Advanced Paternal Age and Future Generations.

Paternal age at conception has been increasing. In this review, we first present the results from the major mammalian animal models used to establish that increasing paternal age does affect progeny outcome. These models provide several major advantages including the possibility to assess multi- transgenerational effects of paternal age on progeny in a relatively short time window. We then present the clinical observations relating advanced paternal age to fertility and effects on offspring with respect to perinatal health, cancer risk, genetic diseases, and neurodevelopmental effects. An overview of the potential mechanism operating in altering germ cells in advanced age is presented. This is followed by an analysis of the current state of management of reproductive risks associated with advanced paternal age. The numerous challenges associated with developing effective, practical strategies to mitigate the impact of advanced paternal age are outlined along with an approach on how to move forward with this important clinical quandary.

Beta-defensin126 is correlated with sperm motility in fertile and infertile men†.

A crucial function of the epididymis is providing a surface glycocalyx that is important for sperm maturation and capacitation. Defensins are antimicrobial peptides expressed in the epididymis. In the macaque epididymis, defensin beta 126 (DEFB126) is important for sperm motility, however, it is not known whether this is the case in humans. The objectives were to determine: (1) if DEFB126 on human ejaculated sperm was correlated with sperm motility in fertile and infertile men, (2) that recombinant DEFB126 could induce immature sperm motility in vitro. Immunofluorescence staining indicated that the proportion of DEFB126-positive sperm was significantly higher in motile sperm. Furthermore, the proportion of DEFB126-labeled sperm was positively correlated with sperm motility and normal morphology. Additional studies indicated that the proportion of DEFB126-positive spermatozoa in fertile volunteers was significantly higher than in volunteers with varicocele, and in infertile volunteers with semen deficiencies. To determine the role of DEFB126 on sperm motility, the DEFB126 gene was cloned and used to generate recombinant DEFB126 in H9C2 cells (rat embryonic heart myoblast cells). Deletion mutations were created into two regions of the protein, which have been linked to male infertility. Immotile testicular spermatozoa were incubated with cells expressing the different forms of DEFB126. Full-length DEFB126 significantly increased motility of co-cultured spermatozoa. However, no increase in sperm motility was observed with the mutated forms of DEFB126. In conclusion, these results support the notion that DEFB126 is important in human sperm maturation and the potential use of DEFB126 for in vitro sperm maturation.

Sperm DNA integrity in adult survivors of paediatric leukemia and lymphoma: A pilot study on the impact of age and type of treatment.

Childhood cancer survivors (CCS) are more likely than siblings to report low sperm count and to use assisted reproductive technologies. Yet, it is still unclear if the sperm produced many years after remission of cancer display DNA and chromatin damage linked to male infertility and poor embryo development. As well, the importance of the age at diagnosis in relation to puberty is poorly understood. In this pilot study, we compared reproductive parameters and sperm damage from adult survivors of childhood leukemia and lymphoma, sub-divided into those diagnosed before or after puberty, to men with no history of cancer. Our data indicate that CCS, independently of the age of diagnosis, have a high risk of low sperm count and when sperm are present, chances of DNA and chromatin abnormalities appear similar to those seen in the general population. Exposure to alkylating agents is correlated with low sperm count whereas exposure to anthracyclines, and doxorubicin in particular, could have long-term consequences on sperm integrity. This study highlights the need for further research on fertility among male CCS and the importance of informing families about the potential long-term impact of chemotherapy on male fertility regardless of age at diagnosis.

Sperm DNA Damage in Cancer Patients.

Fertility is a growing healthcare issue for a rising number of cancer survivors. In men, cancer itself and its treatment can negatively affect spermatogenesis by targeting the dividing spermatogonia and their cellular environment, ultimately leading to a reduction of testicular germ cells and sperm count. Experimental data and prospective longitudinal studies have shown that sperm production can recover after cancer treatment. But despite this, yet unpredictable, recovery in sperm production, cancer survivors are more at risk to produce sperm with aneuploidy, DNA damage, abnormal chromatin structure, and epigenetic defects even 2 years post-treatment. Sperm DNA alteration is of clinical concern, as these patients may father children or seek assisted reproduction technologies (ART) using gametes with damaged genome that could result in adverse progeny outcomes. Interestingly, large cohort studies revealed lower birth rate but no significant impact on the health of the children born from male cancer survivors (naturally or using ART). Nevertheless, a better understanding of how cocktail of chemotherapy and new anticancer agents affect spermatogenesis and sperm quality is needed to reduce side effects. Moreover, developing new fertility preservation strategies is essential as sperm cryopreservation before treatment is currently the only option but does not apply for prepubertal/young postpubertal patients.

