Prediction model for obtaining spermatozoa with testicular sperm extraction in men with non-obstructive azoospermia.

STUDY QUESTION: Can an externally validated model, based on biological variables, be developed to predict successful sperm retrieval with testicular sperm extraction (TESE) in men with non-obstructive azoospermia (NOA) using a large nationwide cohort? SUMMARY ANSWER: Our prediction model including six variables was able to make a good distinction between men with a good chance and men with a poor chance of obtaining spermatozoa with TESE. WHAT IS KNOWN ALREADY: Using ICSI in combination with TESE even men suffering from NOA are able to father their own biological child. Only in approximately half of the patients with NOA can testicular sperm be retrieved successfully. The few models that have been developed to predict the chance of obtaining spermatozoa with TESE were based on small datasets and none of them have been validated externally. STUDY DESIGN, SIZE, DURATION: We performed a retrospective nationwide cohort study. Data from 1371 TESE procedures were collected between June 2007 and June 2015 in the two fertility centres. PARTICIPANTS/MATERIALS, SETTING, METHODS: All men with NOA undergoing their first TESE procedure as part of a fertility treatment were included. The primary end-point was the presence of one or more spermatozoa (regardless of their motility) in the testicular biopsies.We constructed a model for the prediction of successful sperm retrieval, using univariable and multivariable binary logistic regression analysis and the dataset from one centre. This model was then validated using the dataset from the other centre. The area under the receiver-operating characteristic curve (AUC) was calculated and model calibration was assessed. MAIN RESULTS AND THE ROLE OF CHANCE: There were 599 (43.7%) successful sperm retrievals after a first TESE procedure. The prediction model, built after multivariable logistic regression analysis, demonstrated that higher male age, higher levels of serum testosterone and lower levels of FSH and LH were predictive for successful sperm retrieval. Diagnosis of idiopathic NOA and the presence of an azoospermia factor c gene deletion were predictive for unsuccessful sperm retrieval. The AUC was 0.69 (95% confidence interval (CI): 0.66-0.72). The difference between the mean observed chance and the mean predicted chance was <2.0% in all groups, indicating good calibration. In validation, the model had moderate discriminative capacity (AUC 0.65, 95% CI: 0.62-0.72) and moderate calibration: the predicted probability never differed by more than 9.2% of the mean observed probability. LIMITATIONS, REASONS FOR CAUTION: The percentage of men with Klinefelter syndrome among men diagnosed with NOA is expected to be higher than in our study population, which is a potential selection bias. The ability of the sperm retrieved to fertilize an oocyte and produce a live birth was not tested. WIDER IMPLICATIONS OF THE FINDINGS: This model can help in clinical decision-making in men with NOA by reliably predicting the chance of obtaining spermatozoa with TESE. STUDY FUNDING/COMPETING INTEREST: This study was partly supported by an unconditional grant from Merck Serono (to D.D.M.B. and K.F.) and by the Department of Obstetrics and Gynaecology of Radboud University Medical Center, Nijmegen, The Netherlands, the Department of Obstetrics and Gynaecology, Jeroen Bosch Hospital, Den Bosch, The Netherlands, and the Department of Obstetrics and Gynaecology, Academic Medical Center, Amsterdam, The Netherlands. Merck Serono had no influence in concept, design nor elaboration of this study. TRIAL REGISTRATION NUMBER: Not applicable.

A novel cell-processing method 'AgarCytos' in conjunction with OCT3/4 and PLAP to detect intratubular germ cell neoplasia in non-obstructive azoospermia using remnants of testicular sperm extraction specimens.

