A novel ADGRG2 truncating variant associated with X-linked obstructive azoospermia in a large Chinese pedigree.

PURPOSE: In this study, we aimed to identify sterility-related variants in a Chinese pedigree with male infertility and to reveal the different phenotypes and intracytoplasmic sperm injection (ICSI) outcomes of the affected members. METHODS: Physical examinations were performed on male patients. G-band karyotype analysis, copy number variation sequencing, and quantitative fluorescent PCR were conducted to detect common chromosomal disorders in the probands. Whole-exome sequencing and Sanger sequencing were applied to identify the pathogenic genes and the protein expression changes caused by the very mutation were identified by Western Blot in vitro. RESULTS: A novel nonsense mutation (c.908C > G: p.S303*) in the ADGRG2 was identified in all infertile male patients of the pedigree, which was inherited from their mothers. This variant was absent from the human genome databases. This mutation was also unexpectedly found in a male member with normal reproductive capability. Members with the mutation had different genitalia phenotypes, ranging from normal to dilated phenotypes of the vas deferens, spermatic veins and epididymis. There was a truncated ADGRG2 protein in vitro after mutation. Of the three patients' wives treated with ICSI, only one successfully gave birth. CONCLUSIONS: Our study is the first to report the c.908C > G: p.S303* mutation in the ADGRG2 in an X-linked azoospermia pedigree and is the first to report normal fertility in a member with this mutation, expanding the mutation spectrum and phenotype spectrum of this gene. In our study, ISCI had a success rate of only one-third in couples including men with azoospermia with this mutation.

Pathogenic Variants and Genotypes of the CFTR Gene in Russian Men with Cystic Fibrosis and CBAVD Syndrome.

Pathogenic CFTR variants cause cystic fibrosis (CF), and CF-related disorders (CF-RD), including bilateral aplasia of the vas deferens (CBAVD). The spectrum of clinical manifestations depends on the CFTR genotype. The frequency and spectrum of the CFTR variants vary between populations and clinical groups. CFTR variants and genotypes were analyzed in Russian men with CF (n = 546) and CBAVD syndrome (n = 125). Pathogenic variants were detected in 93.95% and 39.2% of the CF and CBAVD alleles, respectively. The most frequent c.1521_1523del (F508del; p.Phe508del) variant was found in 541 (49.5%) CF alleles. A total of 162 CFTR genotypes were revealed in CF patients, including 152 homozygous and 394 compound-heterozygous. The most common CF-genotype was F508del/F508del (24.9%). Other frequent CF-genotypes were F508del/3849+10kbC>T, F508del/CFTRdele2,3, and F508del/E92K. CF-causing variants and/or 5T allele were found in 88% of CBAVD patients: 5T/CFTRmut (48.0%), CFTRmut/N (17.6%), CFTRmut/CFTRmut (6.4%), 5T/5T (10.4%), 5T/N (5.6%) and N/N (12.0%), with the most common CBAVD-genotype being F508del/5T (29.6%). The allele frequencies of F508del, CFTRdele2,3 394delTT, and 3849+10kbC>T were significantly higher in CF patients. L138ins/L138ins, 2184insA/E92K, and L138ins/N genotypes were found in CBAVD, but not in CF patients. The results indicate certain differences in the frequency of some CFTR variants and genotypes in Russian CF and CBAVD patients.

Genetic analysis and intracytoplasmic sperm injection outcomes of Chinese patients with congenital bilateral absence of vas deferens.

PURPOSE: Congenital bilateral absence of the vas deferens (CBAVD) is a major cause of obstructive azoospermia and male factor infertility. CBAVD is mainly caused by mutations in the genes encoding CFTR (cystic fibrosis transmembrane conductance regulator) and ADGRG2 (adhesion G protein-coupled receptor G2). This study aimed to describe CFTR and ADGRG2 variations in 46 Chinese CBAVD patients and evaluated sperm retrieval and assisted reproductive technology outcomes. METHODS: The CFTR and ADGRG2 genes were sequenced and analyzed by whole-exome sequencing (WES), and variations were identified by Sanger sequencing. Bioinformatic analysis was performed. We retrospectively reviewed the outcomes of patients undergoing sperm retrieval surgery and intracytoplasmic sperm injection (ICSI). RESULTS: In total, 35 of 46 (76.09%) patients carried at least one variation in CFTR, but no copy number variants or ADGRG2 variations were found. In addition to the IVS9-5 T allele, there were 27 CFTR variations, of which 4 variations were novel and predicted to be damaging by bioinformatics. Spermatozoa were successfully retrachieved in 46 patients, and 39 of the patients had their own offspring through ICSI. CONCLUSION: There are no obvious hotspot CFTR mutations in Chinese CBAVD patients besides the IVS9-5 T allele. Therefore, WES might be the best detection method, and genetic counseling should be different from that provided to Caucasian populations. After proper counseling, all patients can undergo sperm retrieval from their epididymis or testis, and most of them can have their own children through ICSI.

