High implantation and pregnancy rates with testicular sperm extraction and intracytoplasmic sperm injection in obstructive and non-obstructive azoospermia

Thirty-two infertile couples with obstructive and non-obstructiveazoospermia were included in this study. Testicular sperm extraction(TESE) was performed in 16 obstructive azoospermic cases wheremicrosurgical sperm aspiration (MESA) or percutaneous spermaspiration (PESA) were impossible because of totally destroyedepididymis and 16 non-obstructive azoospermia cases with severespermatogenetic defect where the testicles were the only sourceof sperm cells. A total of 288 oocytes was obtained from 32females and 84% were injected. The fertilization rates (FR)with 2 pronuclei (PN) and cleavage rate were 50.8 and 68.2%respectively. A total of 15 pregnancies was achieved (53% perembryo transfer), nine from the obstructive and six from thenon-obstructive group. Four pregnancies resulted in clinicalabortion (26.6%). The ongoing pregnancy rate was 39.2% per embryotransfer (ET) and 343% per started cycle. A high implantationrate was also achieved (26.6% in non-obstructive and 30% inobstructive azoospermia group). Using testicular spermatozoain combination with ICSI in both obstructive and non-obstructiveazoospermic groups, high implantation and pregnancy rates canbe achieved.     

Outcome of intracytoplasmic sperm injection with testicular spermatozoa in obstructive and non-obstructive azoospermia

From 1 August 1993 until 30 September 1994, 69 couples sufferingfrom azoospermia underwent testicular sperm extraction and intracytoplasmicsperm injection. In 50 couples with obstructive azoospermiaa total of 631 meta-phase-II oocytes were injected after testicularsperm extraction yielding a 2-PN fertilization rate of 57%.In female patients <40 years of age an ongoing pregnancyrate per transfer of 42% (14/33) was obtained. So far, eighthealthy babies have been born, including two singletons andthree twin gestations. In 19 couples with non-obstructive azoospermiaa total of 264 metaphase-II oocytes were injected after testicularsperm extraction, yielding a 2-PN fertilization rate of 58%.An ongoing pregnancy rate per transfer of 31% (5/16) was established.So far, six healthy babies have been born including one singleton,one twin and one triplet gestation.     

Increased sperm mitochondrial DNA content in male infertility

BACKGROUND: There is increasing evidence that mitochondrial DNA (mtDNA) anomalies in sperm may lead to infertility. Point mutations, deletions and the presence of a specific mtDNA haplogroup have been associated with poor sperm quality, but little attention has been paid to the role of mtDNA content. METHODS: Using density gradient separation and swim-up methods, we selected motile sperm from 32 normal and 35 abnormal sperm samples. The mtDNA/beta-globin gene ratio was determined by real-time quantitative PCR. RESULTS: The average mtDNA/beta-globin ratio of sperm collected from 100% density layers was 1.4 for normal sperm, 6.1 for sperm samples presenting at least one abnormal criterion [among the three criteria established by World Health Organization (1999), i.e. sperm count, motility and morphology], and 9.1 for sperm samples presenting two or more of these abnormal criteria. These differences are very highly significant (P < 0.0001). The mtDNA numbers were also much greater in sperm collected from the 40% density gradient layers (mean: 17.1, P < 0.001), known to contain the most abnormal sperm of the sperm samples, than in those collected from the 100% layers known to contain sperm with the best fertilizing ability. CONCLUSION: Our results showed significant mtDNA amplification in sperm collected from abnormal sperm samples.     

Serum inhibin B determination is predictive of successful testicular sperm extraction in men with non-obstructive azoospermia

