Can inhibin-B predict the outcome of microsurgical epididymal sperm aspiration in patients with suspected primary obstructive azoospermia？

Aim： To evaluate whether inhibin-B can predict the outcome of a microsurgical epidymal sperm aspiration （MESA） procedure in patients with suspected primary obstructive azoospermia （OA） and if inhibin-B can replace testicular biopsy in the diagnostic work-up of these patients. Methods： Inhibin-B levels and testicular biopsy scores were related to the outcome of MESA in 43 patients with suspected primary OA. MESA was considered to be successful when epididymal sperm could be identified during the procedure. Results： Spermatozoa were present in the epididymal aspirate in 28 out of the 43 patients （65%）. lnhibin-B values were not significantly different in patients with successful or unsuccessful MESA. The modified Johnsen score, however, was significantly lower in patients with unsuccessful MESA （P = 0.003）. A rete testis obstruction or epididymal malfunctioning was found in 15% of patients with suspected primary OA, reflected by unsuccessful MESA despite normal inhibin-B levels and normal testicular histology. Conclusion： Inhibin-B cannot replace testicular biopsy as a diagnostic tool in the work-up of patients with suspected primary OA. Testicular biopsy is useful in identifying patients with spermatogenic arrest, who might have normal inhibin-B values.

Successful pregnancy and birth after intrauterine insemination using caput epididymal sperm by percutaneous aspiration

<abstract>Aim: To manage male infertility with obstructive azoospermia by means of percutaneous epididymal sperm aspiration (PESA) and intrauterine insemination (IUI). Methods: Ninety azoospermic patients with congenital bilateral absence of the vas deferens (BAVD, n=58) or bilateral caudal epididymal obstruction (BCEO, n=32) requesting for fine needle aspiration (FNA), PESA and IUI were recruited. The obstruction was diagnosed by vasography and determination of the fructose, carnitine and alpha-glucosidase levels in the seminal fluid. Results: The mean sperm motility, density, abnormal sperm and total sperm count of the caput epdidymis were 16 %±22 %, (12±31) ×106/mL, 55 %±36 % and (16±14)×106, respectively. In the 90 couples, a total of 74 PESA procedures and 66 cycles of IUI were performed. Three pregnancies resulted, including one twin pregnancy giving birth to two healthy boys, one single pregnancy with a healthy girl and another single pregnancy aborted at week 6 of conception. The pregnancy rate per IUI cycle was 4.5 %. Conclusion: The birth of normal, healthy infants by IUI using PESA indicates that the caput epididymal sperm possess fertilization capacity. The PESA-IUI programme is a practical and economical procedure for the management of patients with obstructive azoospermia.

Microsurgical epididymal sperm aspiration： indications, techniques and outcomes

Microsurgical epididymal sperm aspiration （MESA） refers to retrieval of sperm-containing fluid from optimal areas of the epididymis that are selected and sampled using high-power optical magnification provided by an operating microscope. Retrieved sperm are subsequently used for intracytoplasmic sperm injection （ICSI） to induce fertilization and pregnancy. MESA is considered by many experts to be the gold standard technique for sperm retrieval in men with obstructive azoospermia given its high yield of quality sperm, excellent reported fertilization and pregnancy rates, and low risk of complications. However, MESA must be performed in an operating room, requires microsurgical skills and is only useful for reproduction using ICSI. Herein we present an overview of the evaluation of candidate patients for MESA, the technical performance of the procedure and the outcomes that have been reported.

