Multidisciplinary consensus on the criteria for fertility preservation in cancer patients

Infertility is one of the main sequelae of cancer and its treatment in both children and adults of reproductive age. It is, therefore, essential that oncologists and haematologists provide adequate information about the risk of infertility and the possibilities for its preservation before starting treatment. Although many international clinical guidelines address this issue, this document is the first Spanish multidisciplinary guideline in paediatric and adult oncological patients. Experts from the Spanish Society of Medical Oncology, the Spanish Fertility Society, the Spanish Society of Haematology and Haemotherapy, the Spanish Society of Paediatric Haematology and Oncology and the Spanish Society of Radiation Oncology have collaborated to develop a multidisciplinary consensus.

Multidisciplinary consensus on the criteria for fertility preservation in cancer patients.

Infertility is one of the main sequelae of cancer and its treatment in both children and adults of reproductive age. It is, therefore, essential that oncologists and haematologists provide adequate information about the risk of infertility and the possibilities for its preservation before starting treatment. Although many international clinical guidelines address this issue, this document is the first Spanish multidisciplinary guideline in paediatric and adult oncological patients. Experts from the Spanish Society of Medical Oncology, the Spanish Fertility Society, the Spanish Society of Haematology and Haemotherapy, the Spanish Society of Paediatric Haematology and Oncology and the Spanish Society of Radiation Oncology have collaborated to develop a multidisciplinary consensus.

Comprehensive investigation in patients affected by sperm macrocephaly and globozoospermia.

The aim of this study was to provide a comprehensive genetic/phenotypic characterization of subjects suffering infertility owing to sperm macrocephaly (n = 3) or globozoospermia (n = 9) and to investigate whether the patients' genetic status was correlated with the alteration of various sperm parameters. AURKC was sequenced in case of sperm macrocephaly while the DPY19L2 status has been analyzed by multiple approaches including a novel qPCR-based copy number assay in case of globozoospermia. Globozoospermic patients were also analyzed for SPACA1, a novel candidate gene herein tested for the first time in humans. The effect of the patients' genetic status was interrogated by implementing the molecular screening with the characterization of several sperm parameters: (i) routine sperm analysis, integrated with transmission electron microscopy; (ii) sperm fluorescent in situ hybridization (FISH) analysis; (iii) sperm DNA fragmentation (DF) analysis. Moreover, for the first time, we performed microsatellite instability analysis as a marker of genome instability in men with sperm macrocephaly and globozoospermia. Finally, artificial reproductive technology (ART) history has been reported for those patients who underwent the treatment. Macrocephalic patients had an AURKC mutation and >89% tetraploid, highly fragmented spermatozoa. DPY19L2 was mutated in all patients with >80% globozoospermia: the two homozygous deleted men and the compound heterozygous showed the severest phenotype (90-100%). The newly developed qPCR method was fully validated and has the potential of detecting also yet undiscovered deletions. DPY19L2 status is unlikely related to FISH anomalies and DF, although globozoospermic men showed a higher disomy rate and DF compared with internal reference values. No patient was mutated for SPACA1. Our data support the general agreement on the negative correlation between macro/globozoospermia and conventional intracytoplasmic sperm injection outcomes. Microsatellites were stable in all patients analyzed. The comprehensive picture provided on these severe phenotypes causing infertility is of relevance in the management of patients undergoing ART.

Altered gene expression signature of early stages of the germ line supports the pre-meiotic origin of human spermatogenic failure.

The molecular basis of spermatogenic failure (SpF) is still largely unknown. Accumulating evidence suggests that a series of specific events such as meiosis, are determined at the early stage of spermatogenesis. This study aims to assess the expression profile of pre-meiotic genes of infertile testicular biopsies that might help to define the molecular phenotype associated with human deficiency of sperm production. An accurate quantification of testicular mRNA levels of genes expressed in spermatogonia was carried out by RT-qPCR in individuals showing SpF owing to germ cell maturation defects, Sertoli cell-only syndrome or conserved spermatogenesis. In addition, the gene expression profile of SpF was compared with that of testicular tumour, which is considered to be a severe developmental disease of germ cell differentiation. Protein expression from selected genes was evaluated by immunohistochemistry. Our results indicate that SpF is accompanied by differences in expression of certain genes associated with spermatogonia in the absence of any apparent morphological and/or numerical change in this specific cell type. In SpF testicular samples, we observed down-regulation of genes involved in cell cycle (CCNE1 and POLD1), transcription and post-transcription regulation (DAZL, RBM15 and DICER1), protein degradation (FBXO32 and TM9SF2) and homologous recombination in meiosis (MRE11A and RAD50) which suggests that the expression of these genes is critical for a proper germ cell development. Interestingly, a decrease in the CCNE1, DAZL, RBM15 and STRA8 cellular transcript levels was also observed, suggesting that the gene expression capacity of spermatogonia is altered in SpF contributing to an unsuccessful sperm production. Altogether, these data point to the spermatogenic derangement being already determined at, or arising in, the initial stages of the germ line.