Recent work indicates that serum inhibin B is a useful marker of spermatogenesis and inhibin B production sufficient to maintain detectable serum concentrations in adults depends on spermatogenic activity. The purpose of the present study was to investigate the usefulness of serum inhibin B measurement to predict the success of testicular sperm extraction (TESE) in 17 men with nonobstructive azoospermia to be treated by intracytoplasmic sperm injection (ICSI) (group 1). Two additional groups were used as positive controls; group 2 comprised 22 infertile men having obstructive azoospermia, and group 3, which included 29 semen donors having normal seminal parameters. Follicle stimulating hormone (FSH) was significantly higher (P < 0.01) and inhibin B significantly lower (P < 0.001), in group 1 as compared with groups 2 and 3. Serum inhibin B concentrations were significantly higher (P < 0.001) among successful TESE men as compared with those having failed TESE. In contrast, no differences were detected between these two groups with respect to serum FSH or testicular size. In addition, serum inhibin B but not FSH discriminated between successful and failed TESE in group 1 subjects as compared with control groups. According to the receiver operating characteristics curve analysis, the best inhibin B value for discriminating between successful and failed TESE was >40 pg/ml (sensitivity 90%, specificity 100%). It is concluded that inhibin B measurement is a useful non-invasive predictor of spermatogenesis and thus, all azoospermic males should have serum inhibin B concentrations determined in addition to FSH measurement and karyotyping prior to undergoing TESE.     

Diagnostic epididymal and testicular sperm recovery and genetic aspects in azoospermic men.

Various procedures for sperm recovery in azoospermic men have been described, from open testicular biopsy to simple needle aspiration from the epididymis and the testis. Fifty-one obstructive and 86 non-obstructive azoospermic men were treated to compare the recovery of spermatozoa obtained by percutaneous aspiration from the epididymis (PESA) and aspiration/extraction from the testis (TESA, TESE) with histopathology. If TESA failed, the work up proceeded with TESE. All patients were karyotyped. Spermatozoa were recovered by PESA or TESA in all obstructive men (51/51 patients). In 22 out of 86 patients with non-obstructive azoospermia, testicular spermatozoa could be successfully recovered by TESA. In five additional patients TESE was successful in recovering spermatozoa where TESA had failed. In 43 patients, neither TESA nor TESE was successful. Sixteen patients chose not to proceed with TESE. Seven out of 86 patients had an abnormal karyotype in the non-obstructive group (8%), none in the obstructive group. In the non-obstructive patient group testicular histopathology showed hypospermatogenesis, incomplete maturation arrest and germ cell aplasia with focal spermatogenesis in cases where spermatozoa were recovered and complete germ cell aplasia, complete maturation arrest and fibrosis in cases where no spermatozoa were found. Spermatozoa were recovered by PESA or TESA from all patients with obstructive azoospermia and from approximately 40% of patients with non-obstructive azoospermia by TESA or TESE. Retrieval of viable spermatozoa in the infertility work-up was highly predictable for sperm recovery in subsequent ICSI cycles. TESA performed under local anaesthesia seems almost as effective as more invasive procedures in recovering testicular spermatozoa, both in obstructive and non-obstructive azoospermic men.     

Serum inhibin B cannot predict testicular sperm retrieval in patients with non-obstructive azoospermia

BACKGROUND: Serum inhibin B, a direct product of the Sertoli cells, may serve as a marker of spermatogenesis. The present retrospective study aimed at evaluating the predictive value of inhibin B for retrieving testicular sperm in non-obstructive azoospermic men. METHODS: The serum inhibin B concentration before sperm retrieval was reviewed in 185 non-obstructive azoospermic patients. RESULTS: Testicular sperm were successfully recovered in 92 of 185 patients (49.7%). The mean inhibin B concentration in these patients was 37.3 pg/ml. No sperm were found in 93 patients (50.3%), and the mean serum inhibin B concentration was 44.9 pg/ml. The discrimination between successful and unsuccessful sperm retrieval was analysed using the receiver operating characteristics (ROC) curve analysis. The best discriminating inhibin B concentration was 13.7 pg/ml (sensitivity 44.6%, specificity 63.4%) with an area under the ROC curve (AUC) of 0.51. Combining both serum FSH and inhibin B did not improve the predictive value: the AUC of inhibin B in men with a serum FSH concentration <25 and > or = 25 IU/l (being the best threshold value in the population studied) was respectively 0.53 and 0.50. The AUC of the inhibin B:FSH ratios was 0.55. CONCLUSIONS: This analysis shows that inhibin B, either alone or in combination with serum FSH, fails to predict the presence of sperm in men with non-obstructive azoospermia undergoing testicular sperm extraction.     