Expression of serum human epididymal secretory protein E4 at low grade and high grade serous carcinomas

Objective:To investigate the value of serum human epididymis protein 4(HE4) in differential diagnosis of patients with low-grade serous(LGSC) and high-grade serous carcinoma(HGSC) serous ovarian cancer.Methods:LGSC and HGSC serous ovarian cancer were diagnosed by the two-tier grade system,serum levels of HE4 and carbohydrate antigen 12S(CA125) were measured by ELBA and radioisotope method,respectively in 60 serous ovarian cancer patients. HE4 and TPS3 protein in cancer tissue were measured by immunohistochemical method. Results:The difference in density of HE4 and TP53 protein was significant between LGSC and HGSC tissue,while serum CA12S did not show significant difference between different serum samples.There was significant difference in serum HE4 levels between LGSC and HGSC and the result was different within FIGO(Ⅰ+Ⅱ) stage,suggesting HE4 was not a reliable biomarker for the discrimination between LGSC and HCSC.HE4 had potential as a biomarker for the discrimination between LGSC and HGSC but the role in early diagnosis was limited.Conclusions:HE4 may be a reliable marker for differential diagnosis of LGSC and HGSC.But its role in early diagnosis of LGSC and HGSC need to be confirmed from the perspective of two-tier grade system.

Age-dependent expression of the cystatin-related epididymal spermatogenic （Cres） gene in mouse testis and epididymis

Aim： To investigate the spatial and temporal expression of the cystatin-related epididymal spermatogenic （Cres） gene in mouse testis and epididymis during postnatal development. Methods： The QuantiGene assay and indirect immunofluorescence technique were used to examine the Cres mRNA and Cres protein level in mouse testis and epididymis on postnatal days 14, 20, 22, 28, 35, 49, 70 and 420. Results： （1） In both the testis and epididymis, Cres mRNA was fast detected on day 20, then it increased gradually from day 20 to day 70, and the high expression level maintained till day 420. （2） In the testis, the Cres protein was exclusively localized to the elongating spermatids and was first detected on day 22. The number of Cres-positive spermatids increased progressively till day 49. From day 49 to day 420, the number of Cres-positive cells was almost stable. （3） The Cres protein was first detected on day 20 in the proximal caput epididymal epithelium. By day 35, the expression level of the Cres protein increased dramatically and the high level was maintained till day 420. Moreover, the luminal fluid of the midcaput epididymis was also stained Cres-positive from day 35 on. No Cres-positive staining was observed in distal caput, corpus and cauda epididymis throughout. Conclusion： The Cres gene displays a specific age-dependent expression pattern in mouse testis and epididymis on both the mRNA and protein level.

Inductively coupled plasma emission spectroscopic and flame photometric analysis of goat epididymal fluid

Aim: The elemental composition of the epididymal luminal fluid (ELF) in adult goat ( Capra indica) was investigat-ed. Methods: ELF was collected by micropuncture from twelve sites along the epididymal duct. The elementalcontents was analyzed with inductively coupled plasma (ICP) emission spectroscopy, a microanalytical technique thatcan simultaneously measure many elements in minute volumes of sample. The Na and K concentrations were determinedby flame photometry. Results: ICP spectroscopy showed the presence of copper, calcium, nickel, iron, magne-sium, chromium, titanium and zinc in ELF, with fluctuating levels at different sites along the length of the epididymis.Cadmium, cobalt, lead and manganese were not fotmd. The Na^+ / K^+ ratio was seen to be higher at the initial seg-ments of the epididymis and lower at the distal. Conclusion: It is proposed that the observed characteristic distribu-tion of elements in ELF may have far reaching implications in sperm maturation and storage known to occur in the epi-didymis. (Asian J Androl 2000 Dec; 2: 288-292)

Vlouse sperm acquire a new structure on the apical hoo Juring epididymal maturation

Spermatozoa emerging from the testis undergo a maturation process in the epididymis during which they change morphologically, biochemically and physiologically to gain motility and the ability to fertilize ova. We examined mouse epididymal sperm with immunostaining and transmission electron microscopy （EM） and identified a previously unknown structure on the apical hook. The structure has a coiled configuration around 11 nm in thickness and is present at the tip of each corner of the triangular-rod shaped perforatorium. Surveying sperm isolated from various regions of the epididymis indicated that mouse sperm acquire the hook rim （HR） structure during its passage through the proximal two-thirds of the caput epididymidis. The structure withstands vigorous sonication and harsh chemical treatments and remains intact after the acrosome reaction. Its location and sturdiness suggest a function in protecting the apical hook from mechanical wear during fertilization. Our EM images of epididymal sperm also revealed additional novel structures as well as lateral asymmetry of the sperm head, indicating that mouse sperm head has a structure more complex than previously recognized.

