Improved detection of CFTR variants by targeted next-generation sequencing in male infertility: a case series.

RESEARCH QUESTION: Congenital bilateral absence of vas deferens (CBAVD) is characterized by 'obstructive azoospermia' in male patients with primary infertility. In the routine clinical workup of infertile men, patients with an absence of vas deferens are screened for CFTR variants. However, current genetic testing panels do not cover all variants, missing some CBAVD cases. Here, CFTR testing was explored by targeted next-generation sequencing (NGS) to improve variant detection. DESIGN: Five individuals with heterozygous pathogenic CFTR variants were identified using targeted NGS in a cohort of 1112 idiopathic infertile men with azoospermia or severe oligozoospermia. Pre-screening exclusion criteria were CBAVD by clinical examination with positive CFTR sequence analysis as part of routine fertility workup. RESULTS: Cases 1, 2 and 3 presented with CBAVD after which CFTR screening by mutation panel analysis was negative. Case 4 presented with congenital unilateral absence of vas deferens, after which CFTR panel analysis identified a heterozygous p.(Phe508del) variant. Case 5 presented with a palpable vas deferens so CFTR panel analysis was not offered. In all five men, targeted NGS revealed additional pathogenic variants: p.(Arg117Cys) and p.(Arg1158*) (case 1); p.(Asp110His) and p.(Ser945Leu) (case 2); p.(Arg248Thr) and p.(Phe508Cys) (case 3); p.(Gly463Ser) (case 4); p.(Phe508del) (case 4 and 5); and p.(Arg117His) (case 5). CONCLUSIONS: Targeted NGS led to the detection of five infertile men with CFTR variants who would otherwise have remained undiagnosed after routine genetic screening during the fertility workup for azoospermia or severe oligozoospermia. Given the wide availability of affordable targeted NGS, the data suggest that full gene analysis, and not mutation panels, should be considered to screen CFTR in azoospermic men.

Cervical determinants of anal HPV infection and high-grade anal lesions in women: a collaborative pooled analysis.

BACKGROUND: Cervical cancer screening might contribute to the prevention of anal cancer in women. We aimed to investigate if routine cervical cancer screening results-namely high-risk human papillomavirus (HPV) infection and cytohistopathology-predict anal HPV16 infection, anal high-grade squamous intraepithelial lesions (HSIL) and, hence, anal cancer. METHODS: We did a systematic review of MEDLINE, Embase, and the Cochrane library for studies of cervical determinants of anal HPV and HSIL published up to Aug 31, 2018. We centrally reanalysed individual-level data from 13 427 women with paired cervical and anal samples from 36 studies. We compared anal high-risk HPV prevalence by HIV status, cervical high-risk HPV, cervical cytohistopathology, age, and their combinations, using prevalence ratios (PR) and 95% CIs. Among 3255 women with anal cytohistopathology results, PRs were similarly calculated for all anal HSIL and HPV16-positive anal HSIL. FINDINGS: Cervical and anal HPV infections were highly correlated. In HIV-negative women, anal HPV16 prevalence was 41% (447/1097) in cervical HPV16-positive versus 2% (214/8663) in cervical HPV16-negative women (PR 16·5, 95% CI 14·2-19·2, p<0·0001); these values were 46% (125/273) versus 11% (272/2588) in HIV-positive women (4·4, 3·7-5·3, p<0·0001). Anal HPV16 was also associated with cervical cytohistopathology, with a prevalence of 44% [101/228] for cervical cancer in HIV-negative women (PR vs normal cytology 14·1, 11·1-17·9, p<0·0001). Anal HSIL was associated with cervical high-risk HPV, both in HIV-negative women (from 2% [11/527] in cervical high-risk HPV-negative women up to 24% [33/138] in cervical HPV16-positive women; PR 12·9, 95% CI 6·7-24·8, p<0·0001) and HIV-positive women (from 8% [84/1094] to 17% [31/186]; 2·3, 1·6-3·4, p<0·0001). Anal HSIL was also associated with cervical cytohistopathology, both in HIV-negative women (from 1% [5/498] in normal cytology up to 22% [59/273] in cervical HSIL; PR 23·1, 9·4-57·0, p<0·0001) and HIV-positive women (from 7% [105/1421] to 25% [25/101]; 3·6, 2·5-5·3, p<0·0001). Prevalence of HPV16-positive anal HSIL was 23-25% in cervical HPV16-positive women older than 45 years (5/20 in HIV-negative women, 12/52 in HIV-positive women). INTERPRETATION: HPV-based cervical cancer screening programmes might help to stratify anal cancer risk, irrespective of HIV status. For targeted secondary anal cancer prevention in high-risk groups, HIV-negative women with cervical HPV16, especially those older than 45 years, have a similar anal cancer risk profile to that of HIV-positive women. FUNDING: International Agency for Research on Cancer.

Beneficial value of testicular sperm extraction-AgarCyto in addition to the standard testicular biopsy for diagnosis of testicular germ cell tumors in nonobstructive azoospermia.