Chromosome 16 abnormalities in embryos and in sperm from a male with a fragile site at 16q22.1.

Two fragile sites, FRA16B and FRA16C, are located in the chromosome band 16q22.1. Neither of them is associated with any specific clinical condition. We report the development and outcome of a clinically applied PGD cycle in a couple who had difficulty in achieving pregnancy. The woman was a carrier of a balanced reciprocal translocation, t(11;22)(q23;q11.2), and the man presented high expression of the fragile site 16q22.1 (FRA16B/C) in peripheral blood lymphocytes. Gains and losses of chromosome 16 fragments were detected in sperm and embryos. To our knowledge, this is the first documented case suggesting a link between FRA16B/C and chromosome 16 abnormalities in embryos and sperm from a carrier.

Hidden mosaicism in patients with Klinefelter's syndrome: implications for genetic reproductive counselling.

BACKGROUND: Most individuals with Klinefelter's syndrome (KS) are azoospermic but residual foci of spermatogenesis have been observed in some patients. However, no consistent predictive factors for testicular sperm extraction success have been established and mosaicism could be a factor to investigate. In this study, we have assessed the degree of mosaicism in somatic and germinal tissues in KS, the meiotic competence of 47,XXY germ cells and the aneuploidy rate of post-reductional cells. METHODS: Five patients with KS previously diagnosed as pure 47,XXY have been studied. Samples from four donors were processed as controls. The chromosome constitution of lymphocytes, buccal mucosa and testicular tissue was assessed by interphase fluorescence in situ hybridization for chromosomes X, Y and 18. In meiotic figures, sex chromosome number and pairing was confirmed. RESULTS: 46,XY cell lines were observed in all patients and tissues analysed. The degree of mosaicism (mean ± SD) differed among tissues (lowest in lymphocytes: 4.8 ± 2.5%; highest in Sertoli cells: 42.3 ± 11.1%). Meiotic figures were found in three cases (KS1, KS2 and KS5), all of them showed an XY complement. Hyperhaploid post-reductional cells were found in all patients (range: 3.3-36.4%) and increased rates versus controls (P< 0.05) were observed. CONCLUSIONS: Diagnosis of homogeneous KS based on lymphocyte karyotyping should be contrasted in other tissues. Mucosa cells could help to better approximate the degree of germ cell mosaicism. Our results indicate that 47,XXY germ cells are not meiotically competent. Increased post-reductional aneuploidy rate is related to meiotic errors in 46,XY cells. Appropriate genetic counselling is recommended in KS.

El banco de semen y la supervivencia espermática en un programa de reproducción asistida / The bank of semen and sperm survival in an assisted reproduction program