Influence of reproductive tract obstruction on expression ）f epididymal proteins and their restoration after patency

Vasectomy is a simple and reliable method of male contraception. A growing number of men after vasectomy request vasectomy reversal due to various reasons. The pregnancy rate is lower than the patency rate after vasovasostomy and the pregnancy rate is time dependent. In this study, we evaluated the influence of reproductive tract obstruction on expression of epididymal proteins and their restoration after patency. Adult male Wistar rats were studied 30, 60 and 120 days after vasectomy, 30 days after vasovasostomy or after sham operations. Two-dimensional gel electrophoresis, mass-spectrometric technique, multidatabase search, Western blotting and real-time PCR were used to analyze the expression regulation of epididymal proteins. Total integrated intensity and total spot area of autoradiograms showed a consistent downward trend with time after obstruction, and this trend remained after patency. The intensity of the autoradiographic spots in three patency groups showed three trends： a downward trend, similar intensity and an upward trend compared with the correspondent obstruction group, respectively. Further verified experiments on human epididymis 2 （HE2）, fertilization antigen-1 （FA-1）, clusterin and PH20 demonstrated that compared with the correspondent obstruction group, the translation levels of HE2 and the mRNA transcription levels of HE2 showed an upward trend in patency groups, especially in the groups of obstruction for 60 days where the expression levels of HE2 were significantly upregulated after patency （P〈O.05）. Reproductive tract obstruction provokes a disregulation of gene expression in the epididymis and this disregulation remained after patency. Successful reversal may recover some proteins and the recovery is time dependent, Obstruction differentially alters mRNA transcription of different proteins and the content of proteins seemed to be easier to be influenced than the gene transcription.

Molecular mechanism of modulating the liquefaction epididymal protease inhibitor of human semen

Aim： To study the molecular mechanism of epididymal protease inhibitor （Eppin） modulating the process of prostate specific antigen （PSA） digesting semenogelin （Sg）. Methods： Human Sg cDNA （nucleotides 82-849） and Eppin cDNA （nucleotides 70-423） were generated by polymerase chain reaction （PCR） and cloned into pET-100D/TOPO. Recombinant Eppin and Sg （rEppin and rSg） were produced by BL21 （DE3）. The association of Eppin with Sg was studied by far-western immunoblot and radioautography. In vitro the digestion of rSg by PSA in the presence or absence of rEppin was studied. The effect of anti-Q20E （N-terminal） and C-terminal of Eppin on Eppin-Sg binding was monitored. Results： Eppin binds Sg on the surface of human spermatozoa with the C-terminal of Eppin （amino acids 75-133）. rSg was digested with PSA and many low molecular weight fragments were produced. When rEppin is bound to rSg, then digested by PSA, incomplete digestion and a 15-kDa fragment results. Antibody binding to the N-terminal of rEppin did not affect rSg digestion. Addition of antibodies to the C-terminal of rEppin inhibited the modulating effect of rEppin. Conclusion： Eppin protects a 15-kDa fragment of rSg from hydrolysis by PSA.

Molecular mechanism of epididymal protease inhibitor modulating the liquafication of human semen

To study the molecular mechanism of epididymal protease inhibitor （Eppin） modulating the liquafication of semen. Methods： Human semenogelin cDNA （nucleotides 82-849） and Eppin cDNA （nucleotides 70-423） were generated by PCR and cloned into pET-100D/TOPO.Recombinant Eppin and Sg were produced by BL21 （DE3）. The association of Eppin with Sg was studied by far-western and radioautography.In vitro the digestion of Sg by PSA in the presence or absence of recombinant Eppin was studied. The effect of anti-Q20E （N-terminal） and C-terminal of Eppin on Eppin-Sg binding was monitored. Results： Eppin binds Sg on the surface of human spermatozoa with C-terminal Eppin （aa75-133）.Recombinant Sg was digested with PSA ,many low molecular weight fragments were produced, when Eppin is bound to Sg ,then digested by PSA ,producing incomplete digestion and a 14.5-14.8 kDa fragmen. Antibody binding to the N-terminal of Eppin did not affect Sg digestion. Addition of antibodies to the C-terminal of Eppin inhibited the modulating effects of Eppin. Conclusion： Eppin modulates the digestion activity of PSA through binding Sg.The active site locates at C-terminal.

