The aim of this study was to investigate the frequency of CFTR mutations among CBAVD patients in South China and the impact of CFTR mutations on the outcomes of TESA-ICSI. By conducting whole-exome sequencing on 42 CBAVD patients, we found that 30 cases (71.4%) carried at least one CFTR gene variation, indicating a significant increase in the proportion of CFTR mutations among CBAVD patients. Moreover, comparing 30 CBAVD patients with CFTR gene mutations and 112 non-CBAVD obstructive azoospermia patients undergoing ICSI with testicular sperm extraction, we observed that although the 2PN rate was significantly higher in non-CBAVD obstructive azoospermia, there were no significant differences in fertilization rates, pregnancy rates, miscarriage rates, and live birth rates. Furthermore, by comparing the outcomes of ICSI with testicular sperm extraction between 30 CBAVD patients with CFTR mutations and 11 CBAVD patients without CFTR mutations, we found no significant differences in fertilization rate, 2PN rate, number of usable embryos, pregnancy rate, miscarriage rate, and live birth rate.
Research both domestically and internationally shows that the CFTR mutation spectrum varies significantly among different races and populations, with over 2,000 mutations reported to date [17, 21, 22]. The main types of mutations include codon deletions, splice mutations, missense mutations, non-coding region mutations, frameshift mutations, and nonsense mutations. Among these, the F508del codon mutation is the most common type in Caucasians, showing significant racial differences, with a mutation frequency of up to 70% in Caucasians but is rarely reported in China [8, 23], possibly due to it being a severe mutation with a high occurrence rate in CF, while the incidence rate of CF in China is quite low [17, 24]. Our study did not find any patients with the F508del mutation, nor did we identify any cases of CFTR codon deletion. Splice mutations are most commonly found at the junction of intron 9 and exon 10 with TG repeats and polyT mutations. The study found that the proportion of polyT polymorphisms varies greatly among different countries and populations, currently reported to be between 13% and 43.7%. The 5 T mutation is the most common mutation type among Chinese CBAVD patients [24], with a frequency ranging from 29.35% to 55.26% in Chinese patients [2, 15, 17, 25]. Our study found a 5 T mutation frequency of 50% (18/36). Additionally, research has found that the combination of the 5 T mutation with adjacent TG repeat sequence changes can affect the pathogenicity of 5 T, with shorter poly T and more TG repeats increasing the probability of CFTR gene exon 10 skipping deletion, which is involved in encoding 60 amino acids of the CFTR protein’s NBD1. The absence of these amino acids would result in the loss of Cl- channel function, ultimately leading to CF and CF-related diseases [26]. Our study identified four cases of CFTR gene c.1210-12 T [5]/c.1210-34TG mutation, accounting for 11.11% (4/36). Missense mutations are most frequently found in exons 4, 7, 11, 17, and 20, and aside from F508del, are the most common and numerous mutation type in Caucasians, with R117H being the most common. However, our cases did not reveal any mutations at this site. Current research indicates that the most common mutations among Chinese CBAVD patients are 1556 V, G970A, and Q1352H [2, 27]. Our cases did not find the 1556 V mutation, which may be related to our small sample size or regional differences within China. We identified three cases each carrying the Q1352H mutation and the G970A mutation, suggesting they might be the most common missense variants among Chinese CBAVD patients.
ADGRG2 is considered the second most common mutation gene leading to CBAVD, located at Xp22.13 with 29 exons, producing 10 transcripts, with the longest transcript being 3.1 kb, encoding the adhesion G protein-coupled receptor G2. It is primarily expressed at the apical membrane of the non-ciliated epithelial cells in the human vas deferens [28]. Research has found that some CBAVD patients negative for CFTR mutations carry ADGRG2 mutations, which are considered related to the occurrence of CBAVD [10, 27,28,29]. Studies by Zhang et al. found high expression of ADGRG2 in the proximal epididymis and vas deferens [30]. Additionally, studies have shown that in cases of ADGRG2 mutations, proximal epididymal tissue lacks ADGRG2 protein expression, further indicating that the loss of ADGRG2 protein function due to ADGRG2 mutations is closely related to the occurrence of CBAVD [31]. In our study, we also identified a novel ADGRG2 p.Arg158His mutation among 42 CBAVD patients.