Objetivo: Analizar el efecto de las condiciones de almacenamiento de semen congelado sobre la fragmentación del ADN espermático, los parámetros seminales pre y post congelación, la recuperación espermática tras selección-capacitación de los donantes de semen (DS) utilizados en inseminación intrauterina y los resultados de gestación, teniendo en cuenta los efectos del tiempo de congelación y la ubicación de los viales en el contenedor criogénico. Material y Métodos: Se valoraron muestras de 17 (DS) criopreservadas entre el 2003-2008 y que fueron utilizadas en 77 procesos de inseminación intrauterina. El estudio de la fragmentación espermática se realizó mediante el método SCD (Sperm Cromatin Dispersion) (Halosperm ®, Indas). Se analizó la pérdida relativa de la motilidad progresiva post descongelación y post selección espermática (centrifugación de gradientes).También se evaluó la tasa de gestación/ciclo. Todos los parámetros se analizaron teniendo en cuenta el tiempo de almacenamiento al descongelar (≥ 3 o < 3 años) y la ubicación relativa de almacenamiento dentro del contenedor (1=más superficial, 11=más profundo). Resultados: El IFS (Indice de Fragmentación Espermático) fue en los 17 DS estudiados de 14,47±1,28. Cuando analizamos el IFS según el tiempo de almacenamiento ≥ 3 o < 3 años los resultados fueron similares (13,50±2,63 versus 15,85±2,66%) p < 0,39. Cuando el tiempo de almacenamiento fue superior a 3 años la motilidad progresiva (a+b) post descongelación fue menor que los almacenados menos de 3 años (3,7± 2,6 versus 41,9±2,4%) p<0,05. El mismo efecto se observó post selección espermática (36,9±2,9 versus 46,8±2,9%) p < 0,019. La tasa global de gestación/ciclo fue de 11,16%. No se observó diferencia significativa del IFS entre el nivel de más superficial a más profundo del contenedor criogénico (15,89±2,33 versus 12,88±0,64%) p < 0,25 ni tampoco de los parámetros seminales de motilidad progresiva (33,19±3,97 versus 38,33±4,72%) p < 0,40. Conclusiones: El índice de fragmentación espermático (IFS) no se modificó con el tiempo de criopreservación ni por el nivel de ubicación de las muestras en el contenedor criogénico. La motilidad progresiva (a+b) post descongelación y post selección espermática empeoró con el tiempo de almacenamiento pero no se vio influida por el nivel de almacenamiento (AU)

Effect of the conditions of storage of semen frozen on the fragmentation of the DNA and the pre and post freezing seminal parameters, as well as spermatic selection by density gradient semen donors (SD) used for intrauterine insemination; also to analyse pregnancy outcomes, and the effects of freezing time and vial location inside the cryogenic container. Material and Methods: Samples cryopreserved between 2003 and 2008 from 17 SD and used in 70 intrauterine insemination, were evaluated. The sperm DNA fragmentation was carried out of the SCD (Sperm Cromatin Dispersion) method ( Halosperm® Indas). Post-thaw and postsperm selection relative loss of progressive mobility was analysed (gradient centrifugation). Also the pregnancy/cycle rate was assessed. All parameters were analysed considering the storage time at thawing (≥ 3 or < 3 years), and their relative location within the container (1 = most superficial, 11 = deepest). Results: The time of criopreservation was 978±79 days (media±EEM). The IFS was 14.47±1.28 in 17 studied DS. When we analyze the IFS according to the time of storage (≥ 3 or <3) years the results were similar (13.50±2.63 versus 15.85±2.66 %) p < 0.39. When the time of storage was superior to 3 years the progressive motility (a+b) post thawed was minor, that the stored less than 3 years (34.7 ± 2.6 versus 41.9±2.4 %) p < 0.05. The same effect observed post spermatic selection (36.9±2.9 versus 46.8±2.9 %) p< 0.019. The global rate of gestation / cycle was 11.16 %. Was not observed significant difference of the IFS between the level of more superficial to deeper of the cryogenic container (15.89±2.33 versus 12.88±0.64 %) p <0.25 not neither of the seminal parameters of progressive motility (33.19±3.97 versus 38.33±4.72 %) p < 0.40. Conclusions: The spermatic index of fragmentation (IFS) they were not modified by the time of cryopreservation not by the level of location of the samples in the cryogenic container. The progressive motility (a+b) post thawing and post spermatic selection deteriorated with the time of storage but not see influenced by the level of storage (AU)

Further insights into the role of T222P variant of RXFP2 in non-syndromic cryptorchidism in two Mediterranean populations.