STUDY QUESTION: Can we diagnose intratubular germ cell neoplasia (IGCN) using the immunohistochemical markers placental-like alkaline phosphatase (PLAP) and OCT3/4 using a novel cell-processing method 'AgarCytos', applied to the remnants of testicular sperm extraction (TESE) specimens and what is the prevalence of a testicular germ cell (pre)malignancy in men with a non-obstructive azoospermia (NOA) undergoing TESE for fertility treatment? SUMMARY ANSWER: IGCN can be successfully detected by immunohistochemical evaluation of AgarCytos, made of the remnants of TESE biopsies. The observed prevalence of a germ cell (pre)malignancy in this specific population was found to be 4.4%. WHAT IS KNOWN ALREADY: Infertile men are at higher risk for testicular cancer than the general population. IGCN can be detected by immunohistochemistry using PLAP and OCT3/4 in standard testicular biopsies and, with less accuracy, in semen. STUDY DESIGN, SIZE, DURATION: Between January 2011 and April 2012 a prospective cohort study was conducted at a Dutch tertiary care academic training hospital. All males with NOA (n = 182) undergoing a urological work-up followed by a diagnostic TESE for fertility treatment (n = 251) were included. PARTICIPANTS, SETTING, METHODS: After cryopreservation of sperm, if present, an AgarCyto was made of the remnants of the TESE biopsies. Sections were stained with haematoxylin-eosin for pathological examination as well as PLAP and OCT3/4 for immunohistochemistry to detect IGCN. MAIN RESULTS AND THE ROLE OF CHANCE: Eight men (4.4%) were diagnosed with a germ cell (pre)malignancy: six of them had seminoma, two without and four with concomitant IGCN, and two of them had IGCN only. Microscopic evaluation including immunohistochemical analysis of the AgarCytos diagnosed three (1.6%) more cases of a germ cell (pre)malignancy compared with scrotal ultrasound alone (one case of bilateral seminoma with concomitant IGCN and two cases of IGCN alone). No false-positive cytology results were found upon conventional histological evaluation. LIMITATIONS, REASONS FOR CAUTION: The main limitation of this study is lack of a simultaneously taken standard testicular biopsy, to compare the results of our novel diagnostic method with. Nevertheless, in all but one of our cases orchidectomy followed and the diagnosis was confirmed by histology. In the remaining case repeat TESE showed similar results. WIDER IMPLICATIONS OF THE FINDINGS: Simultaneous screening for IGCN is highly recommended to men with NOA undergoing TESE, because of the increased incidence of germ cell (pre)malignancies in this specific population. The principal advantage of our new method is that all available testicular tissue can be used for both sperm recovery and pathological evaluation, increasing the yield of spermatozoa as well as the chance to find (pre)malignant cells. In those cases where the disease is still in a premalignant stage, early diagnosis will allow for timely treatment and reduction of morbidity and mortality in this group of patients. STUDY FUNDING/COMPETING INTEREST(S): This study was (partially) funded by Merck Serono (the Netherlands). There are no conflicting interests to disclose. TRIAL REGISTRATION NUMBER: N/A.

A de novo paradigm for male infertility.

De novo mutations are known to play a prominent role in sporadic disorders with reduced fitness. We hypothesize that de novo mutations play an important role in severe male infertility and explain a portion of the genetic causes of this understudied disorder. To test this hypothesis, we utilize trio-based exome sequencing in a cohort of 185 infertile males and their unaffected parents. Following a systematic analysis, 29 of 145 rare (MAF < 0.1%) protein-altering de novo mutations are classified as possibly causative of the male infertility phenotype. We observed a significant enrichment of loss-of-function de novo mutations in loss-of-function-intolerant genes (p-value = 1.00 × 10-5) in infertile men compared to controls. Additionally, we detected a significant increase in predicted pathogenic de novo missense mutations affecting missense-intolerant genes (p-value = 5.01 × 10-4) in contrast to predicted benign de novo mutations. One gene we identify, RBM5, is an essential regulator of male germ cell pre-mRNA splicing and has been previously implicated in male infertility in mice. In a follow-up study, 6 rare pathogenic missense mutations affecting this gene are observed in a cohort of 2,506 infertile patients, whilst we find no such mutations in a cohort of 5,784 fertile men (p-value = 0.03). Our results provide evidence for the role of de novo mutations in severe male infertility and point to new candidate genes affecting fertility.

Follow-up of children born after ICSI with epididymal spermatozoa.

BACKGROUND: To evaluate the safety of ICSI with epididymal sperm, this study compared children born after ICSI treatment with epididymal sperm and children conceived after IVF and ICSI with ejaculated sperm. Additionally, the results of a multidisciplinary, multicentre follow-up of the children conceived with epididymal sperm at 2 years of age are described. METHODS: This follow-up study included 378 children conceived after ICSI with epididymal sperm (percutaneous epididymal sperm aspiration: PESA group) and a control group of 1192 IVF and 1126 ICSI (with ejaculated sperm) children, all with a gestational age of 20 weeks or more. Questionnaires were sent at birth, 1 year and 4 years of age, collecting data on parental, pregnancy and child factors. A total of 148 PESA children were assessed at 2 years of age for motor performance, mental- and language development and compared with the Dutch norms. RESULTS: PESA children showed no increased risks for stillbirths, total deaths and malformations. They also did not differ from IVF and ICSI children in gender rate, birthweight and gestational age. The mental Bayley score was higher (P < 0.05) for PESA singletons and parents reported fewer (P < 0.05) behavioural problems in the PESA group than the Dutch reference group. The scores for syntactic and lexical development for the PESA singletons were better (P < 0.05) than the Dutch standards. CONCLUSIONS: ICSI with epididymal sperm does not lead to more stillbirths or congenital malformations in comparison to IVF and ICSI with ejaculated sperm and does not lead to poor development in comparison with the Dutch reference group.

No primary role of ambulatory urodynamics for the management of spinal cord injury patients compared to conventional urodynamics.