Andrological findings in infertile men with two (biallelic) CFTR mutations: results of a multicentre study in Germany and Austria comprising 71 patients.

STUDY QUESTION: When should cystic fibrosis transmembrane conductance regulator (CFTR) mutation analysis be recommended in infertile men based on andrological findings? SUMMARY ANSWER: CFTR mutation analysis is recommended in all men with unexplained azoospermia in the presence of normal gonadotropin levels. WHAT IS KNOWN ALREADY: While 80-97% of men with congenital bilateral absence of the vas deferens (CBAVD) are thought to carry CFTR mutations, there is uncertainty about the spectrum of clinical and andrological abnormalities in infertile men with bilallelic CFTR mutations. This information is relevant for evidence-based recommendations to couples requesting assisted reproduction. STUDY DESIGN, SIZE, DURATION: We studied the andrological findings of patients with two CFTR mutations who were examined in one of the cooperating fertility centres in Germany and Austria. In the period of January till July 2019, the completed and anonymized data sheets of 78 adult male patients were returned to and analysed by the project leader at the Institute of Human Genetics in Innsbruck, Austria. PARTICIPANTS/MATERIALS, SETTING, METHODS: Minimum study entry criteria were the presence of two (biallelic) CFTR mutations and results of at least one semen analysis. Andrological assessments were undertaken by standardized data sheets and compared with normal reference values. Seventy-one patients were eligible for the study (n = 30, 42% from Germany, n = 26, 37% from Austria, n = 15, 21% other nations). MAIN RESULTS AND THE ROLE OF CHANCE: Gonadotropin levels (FSH, LH) were normal, 22% of patients had reduced testosterone values. Mean right testis volume was 23.38 ml (SD 8.77), mean left testis volume was 22.59 ml (SD 8.68) and thereby statistically increased compared to normal (P < 0.01). although the means remained in the reference range of 12-25 ml. Semen analysis revealed azoospermia in 70 of 71 (99%) patients and severe oligozoospermia <0.1 × 106/ml in one patient. Four semen parameters, i.e. ejaculate volume, pH, α-glucosidase and fructose values, were significantly reduced (P < 0.01). Only 18% of patients had a palpatory and sonographically diagnosed CBAVD, while in 31% the diagnosis of CBAVD was uncertain, in 12% patients, the vas deferens was present but hypoplastic, and in 39% the vas deferens was normally present bilaterally. Seminal vesicles were not detectable in 37% and only unilaterally present in 37% of patients. Apart from total testes volume, clinical findings were similar in patients with two confirmed pathogenic CFTR mutations (Group I) compared with patients who carried one pathogenic mutation and one CFTR variant of unknown significance (Group II). LIMITATIONS, REASONS FOR CAUTION: We could not formally confirm the in trans position of genetic variants in most patients as no family members were available for segregation studies. Nonetheless, considering that most mutations in our study have been previously described without other rare variants in cis, and in view of the compatible andrological phenotype, it is reasonable to assume that the biallelic genotypes are correct. WIDER IMPLICATIONS OF THE FINDINGS: Our study reveals that CFTR mutation analysis has a broader indication than just the absence of the vas deferens. We recommend to completely sequence the CFTR gene if there is a suspicion of obstructive azoospermia, and to extend this analysis to all patients with unexplained azoospermia in the presence of normal gonadotropin levels. STUDY FUNDING/COMPETING INTEREST(S): German Research Foundation Clinical Research Unit 'Male Germ Cells: from Genes to Function' (DFG CRU326, grants to F.T.). There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Genetics of Azoospermia.