Epididymal sperm aspiration in conjunction with in-vitro fertilization and embryo transfer in cases of obstructive azoospermia.

Epididymal sperm aspiration is a new treatment for vasal agenesis. In previous reports, epididymal spermatozoa resulted in pregnancy by utilizing in-vitro fertilization (IVF) or gamete intra-Fallopian transfer (GIFT). We sought to investigate the efficacy of epididymal sperm aspiration in conjunction with IVF in patients with congenital absence of the vas deferens or with secondary extended obstruction of spermatic ducts. Fifty-eight attempts were performed in 23 patients (25-50 years). Eight patients (34.7%) had vasal agenesis and 14 (60.8%) presented with vasal secondary extended obstruction. The sperm count was adequate (greater than or equal 20 x 10(6)/ml) in 13.8% of sperm retrievals and sperm motility of 20% was obtained in 15.5% of sperm retrievals. Fourteen attempts at IVF were performed with epididymal sperm counts of 2-44 x 10(6)/ml and motilities of 0-45%. A mean of six mature oocytes (0-13) were inseminated in each case. Five embryo transfers were performed in five patients' wives (35.7%) and two couples had an early pregnancy loss (14.2%). Epididymal sperm aspiration is an advance in treating such patients, as an adequate number of mature spermatozoa can be obtained and used for IVF. However, spermatozoa directly aspirated from the proximal epididymis and with fertilizing capacity in vitro, gave a high rate of embryo degeneration (greater than 50%) after embryo transfer.     

Establishment of predictive variables associated with testicular sperm retrieval in men with non-obstructive azoospermia

Although testicular biopsy for sperm extraction is a procedure with a potential for complications, sperm retrieval is successful in 30-70% of patients with non-obstructive azoospermia. In order to predict the probability of retrieving at least one testicular spermatozoon we conducted a prospective study of a set of variables in 40 patients with non-obstructive azoospermia. Using the receiver operating characteristic curves, we determined the probability estimates of testicular volume, plasma follicle stimulating hormone (FSH) concentration, Johnsen score and visualization of testicular spermatids in discriminating between patients with successful and failed testicular sperm extraction. Visualization of testicular spermatids provided the best estimate of success of testicular sperm extraction. Of the factors studied using logistic-regression analysis (age, maternal and paternal age at birth, body mass index, luteinizing hormone, testosterone, FSH, testicular volume, the presence of testicular spermatids and Johnsen score), only the presence of spermatids and Johnsen score were independent variables able to predict the success of testicular sperm extraction. The visualization of the presence of spermatids gave a correct prediction of 77% and Johnsen score of 71%. The diagnostic model derived from these independent predictors when validated in 40 patients using the Jackknife technique gave a correct overall prediction of 87%. The probability of successful testicular sperm extraction in patients with non-obstructive azoospermia could be objectively predicted on the basis of simple histopathological criteria represented by the visualization of testicular spermatids and Johnsen score.     

Microsurgical epididymal sperm aspiration: aspirate analysis and straws available after cryopreservation in patients with non-reconstructable obstructive azoospermia. MESA/TESE Group Giessen

Microsurgical epididymal sperm aspiration (MESA) combined with intracytoplasmic sperm injection (ICSI) represents a great advance in the therapy of non-reconstructable obstructive azoospermia. For procedure synchronization, a great number of organizational facilities are needed. Intentional cryopreservation of the aspirate may reduce these problems, therefore the aim of this study was to analyse the amount and quality of aspirate fluid obtained by means of MESA and the quality of the vials after thawing. Furthermore, the available cryopreserved straws were calculated. A total of 93 consecutive MESA procedures were performed and epididymal spermatozoa were obtained in 88 patients. Mean sperm concentration was 40.9 x 10(6) spermatozoa/ml. Global and progressive motility were 24.8 and 7.5% respectively. In one-third of the aspirates, no progressive motile spermatozoa were found. The mean number of straws available was 7.6. In 33 ICSI cycles with frozen-thawed epididymal spermatozoa, a pregnancy rate of 42.4% was achieved. In conclusion, these data show that enough spermatozoa are available for various ICSI cycles following a single MESA procedure in men with non-reconstructable obstructive azoospermia. Furthermore, ICSI with cryopreserved spermatozoa leads to excellent pregnancy rates     