This study conducted WES on 42 Chinese CBAVD patients and found that CBAVD is primarily caused by CFTR mutations and is also associated with ADGRG2 mutations. These results are highly consistent with other WES studies on Chinese CBAVD patients [14, 15, 17]. Therefore, targeted gene testing for CFTR and ADGRG2 is more suitable for CBAVD patients compared to WES.Considering that approximately 30% of CBAVD patients have unknown genetic factors, and WES cannot cover the entire genome, whereas massive parallel sequencing, particularly whole-genome sequencing (WGS), can cover the entire genome. This allows it to detect more potential pathogenic variants, including those in regions not covered by WES. it is recommended to Massive parallel sequencing for patients whose initial screening does not identify the cause. This approach not only helps discover new pathogenic genes and mutations but also provides a more comprehensive basis for genetic counseling and personalized treatment.
CFTR mutations are the most common cause of congenital obstructive azoospermia (OA) in patients. In addition to this, current research suggests that CFTR mutations may also lead to decreased sperm vitality and fertilizing capacity, as well as reduced spermatogenic function. However, there is no clear consensus on whether CFTR mutations affect the outcomes of intracytoplasmic sperm injection (ICSI). Studies have shown that patients with cystic fibrosis (CF) have lower sperm vitality and fewer sperm compared to patients with only the CBAVD phenotype, and ICSI outcomes indicate a significantly reduced fertilization rate for CF patients [12]. Research by Lu et al. suggests that CBAVD patients have a higher miscarriage rate and significantly lower live birth rate compared to non-CBAVD patients, with similar results observed between the CFTR mutation carrier group and the non-CFTR mutation group [13]. Meanwhile, other studies report no significant differences in fertilization rates, pregnancy rates, and live birth rates between CBAVD patients carrying CFTR mutations and those not carrying mutations [13]. Wang et al.’s research shows that OA patients carrying two CFTR mutations, compared to other OA patients undergoing ICSI, found no statistical difference in either laboratory or clinical outcomes [15]. Our study first compared the ICSI outcomes of CBAVD patients with CFTR mutations to those of non-CBAVD OA patients. Although non-CBAVD OA patients may also carry CFTR mutations, literature reports a lower carrier frequency of CFTR mutations in the Chinese population [18]. This grouping can, to some extent, reveal whether CFTR mutations affect ICSI outcomes. Ultimately, our study confirmed that there were no significant differences between the two groups in terms of fertilization rate, number of usable embryos, pregnancy rate, miscarriage rate, and live birth rate.Meanwhile, to further illustrate the impact of the CFTR gene on ICSI outcomes, we compared the ICSI outcomes of 30 CBAVD patients with CFTR mutations to those of 11 CBAVD patients without CFTR mutations. We basically obtained the same conclusions. Therefore, Our study indicates that TESA-ICSI is a reliable method for achieving parenthood in both CBAVD patients and non-CBAVD OA patients, and CFTR mutations do not affect the final clinical outcomes of ICSI.
Our study has certain limitations; firstly, it is a single-center retrospective study, and the conclusions may be influenced by the small sample size and regional characteristics. Secondly, we did not screen non-CBAVD OA patients for CFTR mutations. Although the carriage rate of CFTR mutations is low in the Chinese population, this could still affect our final conclusions. Therefore, there is an urgent need for larger-scale, multicenter studies.
In summary, the genetic heterogeneity of Chinese CBAVD patients differs from the hotspot mutation pattern of Caucasians.We recommend targeted gene testing for the CFTR and ADGRG2 genes in CBAVD patients. For patients who test negative for CFTR and ADGRG2, we suggest conducting massive parallel sequencing. Based on the test results of the patients and their partners, consideration should then be given to whether to proceed with ICSI or PGS.. Additionally, although our study indicates that CFTR mutations do not affect the final ICSI clinical outcomes, considering that no pathogenic variants were found in some CBAVD patients, there are other unknown genetic factors that require further research. Genetic counseling for CBAVD patients before ART treatment is advised.
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