Azoospermia affects 1% of men, and it can be due to: (i) hypothalamic-pituitary dysfunction, (ii) primary quantitative spermatogenic disturbances, (iii) urogenital duct obstruction. Known genetic factors contribute to all these categories, and genetic testing is part of the routine diagnostic workup of azoospermic men. The diagnostic yield of genetic tests in azoospermia is different in the different etiological categories, with the highest in Congenital Bilateral Absence of Vas Deferens (90%) and the lowest in Non-Obstructive Azoospermia (NOA) due to primary testicular failure (~30%). Whole-Exome Sequencing allowed the discovery of an increasing number of monogenic defects of NOA with a current list of 38 candidate genes. These genes are of potential clinical relevance for future gene panel-based screening. We classified these genes according to the associated-testicular histology underlying the NOA phenotype. The validation and the discovery of novel NOA genes will radically improve patient management. Interestingly, approximately 37% of candidate genes are shared in human male and female gonadal failure, implying that genetic counselling should be extended also to female family members of NOA patients.

A novel hemizygous loss-of-function mutation in ADGRG2 causes male infertility with congenital bilateral absence of the vas deferens.

PURPOSE: Cystic fibrosis transmembrane conductance regulator (CFTR) and adhesion G protein-coupled receptor G2 (ADGRG2) have been identified as the main pathogenic genes in congenital bilateral absence of the vas deferens (CBAVD), which is an important cause of obstructive azoospermia. This study aimed to identify the disease-causing gene in two brothers with CBAVD from a Chinese consanguineous family and reveal the intracytoplasmic sperm injection (ICSI) outcomes in these patients. METHODS: Whole-exome sequencing and Sanger sequencing were used to identify the candidate pathogenic genes. Real-time polymerase chain reaction, immunohistochemistry, and immunofluorescence were used to assess the expression of the mutant gene. Moreover, the ICSI results from both patients were retrospectively reviewed. RESULTS: A novel hemizygous loss-of-function mutation (c.G118T: p.Glu40*) in ADGRG2 was identified in both patients with CBAVD. This mutation is absent from the human genome databases and causes an early translational termination in the third exon of ADGRG2. Expression analyses showed that both the ADGRG2 mRNA and the corresponding protein were undetectable in the proximal epididymal tissue of ADGRG2-mutated patients. ADGRG2 expression was restricted to the apical membranes of non-ciliated epithelia in human efferent ducts, which was consistent with a previous report in mice. Both ADGRG2-mutated patients had normal spermatogenesis and had successful clinical outcomes following ICSI. CONCLUSIONS: Our study verifies the pathogenic role of ADGRG2 in X-linked CBAVD and broadens the spectrum of ADGRG2 mutations. In addition, we found positive ICSI outcomes in the two ADGRG2-mutated CBAVD patients.

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.

A rare frameshift variant in trans with the IVS9-5T allele of CFTR in a Chinese pedigree with congenital aplasia of vas deferens.

PURPOSE: Congenital aplasia of vas deferens (CAVD) is an atypical form of cystic fibrosis (CF) and causes obstructive azoospermia and male infertility. Compound heterozygous variants of CFTR are the main cause of CAVD. However, most evidence comes from genetic screening of sporadic cases and little is from pedigree analysis. In this study, we performed analysis in a Chinese pedigree with two CAVD patients in order to determine the genetic cause of this familial disorder. METHODS: In the present study, we performed whole-exome sequencing and co-segregation analysis in a Chinese pedigree involving two patients diagnosed with CAVD. RESULTS: We identified a rare frameshift variant (NM_000492.3: c.50dupT;p.S18Qfs*27) and a frequent CBAVD-causing variant (IVS9-TG13-5T) in both patients. The frameshift variant introduced a premature termination codon and was not found in any public databases or reported in the literature. Co-segregation analysis confirmed these two variants were in compound heterozygous state. The other male members, who harbored the frameshift variant and benign IVS9-7T allele, did not have any typical clinical manifestations of CF or CAVD. CONCLUSION: Our findings may broaden the mutation spectrum of CFTR in CAVD patients and provide more familial evidence that the combination of a mild variant and a severe variant in trans of CFTR can cause vas deferens malformation.

Validation and application of a novel integrated genetic screening method to a cohort of 1,112 men with idiopathic azoospermia or severe oligozoospermia.