OBJECTIVE: To study whether immunohistochemical detection of germ cell neoplasia in situ (GCNIS) in AgarCytos, made of the remnants of the testicular sperm extraction (TESE) specimen, is equally accurate as in a standard testicular biopsy. DESIGN: Prospective cohort study performed between January 2013 and May 2014. SETTING: University hospital. PATIENT(S): All men with nonobstructive azoospermia (n = 197) undergoing a urological work-up followed by a unilateral or bilateral TESE for fertility treatment were consecutively included. INTERVENTION(S): An AgarCyto was made of the remnants of these TESE biopsies. Simultaneously a standard testicular biopsy was performed. For all cases a routine hematoxylin-eosin (H & E) staining was performed as well as immunohistochemistry (PLAP and OCT3/4) to detect GCNIS. MAIN OUTCOME MEASURE(S): The presence or absence of GCNIS in the TESE-AgarCyto and standard testicular biopsy. RESULT(S): Six men (3.0%) were diagnosed with a germ cell (pre)malignancy by immunohistochemistry. No cases were encountered in which the TESE-AgarCyto was negative, whereas the standard testicular biopsy was positive for GCNIS. In one case the TESE-AgarCyto detected a premalignancy that was missed by standard testicular biopsy. Unfortunately a standard testicular biopsy was not available for direct comparison in 50% of the GCNIS-positive patients due to various reasons. CONCLUSION(S): Because GCNIS is heterogeneously distributed in the testis, the TESE-AgarCyto can diagnose GCNIS even when the standard testicular biopsy is negative. Direct comparison of accuracy, however, is not reliable due to the low prevalence of GCNIS and the lack of a standard biopsy when an orchidectomy was performed simultaneously with TESE.

Anal human papillomavirus DNA in women at a colposcopy clinic.

OBJECTIVES: To describe the type-specific prevalence of anal and cervical human papillomavirus (HPV) infections and the cytology in HIV-negative women without a history of cervical cancer, attending a colposcopy clinic. To examine if an HPV positive anal smear is related to anal pathology and consequently indicative for further examinations (high resolution anoscopy, anal biopsy). STUDY DESIGN: From 149 consecutive women an anal swab and a cervical swab were taken, using the Cervex-Brush. The presence of 18 different HPV genotypes was determined using TaqMan-based real-time quantitative PCR targeting type-specific sequences of viral genes. From the fluid containing the cellular material, a liquid-based cytology sample was prepared of both collections with the robotic BD PrepStain Slide Processor. All slides were pre-screened by BD FocalPoint system and categorized from quintiles 1 to 5 and afterwards screened using targeted microscopic interpretation of selected suspicious fields using FocalPoint guided screening review stations. The 2001 Bethesda System Terminology was used for the anal slides. RESULTS: Ninety-six anal samples and all 149 cervical samples were adequate. Overall presence of HPV in the anus was 56.3% and in the cervix 53.7%. Overall, cytological abnormalities were found in 10.8% of anal smears and in 32.8% of cervical smears. HPV genotypes were identified in 47 samples on both sites: partial or complete concordance was found in 85.1%. HPV types 6, 16 and 18 were found in 27.9% and in 26.6% of the anal and cervical samples, respectively. The top three HPV types in the anus were 16, 51 and 39; in the cervix 16, 39, 51 and 56 (a shared 3(rd) place). HPV type 11 was not found. CONCLUSIONS: The presence of HPV genotypes is clearly multifocal in this study population of women attending a colposcopy clinic, with high concordance of genotypes. The number of anal HPV infections is high. Although cytological abnormalities are rare, the presence of HPV may lead to anal lesions later in life. From this perspective, complementary medical history and clinical examination of the anal region are advised.

Bladder exstrophy and male fertility: pregnancies after ICSI with ejaculated or epididymal sperm.

OBJECTIVE: To define the additional value of intracytoplasmatic sperm injection (ICSI). DESIGN: Descriptive clinical study. SETTING: Male patients with bladder exstrophy in an academic setting. PATIENT(S): Three male patients in a stable relationship, desirous to have their own children. They were born with bladder exstrophy and had undergone surgical reconstruction. INTERVENTION(S): The ICSI procedure. MAIN OUTCOME MEASURE(S): Number of pregnancies. RESULT(S): Each of the three men presented a different way of producing sperm. The first male patient had no ejaculation, and sperm cells were retrieved by percutaneous sperm aspiration (PESA). The second could ejaculate with the production of sperm cells, and the third had no ejaculation but collected prostatic fluid by catheterization of a cutaneous fistula; this fluid contained sperm cells. Their partners all had undergone a successful ICSI procedure. CONCLUSION(S): Nowadays, men with bladder exstrophy reach adult age and therefore express the desire to parent their own children. Careful attention to genital reconstruction has to be given to enhance the possibility to antegrade production of sperm. In cases when this is not possible, PESA/testicular sperm extraction in combination with ICSI offer an added opportunity for these couples.