Factors influencing the outcome of ICSI in patients with obstructive and non-obstructive azoospermia: a comparative study

BACKGROUND: Factors influencing success of sperm retrieval in azoospermic patients and outcome of ICSI were evaluated. METHODS AND RESULTS: Uni- and multifactorial analysis were performed using logistic and stepwise analysis, following surgical sperm retrieval by percutaneous epididymal sperm aspiration (55 cycles) or testicular sperm extraction (142 cycles) in 52 and 123 patients with obstructive azoospermia (OA) and non-obstructive azoospermia (NOA) respectively. ICSI cycles using fresh or cryopreserved-thawed sperm were included. Sperm were retrieved to allow ICSI in 100 and 41% of OA and NOA patients, with no significant correlation with patients' age or FSH level. Occurrence of pregnancy was significantly correlated with female age (90th quantile: 38 years), number of oocytes retrieved (10th quantile: five oocytes) and number of oocytes injected (10th quantile: four oocytes). Sperm origin (epididymal versus testicular), status (fresh or thawed), male partner's age, and serum FSH had no significant effect upon implantation rate, pregnancy rate per embryo transfer or spontaneous miscarriage rate. CONCLUSIONS: In OA patients ICSI should be planned in conjunction with surgical sperm retrieval. In contrast, the lack of efficient non-invasive parameters to predict sperm retrieval in NOA suggests that elective surgical sperm retrieval may be offered to these patients prior to ovarian stimulation of their partners, especially when donor back-up is not an alternative. Female factors such as age and ovarian reserve have significant impact upon clinical success rates.     

Mitochondrial DNA deletions and nuclear DNA fragmentation in testicular and epididymal human sperm

BACKGROUND: There are still concerns about the safety of intracytoplasmic sperm injection (ICSI) due to its brief clinical record and lack of animal testing. Testicular and epididymal sperm are now used routinely for ICSI in patients with obstructive azoospermia. The use of such immature sperm compounds fears, since little is known of their mitochondrial and nuclear DNA quality. METHODS: A modified long polymerase chain reaction (LPCR) was employed to study mitochondrial DNA (mtDNA) and a modified alkaline Comet assay to determine nuclear DNA (nDNA) fragmentation in testicular and epididymal sperm from men with obstructive azoospermia (n = 25) attending the Regional Fertility Centre. RESULTS: Testicular sperm displayed significantly more wild-type mtDNA (45% of patients) than epididymal sperm (16% of patients). They also had a lower incidence of multiple deletions and smaller mtDNA fragments. Epididymal sperm harboured more large-scale deletions (P < 0.05). There was a strong correlation between nuclear DNA fragmentation, the number of mtDNA deletions (r = 0.48, r = 0.50, P < 0.001) and their size (r = 0.58, r = 0.60, P < 0.001) in both epididymal and testicular sperm. CONCLUSION: This study suggests that mtDNA and nDNA of testicular sperm have fewer mutations and fragmentation than epididymal sperm and should be used in preference for ICSI in clinical treatment.     

Fertility with testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermic men

In non-obstructive azoospermia spermatozoa can usually onlybe isolated from the testicles, and thus the most promisingtreatment model is testicular sperm extraction (TESE). Hormoneconcentrations, testicular volume determinations and testicularbiopsy results are not uniform enough to select potential candidatesfor successful TESE and intracytoplasmic sperm injection (ICSI)approaches in advance. The aim of this study was to assess theefficacy of using ICSI with testicular spermatozoa in casesof non-obstructive azoospermia and to compare the inclusioncriteria and sperm existence in the testicles in sperm obtainableand non-obtainable groups. All men showed either complete orincomplete (n = 14) maturation arrest in spermatogenesis, severehypospermatogenesis (n = 10) or Sertoli cell-only syndrome (n= 5) in their testicular biopsies. Only 14 out of a total of29 men provided enough spermatozoa for the ICSI procedure, whileno spermatozoa were found in the testicular samples of the remaining15 men. Out of 123 oocytes obtained from 14 females, 101 wereinjected with the husbands' testicular sperm cells. Total fertilizationfailure was observed in three cases. Of 39 oocytes fertilized,38 cleaved. The fertilization and cleavage rates were 38.6 and97.4% respectively. The pregnancy rate was 20.7% per initiatedcycle. In the group from whom spermatozoa were obtainable, thepregnancy rate was 42.9% per initiated cycle and 54.5% per embryotransfer. A total of six pregnancies were achieved, of whichtwo Were twins and four were singletons. One singleton pregnancyresulted in abortion in the first trimester. There was no statisticaldifference concerning the serum follicle stimulating hormoneconcentration, testicular volume and biopsy results in groupsin which spermatozoa were obtainable or not. In conclusion,although the association of TESE with ICSI obtained pregnanciesfor some patients with non-obstructive azoospermia, furtherstudies are needed to determine the inclusion criteria for successfulTESE.     