Microdeletions of the Y chromosome (YCMs), Klinefelter syndrome (47,XXY), and CFTR mutations are known genetic causes of severe male infertility, but the majority of cases remain idiopathic. Here, we describe a novel method using single molecule Molecular Inversion Probes (smMIPs), to screen infertile men for mutations and copy number variations affecting known disease genes. We designed a set of 4,525 smMIPs targeting the coding regions of causal (n = 6) and candidate (n = 101) male infertility genes. After extensive validation, we screened 1,112 idiopathic infertile men with non-obstructive azoospermia or severe oligozoospermia. In addition to five chromosome YCMs and six other sex chromosomal anomalies, we identified five patients with rare recessive mutations in CFTR as well as a patient with a rare heterozygous frameshift mutation in SYCP3 that may be of clinical relevance. This results in a genetic diagnosis in 11-17 patients (1%-1.5%), a yield that may increase significantly when more genes are confidently linked to male infertility. In conclusion, we developed a flexible and scalable method to reliably detect genetic causes of male infertility. The assay consolidates the detection of different types of genetic variation while increasing the diagnostic yield and detection precision at the same or lower price compared with currently used methods.

Molecular screening of CFTR gene in Egyptian patients with congenital bilateral absence of the vas deferens: a preliminary study.

In the current study, we enrolled 14 Egyptian infertile males with isolated congenital bilateral absence of the vas deferens (CBAVD). Screening for the most commonly reported 36 CFTR mutations, and the intron 8 (T)n splice variant was performed by multiplex PCR followed by reversed hybridisation. Samples with the 5T variant were picked for DNA sequencing of intron 8/exon 9 region to identify the number of adjacent TG repeats. The p.Phe508del and the p.Ser1251Asn mutations were detected in heterozygous state in three patients (10.7% of alleles) and in one patient (3.6% of alleles), respectively, while the 5T variant was detected in five patients (28.6% of alleles). Among those five patients, four had TG12 repeats and one had TG13 repeats confirming the pathogenic penetrance of all 5T alleles in Egyptian CBAVD patients. The allelic frequencies of the mutations p.Phe508del, p.Ser1251Asn and the 5T variant in 60 Egyptian cystic fibrosis patients were 24.2%, 3.3% and 2.5% respectively. The mutation p.Ser1251Asn was detected for the first time in isolated CBAVD patient in our study. Due to the high prevalence of p.Phe508del mutation and 5T variant in Egyptian CBAVD patients, we recommend their screening initially, ideally followed by full CFTR gene sequencing in unidentified patients.

Optimizing sexual reproductive health of men and women with cystic fibrosis: A systematic review.

This systematic review summarizes the impact of cystic fibrosis (CF) on sexual and reproductive health (SRH) in males and females, covering pubertal development, hormonal function, family planning, and fertility. Included articles featured historical CF diagnostic criteria, preclinical or clinical data (retrospective cohorts or open label trials), while excluded articles lacked full text availability, explicit methodology, or comparisons between CF and non-CF patients. Genotype differences in CFTR mutations influenced symptom severity. Males with CF experienced delayed puberty, hypogonadism, infertility from obstructive azoospermia, and semen parameter issues. Female CF patients showed decreased fertility, possibly linked to disrupted ionic balance and ovarian cystic disease. Assistive reproductive technologies addressed fertility issues, but success varied based on disease severity and genotype. CFTR modulators aided pulmonary function and sexual health but require further assessment for fertility benefits.

Compound heterozygous mutations in CFTR causing congenital bilateral absence of the vas deferens in a Chinese pedigree.

BACKGROUND: Cystic fibrosis (CF) is an autosomal recessive disorder rarely found in Asian populations. Most males with CF are infertile because of obstructive azoospermia (OA) caused by congenital bilateral absence of the vas deferens (CBAVD). Compound heterozygous mutations of cystic fibrosis transmembrane conductance regulator (CFTR) are among the most common pathogenic factors in CBAVD. However, few genealogical analyses have been performed. METHODS: In this study, whole-exome sequencing and cosegregation analysis were performed in a Chinese pedigree involving two siblings with CBAVD. Moreover, in vitro gene expressions were used to analyze the pathogenicity of a novel CFTR mutation. RESULTS: We identified compound heterozygous mutations of CFTR comprising the known disease-causing variant c.1210-11T>G (also known as IVS9-5 T) and c.2144delA;p.q715fs in two siblings with CBAVD. To verify the effects in vitro, we transfected vectors expressing wild-type and mutated CFTR into 293T cells. The results showed that the CFTR protein containing the frameshift mutation (c.2144delA) was 60 kD smaller. With testicular sperm aspiration/intracytoplasmic sperm injection-embryo transfer (TESA/ICSI-ET), both CBAVD patients fathered healthy offspring. CONCLUSION: Our study revealed that compound heterozygous mutations of CFTR are involved in CBAVD, expanding the known CFTR gene mutation spectrum of CBAVD patients and providing more evidence that compound heterozygous mutations can cause familial CBAVD.