AIMS: Adequate urodynamic assessment of bladder behavior is essential in spinal cord injury (SCI) patients. Ambulatory urodynamics are more sensitive to detect detrusor overactivity (DO) than conventional urodynamics. The primary objective of this study was to determine the value of ambulatory urodynamics for the diagnosis of DO in SCI patients compared to conventional urodynamics. METHODS: Twenty-seven SCI patients who were suspected of DO underwent both conventional and ambulatory urodynamics at one day. A single involuntary detrusor contraction (IDC) was defined as a detrusor pressure rise of at least 10 cmH(2)O. DO according to the ICS definition was used in addition to minimize the influence of catheter artifacts. Outcome of urodynamics was used for decisions on treatment. RESULTS: Ambulatory urodynamics were more sensitive to diagnose IDC and DO. Conventional urodynamics had a sensitivity of 82% and specificity of 75% for DO diagnosis compared to ambulatory urodynamics. Mean maximum detrusor pressures did not differ significantly between both urodynamics. When the maximum detrusor pressure at conventional urodynamics did not exceed 40 cmH(2)O, 83% (10/12) of patients had a mean maximum detrusor pressure under 40 cmH(2)O at ambulatory urodynamics. Although the inter-individual DO diagnostic agreement was lower for ambulatory than conventional urodynamics (58%, K = 0.201 vs. 77%, K = 0552), the treatment agreement was higher for ambulatory urodynamics (58% vs. 42%). CONCLUSIONS: Ambulatory urodynamics do not seem necessary for diagnosis and risk assessment in SCI patients suspected for DO when conventional urodynamics are done properly. The exact role of urodynamics in treatment decision remains to be determined.

Screening for human papillomavirus: is urine useful?

Persistent infection with high-risk Human papillomavirus (hr-HPV 16, 18, 31, 33, and 45) is the main risk factor for developing malignant genital lesions. Screening methods and follow-up schedules for cervical cancer are well known. A golden standard to screen and monitor men does not exist yet, because HPV-related, life threatening malignancies in men are rare. The importance of male HPV screening lies mainly in HPV vaccination. Young females are the target group for HPV, but men are considered to be the reservoir for HPV and to have a role in the perpetuation of the infection in the general population. We looked at the usefulness of urine as a tool for HPV screening. Pubmed was searched with the words ''HPV'', ''Urine,'' and ''HPV-DNA''. The chance of finding HPV-DNA in urine is higher in men with lesions in the urethra than outside the urethra, and in women with abnormal cervical cytology. In general, the results of testing urine for HPV-DNA are better for women than for men, probably because of the anatomical position of the urethra to the vagina, vulva, and cervix. In both genders, urine HPV prevalence is higher in HIV pos patients and in high-risk populations. Urine, to screen asymptomatic low-risk-profile (wo)men seems less useful because their urine samples are often inadequate. If urine proves to be the best medium to screen, a low-risk population remains controversial.

HPV in men.

PURPOSE: To collect information about HPV in men and the (possible) correlation with HVP infection in women. METHODS: Review of the literature. RESULTS: An overview of HPV-related penile and anal malignancies in men and the risk factors of acquiring HPV. CONCLUSION: In men HPV is also partially responsible for anogenital malignancies. Although the prevalence of HPV-related malignancies in men is much lower than in women, it is useful to gain more knowlege. Especially knowing if men are really the HPV reservoir and transmitters for women can make a difference in deciding whether men should also be screened for HPV and if they are good candidates for vaccination.

Cytological evaluation of spermatogenesis: a novel and simple diagnostic method to assess spermatogenesis in non-obstructive azoospermia using testicular sperm extraction specimens.

Most of the non-obstructive azoospermia (NOA)-patients have only focal spermatogenesis which results in insufficient numbers of spermatozoa to reach the ejaculate. In ≈50% of these NOA-patients testicular sperm extraction (TESE) is successful and intracytoplasmic sperm injection (ICSI) is pursued. We studied whether (i) spermatogenesis can be evaluated by defining the ratios between Sertoli cells, pachytene spermatocytes and spermatozoa in a testicular cell suspension, and (ii) these ratios are associated with the outcome of fertility treatment. A retrospective cohort study was conducted between June 2007 and August 2012. In this period, 441 consecutive ICSI-TESE cycles were performed in 212 couples. For each TESE biopsy, the ratios between Sertoli cells, pachytene spermatocytes and spermatozoa were calculated. A control population of 32 vasectomized men was used to define cut-off values for complete spermatogenesis. Based on the pachytene to sperm ratio (P/Sp) and number of spermatozoa per 100 Sertoli cells (#Sp/100SC) groups were defined as complete spermatogenesis, hypospermatogenesis and partial maturation arrest (MA). Validation of the cytological diagnoses was performed by comparing the results of cytology to the histological evaluation of spermatogenesis in 40 cases. In 92.5%, a perfect match was observed and in the three remaining cases cytology corresponded well with the results of TESE. Couples with complete spermatogenesis have a higher ongoing pregnancy rate after the first treatment cycle compared to couples with hypospermatogenesis (34 vs. 16%; p = 0.02) and partial MA (34 vs. 19%; p = 0.11). In conclusion, pachytene spermatocytes, spermatozoa and Sertoli cells can be easily identified and counted in a cell suspension and their ratios can be successfully used to diagnose the level of spermatogenic impairment. This pilot study indicates that once successful spermatozoa retrieval is achieved, treatment outcome declines when spermatogenesis is impaired in NOA. The predictive value of cytological evaluation of spermatogenesis has to be established in a future prospective trial.

