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Objective: To assess if epididymal sperm cryopreserved 24 hours after exposure to oocytes in conventional IVF can be successfully used for intracytoplasmic sperm injection (ICSI) in a subsequent cycle., Design: Case report., Setting: University of California, Irvine, Center for Reproductive Health., Patients: Two men with obstructive azoospermia requiring microsurgical epididymal sperm aspiration, IVF, and ICSI., Interventions: Freezing of epididymal sperm 24 hours after egg exposure in conventional IVF and subsequent use for assisted fertilization in a new cycle., Main Outcome Measure: Frozen-thawed epididymal sperm survivability and maintenance of fertilization and pregnancy capacity., Results: At the time of sperm aspiration procedure (cycle I) a total of 30 oocytes were available for insemination. Of these, 15 were used for conventional IVF resulting in 2 embryos (13%) and 15 were used for ICSI, resulting in 3 embryos (20%). Sperm was cryopreserved 24 hours after conventional IVF and thawed 6 months later in a new cycle. Upon thawing, sperm were still found to be motile and at this time (cycle II) only assisted fertilization was used. Of 27 oocytes injected, 12 (44%) produced normal, cleaving embryos. One singleton pregnancy with the birth of a healthy infant girl was achieved after the tubal transfer of 5 embryos., Conclusion: The birth of a normal, healthy infant girl with epididymal sperm frozen 24 hours after exposure to oocytes in conventional IVF emphasizes the value of freezing any aliquot of epididymal sperm, even if the motility is very low, to avoid additional surgery in the male. From a basic science standpoint, this observation may renew interest in the study of sperm cryopreservation after occurrence of acrosome reaction and hyperactivation.

Microsurgical epididymal sperm aspiration (MESA) and in vitro fertilization (IVF) is primarily offered to men with congenital absence of the vas deferens (CAVD). However, the IVF capacity of these epididymal sperm is low ( < 15%) and unpredictable. In this study, IVF and intracytoplasmic sperm injection (ICSI) results in patients with non-congenital, irreparable obstructive azoospermia were analysed. Thirty-three patients were evaluated for a total of 37 cycles of MESA and IVF. Most had obstruction secondary to failed vasectomy reversal and to epididymal blockage. The overall fertilization rate was 30% with regular IVF and 26% with ICSI, and six clinical pregnancies were obtained. Both rates are significantly higher than the IVF rate previously reported with sperm from men with CAVD (13%, P < 0.00001). In men with non-congenital obstructive azoospermia, a significant difference was found in the average sperm count (56.9 x 10(6) v. 12.3 x 10(6), P < 0.04) and total motile count (16.6 x 10(6) v. 1.6 x 10(6), P < 0.01) respectively for patients who achieved IVF and those who did not. It is concluded that (a) the real IVF capacity of human epididymal sperm is 30%, or 42%, if calculated only for patients who achieved fertilization, (b) this higher rate is an indirect support of the hypothesis that sperm from men with CAVD have intrinsic biochemical defects, related to cystic fibrosis mutations, responsible for their low and unpredictable IVF rate, and (c) MESA and IVF can be offered at the same time or as an alternative to patients requesting vasectomy reversal.

Objective: To assess the relative escape force of human epididymal sperm using a laser generated optical trap and compare it with that of human ejaculated sperm., Design: Evaluation of the relative force generated by epididymal and ejaculated sperm using an 800-nm laser-generated optical trap system (titanium-sapphire, model 899-01; Coherent Innova, Palo Alto, CA)., Setting: University-based facility at the Beckman Laser Institute and Medical Clinic and Center for Reproductive Health, University of California, Irvine., Interventions: A total of 2,720 sperm from 28 samples were randomly analyzed. Fifteen were ejaculated samples (1,650 sperm) obtained from men with proven fertilization, and 13 were epididymal samples (1,070 sperm) aspirated microsurgically from patients with obstructive azoospermia. An optical trap equipped with the 100x Neofluar objective was used to analyze an average of 100 sperm per patient., Main Outcome Measures: Determination of mean relative escape force values in milliwatts for epididymal and ejaculated sperm samples., Results: The mean relative escape force for epididymal sperm was 32.4 mW, significantly lower than ejaculated sperm, which was 85.1 mW. By correlating epididymal sperm relative force with fertilization in vitro at an arbitrary cutoff value of 30 mW, it was found that no fertilization occurred if a sample had < 13% of sperm at that value., Conclusions: [1] The average relative escape force of the epididymal sperm was found to be 60% weaker than that of ejaculated sperm. [2] It is demonstrated that the noncontact laser optical trap is a sensitive tool that can evaluate single sperm force as a new physiological parameter.