The aetiopathogenesis of isolated cryptorchidism remains largely unknown. Mutation screenings in the most relevant candidate genes for testicular maldescent lead to controversial data in the literature. In particular, the role of the T222P genetic variant of the RXFP2 gene is still debated. Given the controversies, the aim of this study was to provide further data on this genetic variant in two Mediterranean populations. A total of 577 subjects from Spain and 550 from Italy (with and without a history of cryptorchidism) were analysed. The T222P substitution was found in both unilateral and bilateral cases and in a total of 12 controls. These data exclude a clear-cut cause-effect relationship between T222P variant and testicular maldescent. The T222P variant was found at a similar frequency in both cases and controls in the Spanish population, whereas in Italy, the frequency of T222P resulted significantly higher in the cryptorchid group (p = 0.031). The observed difference between the two countries and the highly variable phenotypic expression of the T222P variant may depend on the genetic background or on environmental conditions. The haplotype analysis of the RXFP2 gene in T222P carriers and their parents showed that this variant is linked to the previously inferred C-C-G-A-13 haplotype and consequently provides further support to the 'founder effect' hypothesis. In conclusion, our data indicate that T222P is a frequent variant in the Spanish population with no pathogenic effect. Although in Italy it seems to confer a mild risk (odds ratio = 3.17, 95% confidence interval: 1.07-9.34) to cryptorchidism, the screening for this variant for diagnostic purposes is not advised because of the relatively high frequency of control carriers (1.4% of Italian men without a history of cryptorchidism).

Cancer testicular y criopreservación de semen / Testicular cancer and criopreservation of semen

A pesar de los numerosos estudios que demuestran una disminución en la calidad seminal de los pacientes con cáncer testicular todavía no se conocen todos los mecanismos exactos que puedan explicar su etiopatogenia: defectos preexistentes en la espermiogénesis, efectos sistémicos del cáncer, factores endocrinos y autoinmunes. La criopreservación de espermatozoides se debe proponer a todo paciente en edad fértil y con deseo genésico potencial, antes de comenzar los tratamientos oncológicos

The causes of poor seminal quality in testicular cancer patients are not web understood: múltiple factors are likely involved, including preexisting defects in germ cells, systemic effects of cancer, and endocrine and inmunological disturbances. The cryopreservation of spermatozoa is a simple and practical approach available to all patients with cancer who wish to preserve their fertilizing potential before cancer therapy

Molecular evaluation of CFTR sequence variants in male infertility of testicular origin.

Although the involvement of the CFTR gene has been well established in congenital agenesia of vas deferens, its role in non-obstructive (NOb) infertility is still a matter of debate. In order to definitively define the involvement of the CFTR gene in spermatogenic impairment and a potential synergistic contribution to known genetic and clinical factors, genetic variants in the entire coding sequence and the immediately flanking regions of the CFTR gene, along with a thorough clinical evaluation, were analysed in 83 NOb infertile patients and 87 clinically well-defined fertile individuals as controls. The results of our study showed no statistical difference between CFTR carrier frequency in the infertile and fertile population. Specifically, the IVS8-6(5T) allele carrier frequency was similar in NOb infertile patients when compared with fertile men, but it is noteworthy that, when fertile men were classified into having optimal and suboptimal fertility, no 5T allele was found among the 35 men with optimal fertility parameters. In conclusion, extensive CFTR analysis in infertile individuals and fertile population as adequate control definitively excludes the involvement of the CFTR gene variants in sperm production and stresses the importance of carefully identifying those individuals with obstructive defects, in whom CFTR screening will be beneficial.

Cyclamate intake and cyclohexylamine excretion are not related to male fertility in humans.

Cyclamate and its metabolite cyclohexylamine affect male fertility in high dose animal studies, but this affect has not been investigated in epidemiological studies. This paper reports the first epidemiological study designed to investigate the possibility of a relationship between cyclamate and cyclohexylamine and male fertility in humans, in which 405 cases of clinically defined infertility in men and 379 controls were surveyed. Semen evaluation, urine analysis for cyclamate and cyclohexylamine and dietary questionnaires were compared between cases and controls. No evidence was found of a significant association between cyclamate intake and male infertility; neither high cyclamate nor high cyclohexylamine excretion were associated with elevated risk. The lack of association remained after adjusting by age, area of residence, education, total energy intake and other variables. No significant correlations were observed between cyclamate intake, metabolism or excretion, and sperm count and motility. The results demonstrate no effect of cyclamate or cyclohexylamine on male fertility at the present levels of cyclamate consumption.

[Response to intracavernous administration of 3 different drugs in the same group of patients with erectile dysfunction]. / Respuesta a la administración intracavernosa de tres fármacos diferentes en el mismo grupo de pacientes con disfunción eréctil.