Pregnancy outcome and neonatal data of children born after ICSI using testicular sperm in obstructive and non-obstructive azoospermia

BACKGROUND: Registries on outcome of ICSI pregnancies obtained with testicular sperm do not differentiate between obstructive (OA) and non-obstructive azoospermia (NOA). We evaluated the pregnancy outcome and neonatal data on children born after ICSI using testicular sperm of men with histologically proven OA or NOA. METHODS: Pregnancies obtained after ICSI using testicular sperm of men with defined NOA (n = 70) were compared with those of men with OA (n = 204). RESULTS: Multiple birth rates in NOA and OA couples, respectively, were 21 versus 27% (P = NS), overall preterm delivery rates were 38 versus 26% (NS), and prematurity rates were 24 versus 13% for singletons (NS) and 86 versus 54% for twins (relative risk 1.59, 95% confidence interval 1.04-2.42). Median gestational age for singletons was 38.3 versus 39.3 weeks, respectively (P < 0.05). The low birth weight rates were 34 versus 31%, respectively (NS). The early perinatal mortality rate was 66 versus 15 per 1000 births, respectively, (NS). Major congenital malformations were observed in 4 versus 3%, respectively, of the live born babies (NS). Prenatal karyotypes showed 7% de-novo abnormalities in the NOA group versus 1% in the OA group (NS). CONCLUSIONS: Our data do not show differences between NOA and OA pregnancies except for a strong tendency towards a lower gestational age in singletons and a higher percentage of premature twins in the NOA group. Although our data are based on a limited sample, the differences observed call for further analysis. Given the low pregnancy rates after ICSI with NOA, a multicentre study, differentiating NOA and OA patients, would be recommended.     

Increased sperm motility after in-vitro culture of testicular biopsies from obstructive azoospermic patients results in better post-thaw recovery rate

The objective of this study was to optimize the use of testicular biopsies in 14 patients with obstructive azoospermia. Testicular specimens were retrieved from six patients (group I) and cultured at 32 and 37 degrees C for up to 20 days; changes in percentage motile spermatozoa were compared. In four men of group I, one portion of the specimen was frozen at retrieval, and changes in post-thaw motility after 24 h of culture at 37 degrees C were recorded. In the other eight patients (group II), testicular specimens were frozen at retrieval and after 72 h culture at 37 degrees C. Pre and post-freezing motility and post-thaw recovery rate were compared. No significant differences were observed until day 8 in the improvement of motility between 32 and 37 degrees C in-vitro culture. Maximum motility was reached, under both conditions, between 48 h and 72 h. Post-thaw 24 h culture at 37 degrees C of specimens frozen at retrieval did not improve motility; however, 72 h pre-freezing culture significantly improved initial motility (P: < 0.01), post-thaw motility (P: < 0.01) and post-thaw recovery rate (P: < 0. 001). The higher recovery rate of samples frozen 3 days after retrieval allows more economical use of the tissue that is available.     