Fertilization and embryo development with spermatozoa obtained from testicular sperm extraction into oocytes generated from human chorionic gonadotropin-primed in vitro maturation cycles.

OBJECTIVE: To evaluate the fertilization rate and embryo development resulting from intracytoplasmic sperm injection (ICSI) of spermatozoa retrieved by testicular sperm extraction (TESE) in hCG-primed in vitro maturation (IVM) cycles. DESIGN: Case-control study. SETTING: University teaching hospital. PATIENT(S): Twenty-four IVM cycles were performed in 21 patients (mean age, 32.3 ± 2.4 years) with polycystic ovaries (PCO) whose partners were nonobstructive azoospermic. Twelve cycles where IVM oocytes were also retrieved were compared with a control group consisting of age-matched IVM cycles with ICSI using ejaculated spermatozoa (n = 12). INTERVENTION(S): In vitro maturation treatment with TESE sperm. MAIN OUTCOME MEASURE(S): Fertilization and embryo development between sibling oocytes matured in vivo and in vitro. RESULT(S): Eight singleton pregnancies and one twin pregnancy were obtained after ET (9/24, 37.5%). In the 12 IVM cycles where in vivo-matured oocytes were also obtained, the fertilization rate after TESE-ICSI was significantly higher in in vivo-matured oocytes than in sibling in vitro-matured oocytes (84.2% vs. 53.2%). The proportion of good quality embryos was also higher (63.5% vs. 40.2%). In the control group of cycles with ejaculated spermatozoa, there was no difference in fertilization rates between sibling oocytes matured in vivo and in vitro (84.6% vs. 79.6%). CONCLUSION(S): Our results suggest that IVM of immature oocytes combined with TESE-ICSI is an option for couples with PCO and azoospermia. However, there are lower fertilization and good quality embryo rates achieved when TESE-ICSI was done with in vitro-matured oocytes. Additional studies are necessary to determine the role of this treatment combination.

Epidermal growth factor regulates connexin 43 in the human epididymis: role of gap junctions in azoospermia.

BACKGROUND: Gap junctions (GJs) allow for direct communication between adjacent cells. They are composed of connexons consisting of transmembrane proteins, connexins (Cxs). The objectives of this study were to determine if GJ proteins GJA1 (Cx43), GJB1 (Cx32) and GJB2 (Cx26) are present in the epididymis of men with a normal epididymis, to assess whether or not Cx expression and localization are altered in azoospermic patients, and to determine if epidermal growth factor (EGF) regulates GJA1 expression. METHODS: Epididymides were obtained from men with localized testis cancer with active spermatogenesis and histologically normal epididymal tubule (group 1), men with non-obstructive azoospermia secondary to Sertoli-cell only syndrome (group 2) and from azoospermic men with normal spermatogenesis and epididymal obstruction (group 3). Epididymides were subdivided into three segments: caput, corpus and cauda. Quantitative real-time RT-PCR was performed to assess GJA1, GJB1, GJB2 and EGF receptor (EGFR) mRNA levels in epididymides from patients from each group (all n=3, except n=1 for caput blockage). A human caput epididymal cell line was then used to determine the role of EGFR signaling on the regulation of human epididymal GJA1. RESULTS: Real-time RT-PCR analysis revealed that GJA1, GJB1, GJB2 and EGFR were expressed along the human epididymis. In the cauda epididymidis of group 2 and 3 men, we observed a significant decrease in GJA1 (P=0.0456 and P=0.0465, respectively) and GJB1 (P=0.0450 and P=0.0497, respectively) mRNA levels when compared with group 1 men. We also observed a decrease in EGFR mRNA levels (P=0.0358) in the cauda epididymidis of group 3 men when compared with group 1. Immunocytochemistry revealed that in the epididymis, GJA1 and EGFR were localized between basal and principal cells and between adjacent principal cells. In group 2 and 3 patients, however, we noted a dramatic increase in cytosolic immunostaining for both GJA1 and EGFR in both principal and basal cells. Using a human caput epididymal cell line derived from fertile men, we demonstrated that changes in GJA1 phosphorylation could be regulated by EGF (P=0.015) and the extracellular regulated kinase 1/2 signaling pathway (P=0.03). Furthermore, while the phosphoinositide-3-kinase (PI3K)/AKT signaling pathway did not alter GJA1 phosphorylation, treatment with PI3K/AKT inhibitor LY294002 significantly (P=0.024) inhibited the EGF-stimulated increase in GJA1 total protein levels at 24 h. Immunolocalization indicated that loss of PI3K/AKT signaling was associated with increased cytosolic localization of Cx43 in this cell line. CONCLUSIONS: Together, these data suggest that in azoospermic men decreased expression of EGFR may be responsible for decreasing GJA1 levels and increasing its cytosolic localization via the PI3K/AKT signaling pathway.