L138ins Variant of the CFTR Gene in Russian Infertile Men.

(1) Introduction: Pathogenic variants in the CFTR (Cystic Fibrosis Transmembrane conductance Regulator, OMIM: 602421) gene cause Cystic Fibrosis (CF, OMIM: 219700) and CF-related disorders (CF-RD), often accompanied by obstructive azoospermia due to congenital bilateral aplasia of vas deferens (CBAVD, OMIM: 277180) in male patients. The L138ins (c.413_415dup; p. (Leu138dup)) is a mild variant in the CFTR gene that is relatively common among CF-patients in Slavic populations. The frequency of this variant in Russian infertile men has not been sufficiently studied; (2) Materials and Methods: The sample consisted of 6033 Russian infertile men. The patients were tested for 22 common in Russian populations pathogenic variants of the CFTR gene and the IVS9Tn-polymorphic locus of the intron 9. Molecular-genetic studies were performed using amplified fragment length polymorphism (AFLP-PCR), multiplex ligation-dependent probe amplification (MLPA), and nested PCR (for analysis of the IVS9Tn-polymorphic locus); (3) Results: Pathogenic variants in the CFTR were detected in 3.9% of patients. The most frequent variants were F508del and CFTRdele2.3(21kb), accounted for 61.0% and 7.1% of detected variants, respectively. The L138ins variant was detected in 17 (0.28%) individuals: one of them was homozygous, 10 patients were heterozygous, and 6 patients were compound-heterozygous (F508del/L138ins, n = 4; L138ins/N1303K, n = 1; L138ins/5T, n = 1). Two pathogenic CF-causing variants in the CFTR gene were detected in 8 patients, including 7 compound heterozygous (F508del/L138ins, n = 4; F508del/N1303K, n = 1; 2184insA/E92K, n = 1; 3849+10kbC>T/E92K, n = 1) and one homozygous (L138ins/L138ins). The L138ins variant was found in 7 out of 16 (43.75%) chromosomes in six of these patients. The most common pathogenic variant, F508del, was identified in five out of them, in 5 of 16 (31.25%) chromosomes. The allele frequency (AF) of the L138ins variant in the sample has been found to be 0.0014.; (4) Conclusions: The L138ins variant of the CFTR gene is the third most common variant after F508del and CFTRdele2.3(kb) among Russian infertile men.

Clinical outcomes of a large cohort of individuals with the F508del/5T;TG12 CFTR genotype.

BACKGROUND: In recent years, patients with cystic fibrosis (CF) conductance regulator (CFTR) variant poly(T) sequences have been increasingly reported with a wide spectrum of clinical severity. We describe the long-term clinical outcomes and progression to a CF diagnosis over time in a large Italian cohort of patients carrying the CFTR F508del/5T;TG12 genotype. METHODS: A retrospective analysis of subjects from 10 CF centres in Italy with the F508del/5T;TG12 genotype was performed. Demographic, clinical, microbiological, and biochemical data, as well as information about the follow-ups and complications of the enroled patients, were collected. RESULTS: A total of 129 subjects (54 females; median age: 15.0 years, range: 0-58 years; 59 older than 18 years) were included. In terms of initial diagnoses, 30 were CF (23.3%), 41 were CFTR-related disorder (CFTR-RD) (31.7%), and 58 were CF transmembrane conductance regulator-related metabolic syndrome/cystic fibrosis screen positive, inconclusive diagnosis (CRMS/CFSPID) (45.0%). After a median follow-up of 6.7 years (range 0.2-25 years), 15 patients progressed to CF, bringing the total number of CF diagnoses to 45/129 (34.9%). Most of these patients had mild lung diseases with pancreatic sufficiency and a low prevalence of CF-related complications. CONCLUSIONS: At the end of the study, 34.9% of subjects with the CFTR F508del/5T;TG12 genotype were diagnosed with CF. We suggest including patients with the F508del/5T;TG12 genotype in long-term follow-ups.

Congenital Bilateral Absence of the Vas Deferens.