Protective effect of Scrophularia striata combined with trehalose and cysteine added to diluents on cryopreservd goat epididymal sperm

Objective:To evaluate antioxidant effects of Scrophularia(S.)striata ethanol extract,trehalose and cysteine added to diluents on cryopreserved goat epididymal sperms.Methods:Motility and standard motion parameters of sperm were assessed by using computer assisted sperm motility analysis system.Sperm viability was evaluated by eosin-nigrosin staining method.Hypo-osmotic swelling test was used to evaluate membrane health.Thiobarbituric acid testing was used to measure malondialdehyde(MDA)concentrations.To assess DNA fragmentation,sperm chromatin dispersion test was used.In Experiment 1,treatments consisting of basal Tris diluent supplemented with 25,50 or 100µg/mL of S.striata ethanol extract gave the best concentration to the freezing diluents.Experiment 2 was carried out to compare the best concentration of S.striata ethanol extract(50µg/mL)resulting from the first experiment with 150 mM trehalose and/or 5 mM cysteine alone or in combination.Results:S.striata ethanol extract(50µg/mL)significantly increased sperm viability,motility and progressive motility and at the same time decreased MDA concentration and DNA fragmentation compared to other treatments(P<0.05).In addition,all treatment groups resulted in viability,membrane health,total motility,progressive motility,curvilinear velocity,straightline velocity higher and MDA lower compared to the control group(P<0.05).Acrosome integrity was significantly higher in 50µg/mL of S.striata ethanol extract combined with cysteine,trehalose,or cysteine+trehalose groups than those in the control,trehalose,cysteine,and 50µg/mL of S.striata ethanol extract groups(P<0.05).Regarding DNA,extenders supplemented with 50µg/mL of S.striata ethanol extract,50µg/mL of S.striata ethanol extract+trehalose,and 50µg/mL of S.striata ethanol extract+trehalose+cysteine were superior to other treatments.Conclusions:Adding 50µg/mL of S.striata ethanol extract alone or in combination with trehalose and cysteine can improve the quality of cryopreserved epididymal sperms of goats.

In-vitro effect of Peganum harmala total alkaloids on spermatozoa quality and oxidative stress of epididymal ram semen

Objective:To determine the in-vitro effect of the total alkaloid extract of Peganum(P.)harmala seeds on ram epididymal sperm.Methods:Semen was divided into six groups according to the following concentrations of the P.harmala total alkaloids:1,5,10,50,and 100μg/mL,and the control group.The samples were incubated at ambient temperature(21℃-24℃)for 24 h,and analyzed in terms of motility,membrane integrity,and oxidative status.Results:The sperm kinematic parameters,i.e.straight-line velocity,curvilinear velocity,average path velocity,were significantly higher when treated with P.harmala at concentrations ranging from 1 to 10μg/mL compared to the control group(P<0.05).In addtion,the highest amplitude of the lateral head displacement value was found in the groups treated with concentrations 1 and 5μg/mL of P.harmala compared to the control group(P<0.05).Total and progressive motilities showed that the extracts at 1,5,and 10μg/mL exhibited a high percentage after 24 h of incubation.The effect of P.harmala extracts on the membrane integrity of ram epididymal sperm was concentration-dependent and significantly different compared to the control group(P<0.05).Non-significantly lower lipid peroxidation levels were observed after 24 h of incubation of ram epididymal sperm treated with concentrations 1,5,and 10μg/mL of P.harmala extracts compared to the control group(P>0.05).Conclusions:Low concentrations(1-10μg/mL)of P.harmala extracts stimulate sperm motility,preserve membrane integrity and protect ram spermatozoa from lipid peroxidation.

