understanding of adverse impact of STDs. The majority of the students did not consider sexual relation- ship acceptable at their age, a viewpoint that was more strongly held by girls （61.6%） than boys （45.1%） （P〈0.001）. Just 35.5% of boys would refuse when the lovers required to have a sex activity, far less than girls （76.1%） （P〈0.001）. The rate of sex intercourses among students was 5%, and a slightly higher proportion （46%） had never used contraception among those who had sexual activity. The demands of students on reproductive health were mainly on how to deal with the relationship with lovers （56. 0%）, knowledge on sexual health and menses （41.7%）, sex moral knowledge （48.3%）, contraception （45.0%）, and preventive measures of STDs/acquired immune deficiency syndrome （AIDS） （47. 0%）. Conclusion The reproductive health knowledge for the students was at very low level There were some differences on the knowledge mastered by boys and girls while the sexual opinion of boys was more open than girls. It was urgent to carry out education among junior high school students to promote their reproductive health.

Changes of testicular ultrastructure of rat after clenbuterol exposure

Objective To investigate effects of clenbuterol （CLB） on testicular ultrastructure of rat. Methods Twenty adult male Sprague-Dawley rats were randomly divided into four groups （5 rats per group）. CLB solved in normal saline solution was given at the dose of O mg/kg body weight （bw） （group A, as control）, 0.4 mg/kg bw （group B）, 2.0 mg/kg bw （group C）, and 18.5 mg/kg bw （group D）for 14 d by garage consecutively, respectively. Transmission electron microscopy was used to observe changes on testicular ultrastructure. Results In group B, some small vacuoles were found in Sertoli cells. In groups C and D, vacuoles were common in Sertoli cells and spermatogonia. The phenomenon of vacuolation in group D was more severe than that in group C. In group D, basal membrane showed some irregular and wrinkled changes, Leydig cells had more vacuoles and increased lipid droplets. Conclusion Testicular ultrastructure of rat had pathological changes after CLB exposure, and the alterations became more severe with the increasing doses.