Congenital bilateral absence of the vas deferens (CBAVD) is clinically characterized by the absence of the bilateral vas deferens; the main clinical manifestation is infertility, accounting for 1-2% of male infertility cases. CBAVD may be accompanied by congenital abnormalities in the urogenital system and cystic fibrosis (CF)-related clinical manifestations. CBAVD can develop as a mild manifestation of CF or can be isolated. The main pathogenic mechanism of CBAVD is gene mutation, and CBAVD and CF have a common genetic mutation background. CFTR mutation is the main pathogenic cause of CBAVD and CF, and ADGRG2 mutation is the second most common cause. Although lack of the vas deferens in CBAVD patients causes infertility due to the inability to release sperm, the testes of CBAVD patients have spermatogenic function. Therefore, CBAVD patients can achieve fertility through sperm retrieval surgery and assisted reproductive technology (ART). However, gene mutations in CBAVD patients can have an impact on the ART outcome, and there is a risk of passing on gene mutations to offspring. For CBAVD patients and their spouses, performing genetic counseling (which currently refers mainly to CFTR mutation screening) helps to reduce the risks of genetic mutations being passed on to offspring and of offspring having CF with concomitant CBAVD.

The prevalence of common CFTR gene mutations and polymorphisms in infertile Iranian men with very severe oligozoospermia.

Due to progress in infertility etiology, several genetic bases of infertility are revealed today. This study aimed to investigate the distribution of mutations in the CFTR gene, M470V polymorphism, and IVS8 poly T. Furthermore, we aimed to examine the hotspot exons (4, 7, 9, 10, 11, 20, and 21 exons) to find a new mutation in cystic fibrosis transmembrane conductance regulator (CFTR) gene among infertile Iranian men very severe oligozoospermia (<1 million sperm/mL ejaculate fluid). In the present case-control study, 200 very severe oligozoospermia (20-60s) and 200 fertile men (18-65s) were registered. Five common CFTR mutations were genotyped using the ARMS-PCR technique. The M470V polymorphism was checked out by real-time PCR, and poly T and exons were sequenced. The F508del was the most common (4.5%) CFTR gene mutation; G542X and W1282X were detected with 1.5% and 1%, respectively. N1303K and R117H were detected in 0.5% of cases. F508del was seen as a heterozygous compound with G542X in one patient and with W1282X in the other patient. Also, in the case of M470V polymorphism, there are differences between the case and control groups (p=0.013). Poly T assay showed statistical differences in some genotypes. The study showed no new mutation in the exons mentioned above. Our results shed light on the genetic basis of men with very severe oligozoospermia in the Iranian population, which will support therapy decisions among infertile men.

Prevalence of CBAVD in azoospermic men carrying pathogenic CFTR mutations - Evaluated in a cohort of 639 non-vasectomized azoospermic men.

BACKGROUND: Men with obstructive azoospermia (OA) due to impaired development of the genital tract often carry at least one Cystic Fibrosis Transmembrane Conductance Regulator CFTR mutation. OBJECTIVE: To determine the frequency of Congenital Bilateral Absence of Vas deferens (CBAVD) in men with azoospermia carrying CFTR gene mutations. MATERIALS AND METHODS: Non-vasectomized men with azoospermia referred to our andrological center were consecutively included. All men underwent palpation of the scrotal parts of the Vasa deferentia, ultrasonography of the testicles and hormone profile, and genetic analyses. Testicular biopsy was usually performed. A panel of 32 of the most important CFTR mutations was examined from genomic DNA isolated from blood lymphocytes. Either multiplex PCR analysis or a next-generation sequencing technique was performed. RESULTS: Among the 639 men with azoospermia, 69 (10.8%) had at least one CFTR mutation. Of the 43 patients with at least one of the two CFTR mutations, ΔF508 and R117H, 19 (44.2%) showed CBAVD, 2 (4.7%) Congenital Unilateral Absence of Vas deferens (CUAVD), and 22 (51.2%) presence of the scrotal parts of the Vasa deferentia. In contrast, only 1/21 men (4.8%) with an isolated IVS8-5T variant showed CBAVD. Among the further 20 men with an isolated IVS8-5T variant, 11 had a history of cryptorchidism. Among the 570 men without CFTR mutations, CBAVD was found in only two men and CUAVD in one. FSH level was higher and testicular volume lower in men with present Vasa deferentia compared to those without (P < .001; Student's t test). Thirty-one men with either ΔF508 or R117H mutations, or both, had a testicular biopsy. Motile spermatozoa were found in 100% of 16 cases with CBAVD but in only 6 out of 15 cases with present Vasa deferentia (P < .01; Fisher's exact test). DISCUSSION AND CONCLUSIONS: CBAVD was found in ~ 44% of men with ΔF508/R117H mutations. The data may support that CFTR mutations might affect male fertility through other mechanisms than obstruction of the genital tract. For a practical, clinical purpose analysis for only ΔF508, R117H and IVS8-5T seems sufficient until further research shows anything else.