Outcome of first and repeated testicular sperm extraction and ICSI in patients with non-obstructive azoospermia

BACKGROUND: It is unclear whether or not testicular sperm extraction (TESE) should be repeated for patients in whom no sperm were found during their first TESE attempt. METHODS AND RESULTS: The outcome of repeated TESE was evaluated in patients with non-obstructive azoospermia (NOA) after failing to obtain sperm in their first extraction attempt, or having used all available cryopreserved testicular tissue. Out of 83 patients with NOA, patients repeated TESE two (n = 22), three (n = 8), four (n = 6) and five (n = 3) times. Distribution of main testicular histology included germ cell aplasia (55%), maturation arrest (29%) and germ cell hypoplasia (16%). The first TESE yielded mature sperm for ICSI in 39% of patients (sp+), and failed in the remaining 61% (sp-). A second TESE yielded mature sperm in 1/4 from the sp- group and in 16/18 from the sp+ group. At the third, fourth and fifth trials, 8/8, 5/6 and 3/3 of the original sp+ patients were sp+ again respectively. Compared with the outcome of the first trial, all further trials did not differ statistically in the rate of fertilization (54 versus 49%), implantation (9.5 versus 5.4%), or clinical pregnancy/cycle (19 versus 15%). No pregnancies were achieved among the three patients after their fifth TESE. Pregnancies occurred in all histological groups, except maturation arrest. CONCLUSIONS: The outcome of repeated TESE cycles, up to the fourth trial, justifies the procedure.     

Chromosome analysis of spermatozoa extracted from testes of men with non-obstructive azoospermia

Infertile men with azoospermia now have the possibility of fathering children by testicular sperm extraction combined with intracytoplasmic sperm injection. However, there are concerns about the risk of chromosomal abnormalities in their spermatozoa. We have studied aneuploidy frequencies for chromosomes 13, 21, X and Y by multicolour fluorescence in-situ hybridization (FISH) in testicular spermatozoa extracted from three men with non-obstructive azoospermia. The men were 34-37 years of age and had normal follicle-stimulating hormone (FSH) concentrations and normal 46,XY somatic karyotypes. A total of 3324 spermatozoa was analysed. The infertile patients had an elevated frequency of disomy for chromosomes 13, 21, XY disomy compared to controls but none of these reached statistical significance. Also there was no significant difference in the sex ratio or the frequency of diploidy in azoospermic patients compared to normal control donors. This first report on chromosomal aneuploidy in spermatozoa extracted from testes of patients with non-obstructive azoospermia suggests that some azoospermic men do not have a substantially increased risk of chromosomally abnormal spermatozoa.     

Frequent polymorphism of the mitochondrial DNA polymerase gamma gene (POLG) in patients with normal spermiograms and unexplained subfertility

BACKGROUND: Male fertility largely depends on the quality of sperm production, which may be affected by environmental and genetic factors. In this study, we explored a possible role of the polymerase gamma (POLG) gene polymorphism, recently reported to be associated with male infertility in some populations. METHODS: The polymorphic CAG repeat (usually 10 codons long) in the POLG gene was studied in 1298 male subjects: 429 patients with infertility/subfertility, and 869 controls (495 men from the general population with unknown fertility and 374 recent fathers). In all subjects, the POLG polymorphism was assessed in relation to their semen quality, and--in the fertile controls--with biological fecundity measured as waiting time-to-pregnancy (TTP) for the couples. In the patients lacking the common POLG allele, the outcome of the assisted reproductive techniques (ART) for the couples was evaluated. RESULTS: The absence of one (10/ not equal to 10) or both common POLG alleles (not equal to 10/not equal to 10) was more frequent among the subfertile patients than among fertile controls (P=0.021 and P=0.04 respectively). The estimated predictive value for infertility in a man homozygous for the POLG polymorphism was 15.5% (95% CI: 4.8-51%). There was a positive association with sperm concentration: 14.3% of the normospermic subfertile patients were homozygous for the absence of the common POLG allele (not equal to 10/not equal to 10), in comparison with 2.3% of unselected controls (P=0.001) and 0.9% of the fertile men (P=0.0001). No association with sperm motility, morphology and TTP was found. Spermatozoa of the three not equal to 10/not equal to 10 patients treated with IVF retained the ability to penetrate the egg, but the fertilization rate was low. Nine homozygous not equal to 10/ not equal to 10 patients were treated with ICSI, resulting in pregnancy in seven couples. CONCLUSIONS: The POLG gene polymorphism should be considered as a possible contributing factor in patients with unexplained subfertility and normal spermiograms. The oocyte penetration ability of sperm may be partially impaired in the not equal to 10/not equal to 10 patients but most of them can be successfully treated with ICSI.     