OBJECTIVE: To compare the subjective and objective penile rigidity in the same group of patients with erectile dysfunction after intracavernous injection of different vasoactive drugs. METHODS: 91 impotent males were randomly assigned to three groups of intracavernous injection: A: 20 micrograms PGE1 B: 30 mg papaverine hydrochloride + 1 mg phentolamine (bimix), and C: 10 micrograms PGE1 + 15 mg papaverine hydrochloride + 0.5 mg phentolamine (trimix). Each patient was scheduled to receive the three modalities of intracavernous injection randomly with an interval of 7-10 days between injections. Penile rigidity equal to or more than 60% was considered positive. Subjective and objective penile rigidity were evaluated with callipers by the same observer. RESULTS: 82 patients completed the study and 9 dropped out after prolonged erection with one of the ICI modalities. Rigidity after treatment with C (66 +/- 15%) was significantly superior to that of B (59 +/- 15%, p = 0.0001) and A (60 +/- 13%, p = 0.0115). No differences were observed between A and B (p = 0.4644). Analysis of only the positive response showed significant differences between A and C, but not between C and B (p = 0.3323). Differences were not found between the response to PGE1 (A) and bimix (B) (p = 0.1275). The order of application of the drug had no effect on response (p = 0.026). CONCLUSIONS: A higher percentage of positive response in patients with erectile dysfunction was achieved with the trimix modality. Choice of more potent ICI regimens can improve the diagnostic and/or therapeutic efficacy in males that do not respond to PGE1 alone.

Adenosine triphosphate-binding cassette superfamily transporter gene expression in severe male infertility.

Cystic fibrosis transmembrane regulator (CFTR), multidrug-resistant (MDR)1, and multidrug resistance-associated (MRP) proteins belong to the ATP-binding cassette (ABC) transporter superfamily. A compensatory regulation of MDR1 and CFTR gene expression has been observed in CFTR knockout rodent intestine and in an epithelial cell line of human colon, whereas a high homology and similar anion binding site are shared by MRP and CFTR proteins. To provide better insight into the relationship among the expression behavior in vivo of the three genes in human testis, analysis of MDR1 and MRP gene expression in testicular biopsies was performed and related to the presence of CFTR gene mutations in congenital absence of the vas deferens (CAVD: n = 20) and non-CAVD (n = 30) infertile patients with azoospermia or severe oligozoospermia. A CFTR mutation analysis performed in both groups of patients supported the involvement of CFTR gene mutations in CAVD phenotype (85%) and in defective spermatogenesis (19%). Quantitative reverse transcription-polymerase chain reaction analysis of testicular tissue showed a CFTR-independent MDR1 and MRP gene expression in human testis, suggesting that the mechanisms underlying CFTR gene regulation in testis are different from those in intestine. These findings should contribute to the understanding of patterns of in vivo expression of CFTR, MDR1, and MRP genes in CFTR-related infertility.

Heterogeneity for mutations in the CFTR gene and clinical correlations in patients with congenital absence of the vas deferens.

Congenital absence of the vas deferens (CAVD) is a heterogeneous disorder, largely due to mutations in the cystic fibrosis (CFTR) gene. Patients with unilateral absence of the vas deferens (CUAVD) and patients with CAVD in association with renal agenesis appear to have a different aetiology to those with isolated CAVD. We have studied 134 Spanish CAVD patients [110 congenital bilateral absence of the vas deferens (CBAVD) and 24 CUAVD], 16 of whom (six CBAVD, 10 CUAVD) had additional renal anomalies. Forty-two different CFTR mutations were identified, seven of them being novel. Some 45% of the CFTR mutations were specific to CAVD, and were not found in patients with cystic fibrosis or in the general Spanish population. CFTR mutations were detected in 85% of CBAVD patients and in 38% of those with CUAVD. Among those patients with renal anomalies, 31% carried one CFTR mutation. Anomalies in seminal vesicles and ejaculatory ducts were common in patients with CAVD. The prevalence of cryptorchidism and inguinal hernia appeared to be increased in CAVD patients, as well as nasal pathology and frequent respiratory infections. This study confirms the molecular heterogeneity of CFTR mutations in CAVD, and emphasizes the importance of an extensive CFTR analysis in these patients. In contrast with previous studies, this report suggests that CFTR might have a role in urogenital anomalies.

Testicular CFTR splice variants in patients with congenital absence of the vas deferens.