Live birth after ICSI of micro-TESE-retrieved spermatozoa into in vitro-matured oocytes.

This case report describes a live birth resulting from intracytoplasmic sperm injection (ICSI) of spermatozoa retrieved by microdissection testicular sperm extraction (micro-TESE) into oocytes produced from human chorionic gonadotropin-primed in vitro maturation (IVM) cycles. In the IVM treatment, a total of 30 oocytes (1 mature and 29 immature oocytes) were retrieved. Following IVM, 9 oocytes had matured. A total of 4 oocytes were fertilized after ICSI with the husband's micro-TESE spermatozoa and 4 embryos were transferred into the uterus on day 3. A healthy boy weighing 2500 g was born at 35.5 weeks of gestation.

Alterations in the human blood-epididymis barrier in obstructive azoospermia and the development of novel epididymal cell lines from infertile men.

Post-testicular sperm maturation requires a specific luminal environment in the epididymis that is created, in part, by the blood-epididymis barrier. There is limited information on gene expression in the epididymis of infertile obstructive azoospermia (OA) patients due to the difficulty in obtaining tissues. The objectives of this study were to determine if epididymal tight junction proteins are altered in OA and to develop cell lines that could serve to elucidate alterations in the epididymis of infertile men. Epididymal claudin (CLDN) 1, CLDN4, and CLDN10 mRNA levels were altered in OA downstream from the obstruction site. Epithelial cell lines derived from the caput epididymidis of one OA patient were developed (infertile human caput epididymal cell line [IHCE]). IHCEs were composed of homogenous populations of diploid cells that ultrastructurally resembled in vivo principal cells. The cells expressed cytokeratin, SPAG11B, CLDN2, CLDN3, desmoplakin, and vimentin. However, the cells did not express several other epididymal markers (CRISP1, SPINLW1, NPC2, CD52, or DCXR) or junctional proteins (CDH1, CDH2, CLDN1, CLDN4, CLDN7, or CLDN8). Further studies using IHCE1 and transepithelial resistance indicated that the cells were unable to form tight junctions. Microarray analyses comparing gene expression in IHCE1 and a recently developed fertile human caput epididymal cell line revealed differential expression of genes encoding junctional proteins, cell junction regulators, and epididymal proteins. Together, these data indicate that epididymal cellular junctions appear to be altered in OA.

Assessing the role of claudins in maintaining the integrity of epididymal tight junctions using novel human epididymal cell lines.