Epididymal metastasis from gastric cancer: a case report

Epididymal metastasis from gastric cancer is extremely rare. We reported one rare case. The 34-year-old male patient was found swelling lumps on his right testicle 6 months after adjuvant chemotherapy. The patient was given another 2 cycles of chemotherapy which followed by DCX regimen after the orchiectomy. The patient is still survival 21 months after radical gastrectomy. Therefore the early correct diagnosis and rational treatment are very important for improving the survival rate of patients.

Medium Term Survival of Bovine Caudal Epididymal Sperm under Conditions Based on Epididymal Fluid Characteristics

Fresh semen has a very limited life span after it leaves the gonad. The relative research shows that the epididymal plasma provides a special environment to spermatozoa for survival. In this study, the osmolarity and protein concentration of caput and caudal bull epididymis were analyzed. Bovine caudal epididymal sperm were incubated in different protein and sorbitol combinations of CEP-2 diluents at 4 ℃ for 120 h and sperm viability was assessed. Under this condition, sperm viability was greater than 60% after 5 d. The osmolarity of caput and caudal epididymis were （287.0 ± 13.7） and （310.8 ± 17.0 ） mOsm, respectively. The protein concentration of caput and caudal epididymis were （ 37.43 ± 12.55 ） and （50.58±11.08） mg/mg, respectively. This may have an application in the animal production industry.

Studies on Pathogenesis and Prophylaxis and Treatment of Postvasectomy Epididymal Stasis Syndrome

Eighty-four hamsters were vasectomized on both sides,Another group of 76 hamsters was used as the control group.The animal experiment demonstrated that the epididymal function was damaged on the basis of the micropuncture and microanalysis of the epididymal plasma.Histologic examination revealed the dilatation of the lumen,infiltration of chronic inflammatory cells in the interstitium and sperm granuloma.Thirty-nine patients with postvasectomy epididymal stasis were treated with microwave radiation and 10 cases with epididymectomy.The B and C ultrasonography and MRI were used for diagnosis.Histologic examination suggested that chronic obstruction of vasectomy mode(both-side electric coagulation or keeping proximal open)may lead to formation of the new dynamic equilibrium, therefore benefiting the spermatogenesis of testis.Others with acute obstruction of the vas(bilateral ligation) may cause epididymal stasis.

Azteca Breed Horse Epididymal Sperm Evaluation: A Comparison of Head, Corpus and Cauda Sperm Quality

The main objective of this study was to determine differences in sperm recovered from the three segments of the epididymis, i.e., head, corpus and cauda, using routine measurements of sperm quality and DNA fragmentation assessment. There was a larger sperm concentration (P < 0.05) in the head (3.34 × 106 sperm/mL) compared with the corpus and cauda. The percentage of normal morphology was higher in the cauda but had lower motility (P < 0.05). The DNA fragmentation index was higher for sperm obtained from the head and body of the epididymis (P < 0.01) compared with the cauda. The DNA fragmentation index values were 12% higher (P < 0.01) using Wright stain compared with DAPI stain in sperm from the head and corpus of the epididymis, but similar in the cauda. Spermatobioscopy parameters match the maturation changes that occur in the epididymal tract with higher quality sperm being from the cauda, but with a lower concentration compared to sperm found in the head.

Morphological Studies on the Epididymal Duct of the One-Humped Camel (Camelus Dromedaries)

The present work was carried out on 20 epididymes of sexually mature one humped camel to elucidate the gross anatomical, scanning electron microscopical and light microscopical features of the epididymal duct. Anatomically, the epididymal duct of camel consists of three parts i.e. head, body and tail. Histomorphologically, the epididymal duct is subdivided into initial, middle and terminal segments, of which the middle segment is further subdivided into proximal, intermediate and distal parts. The epithelium lining the epididymal duct is ciliated pseudostratified columnar comprising mainly five cell types, namely principal, basal, apical, dark and halo cells. It displays numerous intraepithelial glands in the middle segment. In general, there is a gradual increase in the total diameter and decrease in the epithelial height of the epididymal duct from the initial to the terminal segments;that may, mechanically, facilities passage of the sperms toward the terminal segment. The lamina propria of the epididymal duct contains a layer of interlacing elastic fibers which may allow adequate elasticity of the duct that could help in its expansion. Moreover it is surrounded by numerous layers of circularly and obliquely arranged smooth muscle fibers which always increase in thickness toward the terminal segment.