One of the limiting factors for the successful treatment of male sterility due to congenital absence of the vas deferens (CAVD) is the low (< 20%) and extremely unpredictable rate of in-vitro fertilization of their epididymal spermatozoa. The recent demonstration that CAVD is a mild form of cystic fibrosis (CF) disease, almost exclusively involving the genital tract, has prompted us to investigate the hypothesis of whether there could be an association between particular CF genotype mutations and the in-vitro performance of epididymal sperm. In this study 63 patients with surgically confirmed diagnosis of CAVD undergoing microsurgical epididymal sperm aspiration (MESA) and in-vitro fertilization (IVF) were evaluated. The genetic screening was carried out on DNA extracted from peripheral lymphocytes and amplified by the polymerase chain reaction. A total of 12 mutations in the cystic fibrosis transmembrane regulator gene (CFTR), representing approximately 90% of the total known CF mutations, were tested. The presence or absence of mutations, as well as the type of mutation found, was correlated with the IVF and pregnancy rates. Of the 63 patients examined, 40 (64%) were positive to the CF screening and 23 were negative. In three cases no spermatozoa were found because of rete testis blockage. The difference in fertilization rates between patients testing positive and negative was highly significant (P = 0.000003), 13 versus 23%, respectively. In order to pinpoint which mutation could account for the most deleterious effect on the IVF results, we analysed these by keeping the patients separated by CF mutations.(ABSTRACT TRUNCATED AT 250 WORDS)

Objectives: To assess the ability of sperm aspirated from the epididymis of men with congenital absence of the vas deferens to penetrate zona-free hamster oocytes. To directly compare the performance of human epididymal sperm in the zona-free hamster oocyte sperm penetration assay (SPA) with the results of human in vitro fertilization (IVF)., Design: Sperm penetration assay was carried out with epididymal sperm retrieved microsurgically, and with ejaculated sperm obtained from fertile donors (internal controls). For direct comparison, SPA was performed with the same epididymal sperm sample used for IVF., Patients, Participants: Men with congenital absence of the vas deferens undergoing sperm aspiration as part of their infertility treatment and control donors who provided ejaculated sperm., Results: Epididymal sperm penetrated SPA with a score of 0% to 30%. The SPA scores for internal controls using ejaculated sperm was 30% to 71%. Linear regression analysis of the association between penetration scores in SPA and fertilization rate in IVF indicated a positive correlation that was highly significative., Conclusions: These findings using SPA confirm previous reports on the fertilizing potential of human epididymal sperm and its ability to produce normal pregnancies. The good correlation between SPA and human IVF using epididymal sperm suggest that SPA is an excellent bioassay to test laboratory experimental conditions for improving fertilizing capacity of human epididymal sperm.

Objectives: To test, using the immunobead binding technique, for the presence of antisperm antibodies on epididymal sperm, in epididymal fluid, and in serum of men with congenital absence of the vas deferens. To evaluate the in vitro fertilization (IVF) capacity of human epididymal sperm in the presence of antisperm antibodies., Design: Prospective. At the time of oocyte insemination, sperm from the proximal caput epididymis or vasa efferentia were tested by direct immunobinding technique. The epididymal fluid and serum were tested by indirect immunobinding technique., Setting: Center for Reproductive Health, University of California-Irvine., Patients: Forty-five patients with congenital absence of the vas deferens participating in the microsurgical epididymal sperm aspiration and IVF program., Main Outcome Measure: Incidence of antisperm antibodies to epididymal sperm and their relationship with IVF results., Results: Sixteen men (35%) tested positively to the direct immunobead test on epididymal sperm; 7 (16%) were positive in epididymal fluid and 13 (29%) were positive in serum. Five pregnancies (31%) occurred in the positive group of which two were from patients who had sperm binding of 100% for immunoglobulin (Ig)G (all over sperm surface) and 90% (midpiece, tail) and 50% (tail, tiptail), respectively, for IgA. Five pregnancies (18%) were obtained in the negative group. No statistical difference was observed in the overall fertilization rate between the two groups., Conclusion: Human epididymal sperm and epididymal fluid retrieved from men with congenital absence of the vas deferens can react positively to immunobead test. However, the presence of antisperm antibodies do not seem to impair the IVF capacity of epididymal sperm.

This report describes the historical experience of our laboratory in the handling and processing of epididymal sperm from men with congenital absence of the vas deferens. Three different methods of sperm preparation were used: swim-up or resuspension in the first group of patients treated, mini-Percoll in the second group, and mini-Percoll used in conjunction with pentoxifylline and 2' deoxyadenosine in the third group. Twenty-four percent of cases achieved fertilization in group I, whereas in groups II and III the rate was 59% and 57%, respectively. Fertilization rates in each group were 6.5% in group I, 16% in group II, and 35% in group III. Three pregnancies were obtained in group I, 13 in group II, and 2 in group III. Groups II (21%) and III (63%) were able to have excess embryos to cryopreserve.