The epididymis is responsible for posttesticular sperm maturation. Sperm maturation is dependent on the luminal microenvironments along the epididymis. Though the role of the epididymis is well established, the molecular and cellular mechanisms responsible for sperm maturation remain to be elucidated, particularly in the human, as limited biological tools exist. We have established the first stable epithelial cell lines transformed with SV40 large T antigen (LTAg) from two regions of the human adult epididymis. The cell lines are composed of homogenous populations of diploid principal cells that possess ultrastructural characteristics similar to those of human principal cells in vivo. These cells express transcripts for adherens (cadherins CDH1 and CDH2) and tight (claudins CLDN1, CLDN2, CLDN3, CLDN4, CLDN7, and CLDN8) junctions as well as desmosomes (desmoplakin, DSP). Transepithelial resistance (TER) measurements in fertile human caput epididymal cell line 1 (FHCE1) as well as the immunolocalization of tight junctional protein 1 (TJP1), occludin, and CLDN1 indicate that these cells form functional tight junctions. Furthermore, knockdown of CLDN1, CLDN3, CLDN4, or CLDN7 using specific siRNAs resulted in significant decreases in TER, suggesting that these CLDNs are essential for the barrier function of the blood-epididymis barrier. Disruption of CLDN1, CLDN3, CLDN4, and CLDN7 could, therefore, lead to epididymal dysfunction, resulting in male infertility.

Alterations in gene expression in the caput epididymides of nonobstructive azoospermic men.

Spermatozoal maturation in the epididymis is dependent on proteins secreted by the epithelium and those that create the proper ionic composition and pH of the lumen as well as the blood-epididymal barrier. For the human epididymis, little information exists about the regulation of these proteins in male infertility. Our objectives were to assess gene expression profiles in the caput epididymidis from men with normal spermatogenesis and men with nonobstructive azoospermia. With microarrays, we identified 414 genes in the caput epididymidis that were differentially regulated in infertile men by at least 2-fold compared with the fertile men. They were mostly involved in transcription, intracellular signaling, immunity, and fertility. Although the expression of genes encoding tight junctional proteins was not affected, the localization of CLDN10 and TJP1, but not CLDNs 1, 3, and 8, was altered in infertile patients, suggesting that there are changes in the paracellular functions of the blood-epididymal barrier. Differentially regulated genes included several encoding proteins involved in spermatozoal maturation, water and ion channels, and beta-defensins: CRISP1, SPINLW1, FAM12B, and DEFB129 were upregulated, whereas CFTR, AQP5, KCNK4, KCNK17, SLC6A20, SLC13A3, DEFB126, and DEFB106A were downregulated. Furthermore, the immunolocalization of AQP5, but not of CFTR or CRISP1, varied in infertile and fertile patients. The observation that the expression of genes involved in water and ion transport were repressed in infertile patients suggests that these genes are regulated by the presence of testicular products or spermatozoa in the epididymal lumen or are part of a broader syndrome associated with nonobstructive azoospermia.

Orchestration of occludins, claudins, catenins and cadherins as players involved in maintenance of the blood-epididymal barrier in animals and humans.

Although spermatozoa are formed during spermatogenesis in the testis, testicular spermatozoa are immature and cannot swim or fertilize. These critical spermatozoal functions are acquired in the epididymis where a specific luminal environment is created by the blood-epididymal barrier; proteins secreted by epididymal principal cells bind to maturing spermatozoa and regulate the maturational process of the spermatozoa. In the epididymis, epithelial cell-cell interactions are mediated by adhering junctions, necessary for cell adhesion, and by tight junctions, which form the blood-epididymal barrier. The regulation of these cellular junctions is thought to represent a key determinant in the process of sperm maturation within the epididymis. Tight junctions between adjacent principal cells permit the formation of a specific microenvironment in the lumen of the epididymis that is essential for sperm maturation. Although we have made significant progress in understanding epididymal function and the blood-epididymal barrier, using animal models, there is limited information on the human epididymis. If we are to understand the normal and pathological conditions attributable to human epididymal function, we must clearly establish the physiological, cellular and molecular regulation of the human epididymis, develop tools to characterize these functions and develop clinical strategies that will use epididymal functions to improve treatment of infertility.

Innovative single-armed suture technique for microsurgical vasoepididymostomy.

