Cystic fibrosis gene mutations and infertile men with primary testicular failure.

It has been proposed that the gene responsible for cystic fibrosis, called the cystic fibrosis transmembrane conductance regulator (CFTR) gene, may play an important role in the process of spermatogenesis. A group of azoospermic men with primary testicular failure underwent CFTR mutation analysis, including assessment of the intron 8 polythymidine tract (IVS8-T tract). An association was not found between CFTR mutations or the 5T variant of the IVS8-T tract and the primary testicular failure phenotype. This finding suggests that CFTR does not play a significant role in the aetiopathogenesis of primary spermatogenic dysfunction. Therefore, the abnormal testicular histological findings in some post-pubertal men with cystic fibrosis may be a result of nutritional deficiency or testicular obstruction rather than a primary defect in spermatogenesis. In addition, the decreased sperm count in oligozoospermic men with CFTR mutations may be secondary to partial reproductive tract obstruction and not abnormal spermatogenesis. Lastly, routine screening of men with primary testicular failure for CFTR gene mutations is not warranted.

Proportion of cystic fibrosis gene mutations not detected by routine testing in men with obstructive azoospermia.

CONTEXT: Infertile men with obstructive azoospermia may have mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, many of which are rare in classic cystic fibrosis and not evaluated in most routine mutation screening. OBJECTIVE: To assess how often CFTR mutations or sequence alterations undetected by routine screening are detected with more extensive screening in obstructive azoospermia. DESIGN: Routine screening for the 31 most common CFTR mutations associated with the CF phenotype in white populations, testing for the 5-thymidine variant of the polythymidine tract of intron 8 (IVS8-5T) by allele-specific oligonucleotide hybridization, and screening of all exons through multiplex heteroduplex shift analysis followed by direct DNA sequencing. SETTING: Male infertility clinic of a Canadian university-affiliated hospital. SUBJECTS: Of 198 men with obstructive (n = 149) or nonobstructive (n = 49; control group) azoospermia, 64 had congenital bilateral absence of the vas deferens (CBAVD), 10 had congenital unilateral absence of the vas deferens (CUAVD), and 75 had epididymal obstruction (56/75 were idiopathic). MAIN OUTCOME MEASURE: Frequency of mutations found by routine and nonroutine tests in men with obstructive vs nonobstructive azoospermia. RESULTS: Frequency of mutations and the IVS8-5T variant in the nonobstructive azoospermia group (controls) (2% and 5.1% allele frequency, respectively) did not differ significantly from that in the general population (2% and 5.2%, respectively). In the CBAVD group, 72 mutations were found by DNA sequencing and IVS8-5T testing (47 and 25, respectively; P<.001 and P = .002 vs controls) vs 39 by the routine panel (P<.001 vs controls). In the idiopathic epididymal obstruction group, 24 mutations were found by DNA sequencing and IVS8-5T testing (12 each; P=.01 and P=.14 vs controls) vs 5 by the routine panel (P=.33 vs controls). In the CUAVD group, 2 mutations were found by routine testing (P=.07 vs controls) vs 4 (2 each, respectively; P=.07 and P=.40 vs controls) by DNA sequencing and IVS8-5T testing. The routine panel did not identify 33 (46%) of 72, 2 (50%) of 4, and 19 (79%) of 24 detectable CFTR mutations and IVS8-5T in the CBAVD, CUAVD, and idiopathic epididymal obstruction groups, respectively. CONCLUSIONS: Routine testing for CFTR mutations may miss mild or rare gene alterations. The barrier to conception for men with obstructive infertility has been overcome by assisted reproductive technologies, thus raising the concern of iatrogenically transmitting pathogenic CFTR mutations to the progeny.

Fresh and frozen epididymal sperm yield comparable pregnancy rates for intracytoplasmic sperm injection.

To determine if the use of fresh epididymal sperm is superior to frozen-thawed epididymal sperm for intracytoplasmic sperm injection, the authors reviewed the charts on all couples undergoing intracytoplasmic sperm injection at an academic center, using microsurgically aspirated epididymal sperm. Forty-nine couples undergoing intracytoplasmic sperm injection for male factor infertility, due to congenital absence of vas deferens or irreparable post-testicular obstruction were studied. The following parameters were measured: (1) fertilization rate per oocyte injected (two pronuclei at 24 h), (2) chemical pregnancy rate (two consecutively elevated serum b-hCG levels, and (3) clinical pregnancy rate (sonographic identification of fetal heart rate). Fertilization rates were 51 and 41%, chemical pregnancy rates were 27 and 30%, and clinical pregnancy rates were 19 and 27% in the fresh epididymal compared to the frozen epididymal sperm. This study shows no significant difference in outcomes using fresh or frozen epididymal sperm for intracytoplasmic sperm injection. Frozen-thawed sperm guarantees availability of sperm prior to oocyte retrieval.

Detection of eubacteria in interstitial cystitis by 16S rDNA amplification.