The involvement of the five thymidine (5T) variant in intron 8 of the cystic fibrosis membrane regulator (CFTR) gene in congenital bilateral absence of the vas deferens (CBAVD) phenotype has been extensively demonstrated. This variant leads to alternative splicing of the CFTR gene which results in a wild-type transcript and one without exon 9. Little is known about expression of the CFTR gene in the testis. We analysed the level of the aberrantly spliced transcripts in testicular biopsies and correlated it with disease expression. Quantitative RT-PCR analysis in testicular biopsies from control and CBAVD patients showed a correlation between the length of the IVS8-6(T) n tract and the level of alternatively spliced transcripts. Results from histological analysis also suggest an involvement of the alternative transcript in the spermatogenic status of patients, leading to a decreased number of mature sperm forms in the tubule.

Effect of testicular damage induced by cryptorchidism on insulin-like growth factor I receptors in rat Sertoli cells.

The distribution and density of functional insulin-like growth factor I (IGF-I) receptors in cryptorchid and scrotal rat testes and epididymides during gonadal development were studied. Cryptorchidism was induced by unilateral gubernaculectomy in 4-day-old animals, and organs were studied at 15, 30, 60 and 90 days of age. Tissue membranes were assayed for 125I-labelled IGF-I binding. Characterization and specificity of binding sites showed that both normal and contralateral undescended testes and epididymides exhibited typical type 1 IGF receptors. In normal testes, IGF-I receptor density was 20.6 nmol g-1 wet mass at day 15, and decreased to 12.8 nmol g-1 wet mass at adult age (day 90). Cryptorchid testes showed IGF-I receptor concentrations similar to normal testes at day 15 and day 30, but in postpubertal stages displayed a divergent pattern, with a continuous increase at day 60 and day 90, reaching a higher density than those found for immature ages (62 nmol g-1 wet mass). Both normal and cryptorchid epididymides had a similar concentration and a comparable decrease in IGF-I receptors throughout development. In studies with immunohistochemical techniques (alpha IR-3 antibody), IGF-I receptors were found in primary spermatocytes, Sertoli cells and Leydig cells. Cryptorchid tubules showed a lack of germinal epithelium and a marked increase of immunoreactive IGF-I receptors in Sertoli cells, compared with normal tubules from scrotal testes. Intense immunoreactivity for IGF-I receptors was present in the principal cells of epididymal tubules in both normal and cryptorchid organs.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens.

BACKGROUND: Congenital bilateral absence of the vas deferens (CBAVD) is a form of male infertility in which mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been identified. The molecular basis of CBAVD is not completely understood. Although patients with cystic fibrosis have mutations in both copies of the CFTR gene, most patients with CBAVD have mutations in only one copy of the gene. METHODS: To investigate CBAVD at the molecular level, we have characterized the mutations in the CFTR gene in 102 patients with this condition. None had clinical manifestations of cystic fibrosis. We also analyzed a DNA variant (the 5T allele) in a noncoding region of CFTR that causes reduced levels of the normal CFTR protein. Parents of patients with cystic fibrosis, patients with types of infertility other than CBAVD, and normal subjects were studied as controls. RESULTS: Nineteen of the 102 patients with CBAVD had mutations in both copies of the CFTR gene, and none of them had the 5T allele. Fifty-four patients had a mutation in one copy of CFTR, and 34 of them (63 percent) had the 5T allele in the other CFTR gene. In 29 patients no CFTR mutations were found, but 7 of them (24 percent) had the 5T allele. In contrast, the frequency of this allele in the general population was about 5 percent. CONCLUSIONS: Most patients with CBAVD have mutations in the CFTR gene. The combination of the 5T allele in one copy of the CFTR gene with a cystic fibrosis mutation in the other copy is the most common cause of CBAVD: The 5T allele mutation has a wide range of clinical presentations, occurring in patients with CBAVD or moderate forms of cystic fibrosis and in fertile men.

Choosing among different technical variations of Percoll centrifugation for sperm selection.