OBJECTIVES: Vasoepididymostomy outcomes are heavily dependent on the surgeon's microsurgical experience and skill. To avoid back-walling the tubular lumen, the needles are generally placed inside-out through the vasal lumen using double-armed microsutures. These double-armed sutures for infertility microsurgery are very expensive and may be difficult to obtain. We describe a randomized trial that used a novel single-armed suture placement pattern for vasoepididymostomy. METHODS: Male adult Wistar rats underwent vasectomy. Two weeks later, vasoepididymostomies were performed using either a single-armed longitudinal intussusception vasoepididymostomy (n = 6) or a standard double-armed longitudinal intussusception vasoepididymostomy (n = 6) technique. After 9 weeks, patency was assessed functionally by evaluating for motile sperm distal to the anastomosis. If no motile sperm were visible, the mechanical patency of the anastomoses was tested by the ability of methylene blue to pass through the surgical anastomosis. RESULTS: The patency rate for the double-armed vasoepididymostomy group was 100% (6 of 6) compared with 83.3% (5 of 6) for the single-armed vasoepididymostomy group. This difference was not significant (P = 0.50). Sperm granulomas were found in three (50%) of six anastomoses in the double-armed group and five (83%) of six anastomoses in the single-armed vasoepididymostomy group (P = 0.27). The mean operative times for the double and single-armed longitudinal intussusception vasoepididymostomy techniques were similar (35 minutes versus 43 minutes; P = 0.39). CONCLUSIONS: The results of our study have shown that the single-armed suture technique to perform vasoepididymostomy is almost as effective as the double-armed technique. Although we still prefer to use double-armed sutures, we believe that this is a practical and effective alternative when specialized double-armed microsurgical sutures are not available.

Gene expression profiling and its relevance to the blood-epididymal barrier in the human epididymis.

The luminal environment along the epididymal duct is important for spermatozoal maturation. This environment is unique and created by the blood-epididymal barrier, which is formed by tight and adhering junctions. For the human epididymis, little information exists on the proteins that comprise these junctions. Our objectives were to assess the gene expression profiles in the different segments of the human epididymis and to identify the proteins that make up the blood-epididymal barrier. Using microarrays, we identified 2980 genes that were differentially expressed by at least 2-fold between the various segments. Of the many genes involved in diverse functions, were those that encoded adhesion proteins (cadherins and catenins) and tight junctional proteins (claudins [CLDN] and others). PCR analyses confirmed the microarray data. Immunolocalization of CLDNs 1, 3, 4, 8, and 10 revealed that the localization of CLDNs differed along the epididymis. In all three segments, CLDNs 1, 3, and 4 were localized to tight junctions, along the lateral margins of adjacent principal cells, and at the interface between basal and principal cells. CLDN8 was localized to tight junctions in all three segments, in addition to being localized in the caput along the lateral margins of principal cells, and in the corpus, at the interface between principal and basal cells. CLDN10, tight junction protein 1, and occludin were localized exclusively to tight junctions in all three epididymal segments. These data indicate that the epididymis displays a complex pattern of gene expression, which includes genes that are implicated in the formation of the blood-epididymal barrier, which suggests complex regulation of this barrier.

The use of a suprascrotal or 'wink' incision for placing a testicular prosthesis.

OBJECTIVE: To describe a suprascrotal technique of testicular prosthesis insertion that obviates the difficulties and complications associated with either the inguinal or scrotal approach, as although the insertion of a testicular prosthesis is common there are few reports of the various techniques of insertion, associated complication rates, and patient satisfaction. PATIENTS AND METHODS: Eight males (aged 14-26 years) who had had a previous orchidectomy, had a testicular prosthesis inserted using a suprascrotal incision. A 2-cm semilunar incision is made just above the scrotum, 2-3 cm lateral to the penis ('wink' incision). The prosthesis is inserted after developing the intrascrotal space with blunt dissection. All patients tolerated the procedure well and all were outpatient procedures. RESULTS: After a median follow-up of 12 months, all patients were satisfied with the aesthetics of the prosthesis. Incisions were hidden by pubic hair. There were no wound infections at the incision site, and no reports of any pain or discomfort associated with the prosthesis. CONCLUSION: The 'wink' incision is an attractive alternative for inserting a testicular prosthesis; the advantages of the suprascrotal approach include: (i) the incision is hidden by pubic hair; (ii) no difficult dissection through fibrotic tissue in patients who have had previous inguinal surgery; and (iii) avoidance of direct contact between the prosthesis and suture line, minimizing the risk of infection, erosion and postoperative pain, while maintaining a pouch of adequate size.