OBJECTIVE: To determine what role non-culturable microorganisms play in the etiology of interstitial cystitis (IC). MATERIALS AND METHODS: Thirty patients fulfilling NIH criteria for the diagnosis of interstitial cystitis and sixteen control patients with culture negative urine gave written informed consent and underwent bladder biopsy. Polymerase chain reaction (PCR) using two sets of universal primers for bacterial 16S rDNA was performed on urine from the cystoscope and on a cold cup bladder biopsy specimen. Of the PCR positive bladder biopsies, three patients with interstitial cystitis and three controls were randomly selected and cloned. Ten clones from each were sequenced and putative taxonomic assignments made. RESULTS: 12/26 (46%) IC and 5/12 (42%) control urine specimens and 16/30 (53%) and 9/15 (60%) bladder biopsies were PCR positive, respectively. The bacterial populations in the two patient groups tested appeared to be different based upon analysis of the 16S rRNA sequences. CONCLUSIONS: Both IC and control patients had non-culturable bacteria in their bladders. A random sampling of the two populations revealed that the bacterial populations are different, suggesting a possible link between one or more bacterial species and IC.

Higher proportion of intact exon 9 CFTR mRNA in nasal epithelium compared with vas deferens.

The 5-thymidine (5T) variant of the cystic fibrosis transmembrane conductance regulator (CFTR) intron 8 polypyrimidine tract (IVS8-T tract) is the most frequent CFTR gene alteration identified in men with congenital bilateral absence of vas deferens (CBAVD). This alternative splicing variant gives rise to two transcripts, one normal with exon 9 intact and the other with in-frame deletion of exon 9. That CBAVD men usually have none of the other clinical signs of classical cystic fibrosis (CF) suggests less functional CFTR is produced in the reproductive tract than in other CF-associated organs. Nasal epithelia and segments of vas deferens were obtained from healthy, previously vasectomized men who presented for vasectomy reversal. Quantitative RT-PCR was performed on these specimens, with the region of CFTR cDNA spanning exon 9 amplified. For both nasal and vasal tissues, a strong positive correlation was found between the length of the IVS8-T tract and the proportion of mRNA with exon 9 intact. In addition, within the same subject, a significantly higher level of transcripts lacking exon 9 was found in vas deferens than nasal epithelia, regardless of the IVS8-T genotype. These findings suggest that the splicing of CFTR precursor mRNA is less efficient in vasal epithelia compared with respiratory epithelia. Thus, differential splicing efficiency between the various tissues which express CFTR provides one possible explanation for the reproductive tract abnormalities observed in infertile men with CFTR gene alterations but without other clinical manifestations of CF.

The genetics of male infertility.

PURPOSE: We provide an up-to-date summary of the genetic aspects of male infertility. MATERIALS AND METHODS: The literature on male infertility was extensively reviewed. RESULTS: Genetic defects are associated with a variety of clinical presentations by the infertile man ranging from gonadotropin-releasing hormone deficiency to spermatogenic failure to obstructive azoospermia. Microsurgery and micromanipulation of gametes make it possible for many of these men to father children. However, with each successive breakthrough in treatment of male infertility there is an increased risk of transmitting genetic abnormalities to the progeny. CONCLUSIONS: Transmission of genetic defects through assisted reproductive techniques can have serious long-term implications. Assisted reproductive techniques should not be initiated in men with a possible or known genetic cause of infertility without prior genetic counseling and risk assessment. Clinicians and researchers involved in reproductive medicine must recognize that, although these techniques have revolutionalized the treatment of male infertility, they have the risk of passing genetic abnormalities to the progeny. Therefore, researchers must proceed cautiously with development and application of assisted reproductive technologies to avoid creating future generations of genetically abnormal individuals. The first step in accomplishing this goal is through an increased understanding of the genetic basis of male reproductive failure.

The hypo-osmotic swelling test for selection of viable sperm for intracytoplasmic sperm injection in men with complete asthenozoospermia.

OBJECTIVE: To determine the ability of the hypo-osmotic swelling test to select viable sperm from nonmotile sperm samples for intracytoplasmic sperm injection (ICSI). DESIGN: Nonrandomized, sequential comparative study. PATIENTS: Thirteen couples enrolled in our ICSI program had 16 cycles in which sperm preparations with 0% motility were obtained. Five cycles used cryopreserved epididymal sperm with complete asthenozoospermia. INTERVENTIONS: In eight cycles, the semen samples were washed through a Percoll gradient and sperm were selected randomly for ICSI. In another eight cycles, the washed sperm were placed in a hypo-osmotic solution (75 mM fructose; 25 mM sodium citrate dihydrate) and the sperm with curled tails taken up with the microinjection needle, rinsed, and used ICSI. MAIN OUTCOME MEASURES: Fertilization rate per oocyte injected as determined by the presence of two pronuclei at 18 hours after retrieval and embryo cleavage rate per oocyte injected at 48 hours after retrieval. RESULTS: With random sperm injection, the fertilization and cleavage rates were 26% and 23%, respectively. In contrast, after injection of sperm selected using the hypo-osmotic swelling test, fertilization and cleavage rates were significantly greater (43% and 39%, respectively). There were three pregnancies in the eight cycles with the hypo-osmotic swelling test-selected sperm, including two from frozen epididymal sperm. CONCLUSION: Based on these preliminary observations, we believe that the hypo-osmotic swelling test will prove to be valuable for increasing fertilization and cleavage rates and pregnancy rates in ICSI cycles where no motile sperm are recovered.