How do we counsel men with obstructive azoospermia due to CF mutations?-a review of treatment options and outcomes.

Obstructive azoospermia (OA) is a rare cause of male infertility, with Congenital Bilateral Absence of The Vas Deferens (CBAVD) being a major cause. A wealth of literature has established an irrefutable link between CFTR mutations and CBAVD, with CBAVD affecting almost all men with cystic fibrosis (CF) disease and a significant portion of men that are CFTR mutation carriers. In the past two decades, assisted reproductive technologies have made the prospect of fathering children a viable possibility in this subset of men, using a combination of sperm extraction techniques and intracystoplasmic sperm injection (ICSI). In order to assess techniques for sperm retrieval, as well as reproductive outcomes, a systemic search of the MEDLINE database was conducted for all articles pertaining to management options for CBAVD, and also all reports describing outcomes of these procedures in the CBAVD population. Both epididymal and testicular sperm extraction (TESE) are viable options for men with CBAVD, and though rigorous data are lacking, live birth rates range from 8% to 50% in most small retrospective series and subset analyses. In addition, there does not appear to be significant differences in the rate of live birth or complications and miscarriages between the various techniques, though further investigation into other factors that limit reproductive potential of men with CFTR mutations and CBAVD is warranted.

Congenital unilateral absence of the vas deferens with ipsilateral renal agenesis encountered during laparoscopic totally extraperitoneal inguinal hernia repair in an adult patient: A case report.

INTRODUCTION AND IMPORTANCE: Congenital unilateral absence of the vas deferens and ipsilateral renal agenesis is a rare condition in which the vas deferens is absent unilateral due to a congenital anomaly. This report illustrates the clinical relevance of absence of the vasa deferentia. This occurs either unilateral, which is associated with renal anomalies, or bilateral which leads to azoospermia and often has a genetic link to cystic fibrosis. CASE PRESENTATION: We report the first incidental finding of congenital unilateral absence of the vas deferens and ipsilateral renal agenesis during bilateral laparoscopic totally extraperitoneal inguinal hernia repair in a 70 year old patient. CONCLUSION: Surgeons who encounter unilateral absence of the vas deferens should assess bilateral absence of the vasa deferentia by palpation in adult patients or by laparoscopic exploration of the contralateral inguinal canal in pediatric patients. Renal ultrasonography should be performed in all patients to assess renal anomalies. Genetic counselling is advised when absence of the vasa deferentia is present bilaterally of unilaterally with associated infertility.

Extensive CFTR Gene Analysis Revealed a Higher Occurrence of Cystic Fibrosis Transmembrane Regulator-Related Disorders (CFTR-RD) among CF Carriers.

Background: A wide range of cystic fibrosis (CF)-related conditions are reported in CF carriers, but no study has explored the possibility that such subjects may be affected by cystic fibrosis transmembrane regulator-related disorders (CFTR-RD). No data are available so far on the occurrence of CFTR-RD among CF carriers. Methods: We studied 706 CF carriers-first- and second-degree relatives of CF patients that carried the parental mutation; such subjects were divided in two groups: a first group (353 subjects, group A) performed at first only the analysis of the CFTR proband mutation; we retrospectively evaluated the number of cases that had been diagnosed as CFTR-RD based on subsequent symptoms; a second group (353 subjects, group B) performed extensive CFTR molecular analysis in absence of any reported symptoms, followed by a clinical evaluation in cases that carry a second CFTR mutation; we evaluated the number of cases that prospectively were diagnosed as CFTR-RD. Results: We found seven (2.0%) out of 353 subjects of group A and 24 (6.8%) out of 353 subjects of group B as affected by CFTR-RD (chi square, p = 0.002). Conclusions: A percentage of CF carriers are affected by undiagnosed CFTR-RD. Genetic tasting scanning analysis helps to identify CFTR-RD, some of which may benefit from follow-up and specific therapies improving their outcome.