Facilitators and obstacles to sperm banking in young men receiving gonadotoxic chemotherapy for cancer: the perspective of survivors and health care professionals.

BACKGROUND: Testicular cancer and Hodgkin's disease are among the most common malignancies to affect young men of reproductive age. Although both are associated with high rates of infertility, sperm banking (SB) remains underutilized by both diagnostic groups. Reasons for this remain elusive. METHODS: This study used a qualitative design. In-depth interviews were conducted with 20 cancer survivors and 18 health care professionals (HCPs) to examine their perspectives on factors that facilitate or hinder SB. Interview data were analysed using a mixed approach and a three-step process of data reduction, data display and conclusion drawing and verification. RESULTS: Eight factors were identified as having an impact on SB, and findings suggest that effective promotion of SB involves adequate communication around the severity and personal risk for infertility, assessing the importance of patients place on having children, emphasizing the benefits of SB and addressing possible obstacles such as cost, misperceptions or cultural and other factors. In addition, the communicator should be perceived as appealing. CONCLUSIONS: These results are conceptually consistent with both the Health Belief Model and the Elaboration Likelihood Model of health promotion and are useful in informing HCPs on how to better promote SB.

Prospective analysis of outcomes after microsurgical intussusception vasoepididymostomy.

OBJECTIVE: To prospectively analyse the outcomes of microsurgical vasoepididymostomy using the intussusception technique, as vasoepididymostomy is considered the most challenging reconstructive microsurgery in urology. PATIENTS AND METHODS: From 1998 to 2003, of 324 men with obstructive azoospermia who had undergone microsurgical reconstruction of the reproductive tracts, 68 (21%) had intussusception vasoepididymostomy bilaterally or unilaterally in a functionally solitary testis. The outcomes of these patients were analysed prospectively. RESULTS: The mean age was 39.8 years for the men and 31.8 years for their partners. The causes of obstruction were after vasectomy in 31%, infection in 22%, iatrogenic in 19%, trauma in 1.5%, and idiopathic in 27%. The median duration of obstruction was 18.8 years; 37% of patients had had previous failed attempts at reconstruction. The mean (range) follow-up was 15.2 (1-36) months. The overall patency (>10 000 sperm/mL) rate was 84% (53/63). Patency was achieved in 60% (38/63) of men at 1 month after surgery. The mean best sperm count was 12.8 (0.01-80) x 10(6)/mL, with a 21 (0-30)% motility. Among patients with a follow-up of > 1 year, the natural paternity rate was 40%. The median time to achieve a natural pregnancy was 14.3 (3-30) months. Pregnancy was achieved with in vitro fertilization or intracytoplasmic sperm injection in 31% of cases, all using fresh ejaculated sperm. CONCLUSIONS: A favourable patency and pregnancy rate can be achieved using microsurgical intussusception vasoepididymostomy. Even when assisted-reproductive technology is needed, fresh ejaculated sperm can be used without requiring a subsequent sperm retrieval procedure. Thus, microsurgical reconstruction of the reproductive tract should be primary therapeutic method in cases of azoospermia from epididymal obstruction.

Surgical approach of giant testicular cancer. Case report and literature review.

Testicular cancer, which generally presents as a scrotal mass of variable sizes, is amongst the most common malignancies in men in the 15- to 35- year age group. A high inguinal orchiectomy is the standard approach for removal of a scrotal mass suspicious of being malignant. A recent report described a combined use of an inguinal incision, for early clamping of the spermatic cord, and a scrotal incision for orchiectomy of a large size testicular seminoma. We hereby report a case of a large size testis cancer removed using a single oblique inguinoscrotal incision. This approach allows inguinal delivery of a large size of scrotal mass without additional concerns of scrotal tumor spillage and violation of tunica vaginalis.