Leptin Receptor in Ram Epididymal Spermatozoa

This experiment was designed to investigate leptin receptors (Ob-R and Ob-Rb) mRNA in ram epididymal spermatozoa by RT-PCR. Ten testes were obtained from abattoir and epididymal spermatozoa recovery was performed. To purify spermatozoa, motile sperm were isolated by the swim-up procedure. Total RNA was isolated from epididymal spermatozoa and placental cotyledon, as a positive control, and then they were purified. Specific bands (98 and 308 bp) for Ob-R and Ob-Rb were detected after RT-PCR in both epididymal spermatozoa and placental cotyledon. We may conclude that Ob-R and Ob-Rb mRNA are present in ram epididymal spermatozoa and leptin perhaps exerts physiological effects, as already demonstrated in human.

Assessment of the Quality of Frozen-Thawed Semen or Epididymal Sperm in Three Native Vietnamese Pig Breeds

Our aim was to evaluate the quality of ejaculated and epididymal frozen-thawed pig sperm of endangered Vietnam native pig breeds. Ejaculated sperm was collected from live boars and epididymal sperm was collected from slaughtered boars of the MuongTe, Kieng Sat and Co BinhThuan breeds and frozen in 0.25 ml straws using a protocol established earlier for modern pig breeds. We evaluated the sperm quality after thawing in terms of motility and rates of viable and abnormal spermatozoa. Our results revealed that the sperm motility and rates of viable and abnormal frozen-thawed sperm were >30%, >44%, and <14%, respectively. The origin of sperm had an effect on the production of pig embryos in vitro. In the Co BinhThuan breed, ejaculated sperm generated higher cleavage, blastocyst and hatching rates than did the epididymal sperm (60.11% vs 56.02%, 17.23% vs 14.31%, 3.78% vs 2.34%, respectively, P < 0.05). Although no difference in cleavage rate, blastocyst formation rate and the average number of cells/blastocysts, the hatching blastocyst rate was different between the breeds (P > 0.05). In the Co BinhThuan breed, the rate of pregnancy of ejaculated groups was similar to that of the epididymal group. In conclusion, the ejaculated and epididymal sperm of native Vietnamese pigs were successfully frozen. We succeeded in creating embryos in vitro and pregnant pigs after artificial insemination from frozen-thawed semen in three native Vietnamese pig breeds for the first time. The use of the ejaculated sperm improved the production of native pig embryos in vitro efficiency.

Follow-up of children born after intracytoplasmic sperm injection with epididymal and testicular spermatozoa

Background To evaluate the safety of intracytoplasmic sperm injection （ICSI） with epididymal or testicular sperm, this study compared children born after ICSI treatment with epididymal or testicular sperm with children conceived after ICSI with ejaculated sperm. Methods This retrospective study included 317 children born after ICSI with percutaneous epididymal sperm aspiration （PESA）, 103 children born after ICSI with testicular sperm aspiration （TESA）, and a control group of 1008 children born after ICSI with ejaculated sperm. All of the patients received their assisted reproductive treatment in the Reproductive Medicine Center of the First Affiliated Hospital of Zhengzhou University from January 2004 to December 2011. Data, such as the rate of stillbirths, perinatal mortality, gestational age, birth weight, and the rate of congenital malformations of the three groups, were compared. Results PESA and TESA children were not different from ICSI children in the rate of stillbirths, perinatal mortality, infant mortality rate, gestational age, the rate of prematurity, and the rate of malformations （P〉0.05）. A slight increase in birth defects was reported in the TESA group compared with those in the control group, but there was no significant difference between the groups （P〉0.05）. Conclusion ICSI with epididymal or testicutar sperm does not lead to more stillbirths or congenital malformations compared with ICSI using ejaculated sperm.