Impact of medical education on knowledge and attitudes regarding the human papilloma virus and vaccination: comparison before and 6 years after the introduction of the vaccines.

AIM: The lifetime risk for acquiring a human papilloma virus (HPV) infection is 80% for sexually active people. High-risk HPVs are causally related to almost every case of cervical cancer, and to a subgroup of vaginal, vulvar, anal, penile and oral/oropharyngeal cancer. Low-risk HPVs are related to cutaneous, anogenital, and oral warts. Two prophylactic vaccines were launched in 2007: they were included in the national vaccination program in Belgium (2009) and in the Netherlands (2010). The objectives of the present study were to determine and compare knowledge and attitudes regarding HPV and vaccination among a study population in 2006 and in 2012. MATERIALS AND METHODS: Shortly before the introduction, and three years after the inclusion, 715 (2006) and 678 participants (2012) were questioned. Participants were categorised as into non-medics, medics, or paramedics. RESULTS: In general, knowledge about HPV has increased over time (p<0.01). Well-known facts are the relationship of HPV with cervical cancer (>94% in 2006; >96% in 2012), and that an HPV infection might be asymptomatic (>95% in 2006; >99% in 2012). In 2012, versus in 2006, paramedics and non-medics (both p<0.01), were more likely to vaccinate all female teenagers. Medics were less likely to support this (p=0.001). More respondents agreed to vaccinate their daughters (p<0.01), as well as their sons (p<0.01). In 2012, when compared with 2006, less non-medics and medics (both p<0.01) and more paramedics (p=0.001) would accept a free catch-up vaccination. Arguments against catch-up vaccination reflected the belief not being at risk and doubts about the vaccines' safety. CONCLUSION: The facts that vaccination programs are regarded as being important, and that knowledge on HPV increased, do not automatically result in an increase in participation in HPV vaccination programs. To increase participation, information must be provided with arguments that cannot be misinterpreted.

Human papillomavirus, lichen sclerosus and penile cancer: a study in Belgium.

PURPOSE: The prevalence of penile cancer varies between 1.5 (industrialized countries) and 4.5 per 100,000 men (non-industrialized countries). Predominant histological subtype is squamous cell carcinoma (SCC). Human papillomavirus (HPV) is found in 40-46% of cases: penile cancer is considered to behave as vulvar cancer. Non HPV related risk factors are lack of circumcision, phimosis, chronic inflammation, and smoking. The role of lichen sclerosus (LS) is unclear. Clinical diagnosis is difficult and treatment often mutilating. Preventive measures can be taken since the risk factors are known: the use of the prophylactic HPV vaccines may contribute. We measured the prevalence of HPV and LS in penile cancer in Belgium. MATERIALS AND METHODS: We found 76 samples of penile lesions in the archives of the departments of Histology of four university hospitals in Belgium. Real-time PCR of type-specific HPV DNA was performed targeting 18 HPV types. PRINCIPAL RESULTS: Patients with penile intraepithelial neoplasia (PeIN) were 56.1 years of age: patients with invasive penile cancer (IPC) 68.5 (p=0.009). Fifty-five samples (55/76) were adequate for HPV targeting. Overall HPV DNA was 70.9%: 89.5% in samples of PeIN (n=19) and 61.1% in samples of IPC (n=36). Invasive penile cancer samples were less likely to be HPV infected (p=0.028). HPV 16 was most prevalent: 48.3%: 20% PeIN, and 28.3% IPC. HPV DNA of the types, included in the prophylactic vaccines, was found in 33% of PeIN and 31.7% of IPC samples. Thrice, low risk HPV (lrHPV) types 6 (1 IPC) and 11 (1 PeIN, 1 IPC) were solely present. There was no difference in the presence of LS between HPV positive and HPV negative samples (p=0.944). CONCLUSIONS: Prevalence of HPV DNA in penile lesions in Belgium is high. However, the prophylactic vaccines may contribute to primary prevention of only a subset of cases. The role of LS remains unclear.