We compared the efficiency of different methodological variations of the centrifugation through discontinuous Percoll gradients (PC) to improve semen samples in the laboratory. Five different combinations of the number, volume and density of PC layers were assayed in 14 semen samples presenting various qualities. Each specimen was divided into five aliquots and processed simultaneously. The percentage of spermatozoa showing optimal movement (VAP > 30 microns s-1 and STR > 80%) after PC selection when the number of gradients was reduced to three or two (PC-3: 43.3%, PC-2: 41.3%) and when the volume of layers was diminished to 0.5 ml (mPC-3: 44.2%, and mPC-2: 48.1%), was higher than in classical columns with four gradients of 1 ml (PC-4: 26.3%). The absolute recovery of optimal sperm was better with PC-2, mPC-2 and mPC-3. In samples showing low concentration or motility of spermatozoa, mPC-2 was the most effective technique. PC-2 and mPC-2 showed a tendency to eliminate more red blood cells contaminating the samples. Straight line velocity and straightness were similarly improved by all the methods. We conclude that the technique of PC centrifugation with only two gradients is simpler and more effective for sperm selection and in cases of poor samples can be used with low-volume layers.

Extensive analysis of 40 infertile patients with congenital absence of the vas deferens: in 50% of cases only one CFTR allele could be detected.

Mutations in the cystic fibrosis (CF) conductance transmembrane regulator (CFTR) gene have been detected in patients with CF and in males with infertility attributable to congenital bilateral absence of the vas deferens (CBAVD). Thirty individuals with CBAVD and 10 with congenital unilateral absence of the vas deferens (CUAVD) were analyzed by single-strand conformation analysis and denaturing gradient gel electrophoresis for mutations in most of the CFTR gene. All 40 individuals were pancreatic sufficient, but twenty patients had recurrent or sporadic respiratory infections, asthma/asthmatic bronchitis, and/or rhino-sinusitis. Agenesia or displasia of one or both seminal vesicles was detected in 30 men and other urogenital malformations were present in six subjects. Among the 40 samples, we identified 13 different CFTR mutations, two of which were previously unknown. One new mutation in exon 4 was the deletion of glutamic acid at codon 115 (delta E115). A second new mutation was found in exon 17b, viz., an A --> C substitution at position 3311, changing lysine to threonine at codon 1060 (K1060T). CFTR mutations were detected in 22 out of 30 (73.3%) CBAVD patients and in one out of 10 (10%) CUAVD individuals, showing a significantly lower incidence of CFTR mutations in CBAVD/CUAVD patients (P << 0.0001), compared with that found in the CF patient population. Only three CBAVD patients were found with more than one CFTR mutation (delta F508/L206W, delta F508/R74W + D1270N, R117H/712-1G --> T), highlighting L206W, R74W/D1270N, and R117H as benign CF mutations. Sweat electrolyte values were increased in 76.6% of CBAVD patients, but three individuals without CFTR mutations had normal sweat electrolyte levels (10% of the total CBAVD patients), suggesting that factors other than CFTR mutations are involved in CBAVD. The failure to identify a second mutation in exons and their flanking regions of the CFTR gene suggests that these mutations could be located in introns or in the promoter region of CFTR. Such mutations could result in CFTR levels below the minimum 6%-10% necessary for normal protein function.

Critical evaluation of the present experience on microsurgical sperm aspiration.

Microsurgical epididymal sperm aspiration (MESA) and IVF techniques are indicated in those cases of bilateral congenital absence of the vas deferens (BCAVD) and in cases of epididymal obstruction (EO) of other aetiologies were vasoepididymostomy is not possible or has failed. Microsurgical deferential sperm aspiration (MDSA) is indicated in those cases where spermatogenesis and epididymal permeability are conserved but spermatozoa, for different causes, cannot reach the ejaculate and other therapies have failed. We have performed this procedure in 70 patients by two different human reproduction teams. Both teams achieved very similar results regarding fertilization rate: 5.5 and 7.2% in cases of BCAVD, 6.5 and 8.8% in cases of EO and 33.3 and 26.7% in cases of non-epididymal pathology (NEP). However, the fertilization rate per patient presents differences between both teams: 27 and 44.4% in BCAVD, 31 and 11% in EO and 80 and 100% in NEP. In spite of a better fertilization rate per patient in team two, it was in the other where pregnancies were achieved (7% in BCAVD, 15% in EO and 20% in NEP). One pregnancy in EO group was achieved by intracytoplasmatic sperm injection. The pregnancy rates per transfer were 25% in BCAVD, 50% in EO and 25% in NEP.