Andrology: Natural spermatoceles in irreversible obstructive azoospermia--reservoirs of viable spermatozoa for assisted conception

Fine needle aspiration of asymptomatic spermatoceles detectedin five men with obstructive azoospermia was evaluated as aminimally invasive method of sperm retrieval for assisted conception.Sperm preparations adequate for in-vitro fertilization wereinitially obtained from three men, but there was failure offertilization in all three cycles. However, intracytoplasmicsperm injection (ICSI) in three couples resulted in fertilizationof 58% of metaphase II oocytes, with embryo transfers in fiveof six cycles. A successful pregnancy and delivery resultedfrom spermato zoa from a man with an irreversible vasectomy,for whom previous epididymal microaspiration and ICSI had beenunsuccessful. Spermatoceles were found in 4% of men with irreversibleobstructive azoospermia referred for assisted conception. Thesepatients should therefore be examined very carefully for smallcysts near the epididymis, because fine needle aspiration, usuallywithout anaesthesia, can instantly locate a ready source ofviable spermatozoa for LCSI This is less invasive than the alternativesperm retrieval procedures, which are more traumatic and requirelocal or general anaesthesia.     

Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation

Cryopreservation of human spermatozoa is extensively used in artificial insemination and IVF programmes. Despite various advances in cryopreservation methodology, the recovery rate of functional post-thaw spermatozoa remains mediocre, with sperm motility being significantly decreased after freezing. This aim of this study was to investigate the effects of cryopreservation on both DNA integrity and morphology of spermatozoa from fertile and infertile men. Semen samples were obtained from 17 fertile and 40 infertile men. All samples were prepared by discontinuous Percoll density centrifugation (95.0:47.5). Samples were divided into aliquots to allow direct comparison of fresh and frozen spermatozoa from the same ejaculate. Aliquots for cryopreservation were mixed with a commercial cryoprotectant and frozen by static phase vapour cooling before plunging into liquid nitrogen. Thawing was carried out slowly at room temperature. Sperm DNA integrity was determined using a modified alkaline single cell gel electrophoresis (comet) assay and sperm morphology analysed using the Tygerberg criteria. DNA of semen and prepared spermatozoa from fertile men was found to be unaffected by cryopreservation. In marked contrast, spermatozoa from infertile men were significantly damaged by freeze-thawing. Cryopreservation had a detrimental effect on morphology of semen and prepared samples from fertile and infertile men.     

Clinical adaptation of the sperm ubuquitin tag immunoassay (SUTI): relationship of sperm ubiquitylation with sperm quality in gradient-purified semen samples from 93 men from a general infertility clinic population

BACKGROUND: The proteolytic chaperone peptide ubiquitin accumulates in defective human spermatozoa. Immunodetection of ubiquitin in human sperm samples correlates with semen quality and male fertility. METHODS: Semen samples from 93 men from couples seeking infertility treatment were separated on a PureSperm density gradient and screened by immunofluorescence microscopy with anti-ubiquitin antibodies. The percentage of spermatozoa with head ubiquitylation was recorded and compared with clinical semen evaluation and embryo development data after IVF or ICSI. Subjects were divided into the following four groups based on the initial clinical diagnosis of the couples; group 1, male factor; group 2, idiopathic infertility; group 3, female infertility with neither partner having children previously; and group 4, female infertility with male partners having children from previous relationships. RESULTS: The percentage of sperm with ubiquitylated heads remaining after PureSperm separation in the respective groups was 4.0% (male factor), 2.5% (idiopathic infertility), 0.7% (female infertility and presumed fertile male) and 0.9% (female infertility with established fertile male). Negative correlations between sperm ubiquitin and several parameters reflective of embryo development after assisted fertilization were found within the male factor group. CONCLUSIONS: Use of this simplified ubiquitin-based sperm quality assay is feasible in a clinical environment. Since the gradient separation does not completely deplete the defective spermatozoa, the modified light microscopic sperm ubiquitin tag immunoassay could add a new level of stringency to the selection of human spermatozoa for ICSI.