Objective: To investigate, by means of immunobinding technique, the antigenic surface expression of human vasa efferentia sperm as compared with that of ejaculated sperm., Design: Briefly, vasa efferentia sperm, retrieved microsurgically, and donors' ejaculated sperm (controls) were used., Patients, Participants: Men with congenital absence of the vas deferens, undergoing sperm aspiration as part of their infertility treatment, and controls who were donors of our sperm bank were first assayed by direct immunobead test and, if found negative, were exposed to sera containing known high titers of antisperm antibodies and then retested by indirect immunobead test., Results: The data showed no difference in binding titers and class of immunoglobulins between vasa efferentia and ejaculated sperm., Conclusions: These findings suggest that, in humans, sperm acquire those surface antigens commonly detected by immunobinding test within the testes before their transit through the epididymis.

Background: Congenital absence of the vas deferens has been considered a virtually untreatable cause of male sterility. Furthermore, sperm that have not passed through at least the head of the epididymis have been thought to be incapable of causing pregnancy. We attempted to determine whether human sperm that had never passed through the epididymis could fertilize eggs in vitro and whether the technique could be used for men with congenital absence of the vas deferens., Methods: Twenty-eight men with congenital absence of the vas deferens underwent microsurgical aspiration of sperm from the epididymis and vasa efferentia for attempted in vitro fertilization of their wives' oocytes, with subsequent transfer of embryos. Thirty-two treatment cycles were begun (four were repeat cycles)., Results: The most motile sperm were found in the proximal epididymis, at or near the vasa efferentia. Embryos were obtained for transfer in 21 cases (66 percent). Ninety-three embryos resulted from 352 mature oocytes (fertilization rate, 26 percent). Clinical pregnancy was achieved in 10 of the 32 treatment cycles (31 percent). Seven women delivered normal infants, and three miscarried. One of the seven live births was of twins. There were six girls and two boys. When fewer than 10 eggs were retrieved, no pregnancy occurred. When 10 or more eggs were retrieved (20 cases), the pregnancy rate was 50 percent., Conclusions: Sperm from the proximal caput epididymidis and even sperm from the vasa efferentia (which have never passed through the epididymis) can fertilize the human oocyte in vitro and result in pregnancy with live birth.

The development of the single beam gradient force optical trap has made it possible to manipulate cells solely by laser light. A continuous wave Nd:YAG (1.06 microns) laser beam was directed into a conventional microscope and focused onto the viewing plane by the objective lens. The laser beam power at which human sperm were released from the trap was measured and correlated to the sperm's linear velocity before trapping. The mean trapping power readings for slow, medium, and fast motile sperm were 57, 73, and 84 mW, respectively. The analysis of measurements over the total population demonstrated that zig-zag motile sperm had significantly higher mean power readings when compared with straight motile sperm with similar mean linear velocities. In two cases, specimens required significantly less trapping power when the measurements were repeated 24 hours later.

The relative contribution of factors affecting the probability of pregnancy from gamete intrafallopian transfer was estimated using data from 218 cycles performed in San Antonio, Texas, and Orange, California. Mature eggs, all of which contained an expanded cumulus, were subclassified according to the corona radiata; those with a starburst corona were considered the most fully mature. Sixty-two clinical pregnancies were established, representing a pregnancy rate of 28.4%. Excluding 49 cases of male factor infertility, the pregnancy rate was 34.9%. Pregnancy rates were not significantly different according to age, cause of female factor infertility, parity, duration of infertility, stimulation protocol, or estradiol level. The only variables having a strong association with pregnancy were sperm and egg parameters. Sperm motility and morphology were statistically more important than sperm density. Based on odds ratios calculated from multiple logistic analysis, pregnancy was 3.80 times more likely if three or more fully mature eggs were transferred, 0.34 times as likely if motility were less than or equal to 30%, and 0.22 times as likely if normal morphology was less than or equal to 50%, controlling for other egg and sperm factors. If all egg and sperm factors in a given cycle were unfavorable, the probability of pregnancy estimated by the logistic model was 4%, whereas if all were favorable the probability of pregnancy in that cycle was 59%.

The incidence of antisperm antibodies in serum and seminal fluid of 27 azoospermic men with congenital absence of the vas deferens is evaluated. The presence of antisperm antibodies was assessed using the immunobead test, the agglutination test, and immobilization test. Five patients with vasovasostomy or vasoepididymostomy attempts were included in the study and tested for the presence of antisperm antibodies. Contrary to a previous report, a low incidence (11%) of antisperm antibodies has been found in patients with congenital absence of the vas. In agreement with previous studies, in five patients who had failed vasoepididymostomy or vasovasostomy, a high incidence (71%) of antisperm antibodies was found.

The force generated by the radiation pressure of a low power laser beam induces an optical trap which may be used to manipulate sperm. We studied the effect of the optical trap on sperm motility. A Nd:YAG laser beam was coupled to a conventional microscope and focused into the viewing plane by the objective lens. Sperm were caught in the trap and manipulated by a joy stick controlled motorized stage. After different exposure periods, the velocity and patterns were analysed by a computerized image processor. There were minor changes in sperm velocity when exposed to the trap for 30 seconds or less. A gradual decrease in the mean linear velocity was observed after 45 seconds of exposure. This optical micromanipulator may also be useful for studying the force generated by a single spermatozoa and evaluating the influence of drugs on motility.