Glaxo/MRS Young Investigator Medal. Molecular studies on adenosine deaminase deficiency and hereditary haemorrhagic telangiectasia

Factorially arranged experiments were designed to study prefreeze packed cell volume (PCV) changes and associated percentages of motile and unstained bull sperm in simple macromolecule-free Tyrode's solution and egg yolk-Tris (EYT), varying in osmolarity, and with addition of rapidly permeating cryoprotectants, glycerol and 1,2-propanediol, and nonpermeating substances, sucrose and NaCl. The percentage of motile and unstained sperm was assessed after resuspending sperm in 300 mOsm/L Tyrode's solution. At 25 degreesC PCV increased in Tyrode's solution as osmolarity was decreased from 250 to 150 mOsm/L and decreased as Tyrode's solution was increased to 400 mosmol/L. The relationship of PCV to the reciprocal of the osmolarity was essentially linear over the range of 150 to 400 mOsm/L, but PCV did not decrease further in solutions ranging from 500 to 1000 mOsm/L. The percentage of motile sperm declined to zero in Tyrode's solution at 700 mOsm/L, but 40% of the sperm were still unstained in 1000 mOsm/L solutions. The addition of glycerol or 1,2-propanediol had little effect on PCV. With glycerol or 1,2-propanediol added to 308 mOsm/L Tyrode's solution to give a total of 1267 mOsm/L, there were 49 and 56% motile sperm, respectively, compared to 1% with NaCl added to give 787 mOsm/L. The PCV and percentage of motile sperm suspended in EYT responded to osmotic changes similar to those reported for Tyrode's solution at both 25 and 5 degreesC. Some sperm remained motile after initial exposure to 800 mOsm/L solutions. These findings may have application in improving bull sperm cryopreservation.     

A simple method for recovering the motile spermatozoa from extremely low quality sperm samples

Recovery of motile spermatozoa from extremely low quality samples for use in the intracytoplasmic sperm injection (ICSI) procedure is difficult. To solve this problem we developed a simple method to recover the motile spermatozoa using a 3% polyvinylpyrrolidone (PVP) droplet. After depositing a sperm pellet into this slightly viscous droplet, motile spermatozoa readily swam out to the clear area while immotile spermatozoa dispersed to a lesser extent, so that motile and immotile cells became clearly separated from each other. A total of 36 ICSI cycles using spermatozoa with extremely low quality characteristics were performed. We recovered the motile spermatozoa from all sperm samples from two sources of poor quality spermatozoa. Thirty-one cycles of ICSI with ejaculate resulted in fertilization and pregnancy rates of 54 and 29% respectively. Five cycles of ICSI with frozen-thawed epididymal spermatozoa resulted in fertilization and pregnancy rates of 70 and 60% respectively. The 3% PVP droplet method is very simple and easy to perform, so it may be useful for recovering the motile spermatozoa from extremely low quality sperm samples used for ICSI.     

Effect of culture, incubation and acrosome reaction of fresh and frozen-thawed ram spermatozoa for in vitro fertilization and intracytoplasmic sperm injection

This study evaluated different sperm treatments for fertilization of sheep oocytes by intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF). In Experiment 1, fresh and frozen semen was separated by Percoll centrifugation and incubated at 30 degrees C or 39 degrees C in HSOF or BSOF medium for 1 h before use for IVF or ICSI. For IVF, oocytes were inseminated and incubated with sperm for 30 min, 4 h and 19 h. Sperm were assessed for acrosome integrity after Percoll centrifugation and 1 h incubation, and those used for IVF were assessed after each period of exposure to the oocytes. Fertilization rates after ICSI were higher for fresh than for frozen-thawed sperm and were highest 19 h after IVF with fresh or frozen-thawed sperm in the presence of HSOF at 30 degrees C. In Experiment 2, fresh semen was separated by Percoll centrifugation and incubated for 5 h in HSOF, and the acrosome reaction was induced with lysophosphatidylcholine. Acrosome integrity was then assessed. Fertilization rates after ICSI were similar for acrosome-reacted and control spermatozoa. These results suggest that induction of the acrosome reaction in spermatozoa before ICSI is unnecessary, whereas a capacitating treatment of spermatozoa is required before IVF.     

Annual rhythmic variations of follitropin, lutropin, testosterone and sex-hormone-binding globulin in men

Four distinct studies were carried out using two data sets of percutaneous epididymal sperm aspiration (PESA) and intracytoplasmic sperm injection (ICSI) procedures performed from March 1993 to January 1997. In study A, an analysis of 181 ICSI treatment cycles following PESA revealed a successful epididymal sperm retrieval rate of 83%. It confirmed that PESA is an effective sperm retrieval method and the associated ICSI pregnancy rate (35% per embryo transfer) compared favourably with that of other sperm retrieval methods. In study B, the relevance of a prior diagnostic PESA procedure was ascertained by comparing the sperm retrieval rates in two groups of patients having their first ICSI treatment cycle with spermatozoa retrieved through PESA. Group B1 (n = 50) had diagnostic PESA prior to the ICSI treatment cycle PESA procedure, unlike patients in group B2 (n = 64) who did not. The sperm retrieval rate in the treatment cycle procedure was not different at 90 and 82.8% for groups B1 and B2 respectively. However, the discontinuation of diagnostic PESA is fraught with problems including liability to medico-legal sanctions. In study C, analysis of 177 treatment cycles involving PESA and ICSI revealed a successful sperm retrieval rate by PESA of 82% in the first cycle, 93% in the second, 96% in the third and 100% in the fourth cycle. The same trend was evident when sperm retrieval was examined in relation to each of the epididymides. Retrieved spermatozoa were found to be motile in 67-100% of cases and the frequency of samples containing motile spermatozoa did not decrease with increase in the number of PESA attempts. These results show that PESA does not jeopardize future epididymal sperm retrieval. In study D, the outcome of treatment with ICSI using ejaculated spermatozoa (305 cycles) (group D1) was compared with that of ICSI using spermatozoa obtained through PESA (54 cycles) (group D2). The median age of women in the two groups of couples was similar (34 years). In group D1, 70% of metaphase II oocytes were fertilized compared with 61% in group D2 (P < 0.01). The cleavage rate and the median numbers of transferred and cryopreserved embryos were similar in both groups. There was no significant difference between the clinical pregnancy rates (33 and 42% in groups D1 and D2 respectively). Our results show that the outcome of PESA-ICSI treatment compares favourably with that of ICSI using ejaculated spermatozoa.     

Bull sperm motility and membrane integrity in media varying in osmolality

Studies were designed to evaluate bull sperm motility and membrane integrity following exposure to Tyrode's solution varying in osmolality from 100 to 1537 mOsm. Congo red and bisbenzimide (HOECHST 33258) stains were used to distinguish between sperm with intact versus disrupted plasma membranes. Sperm motility was subjectively evaluated. No significant differences were found between motile and unstained sperm (sperm with intact membranes) in solutions with nearly physiologic osmolalities between 200 and 300 mOsm. However, in 100- and 150-mOsm solutions, the percentage of motile sperm (5 and 19%), respectively) was lower than the percentage of sperm unstained with Congo red (18 and 35%). With HOECHST 33258 stain, the corresponding values were 7 and 14% versus 26 and 31%. The percentage of motile sperm declined greatly in the 500-mOsm medium, but the proportion of unstained sperm was affected little until the osmotic pressure exceeded 732 mOsm. Partial recovery of motility occurred when sperm were returned to the isotonic medium. This study indicated that the sperm plasma membrane was more resistant to osmotic damage than were the mechanisms responsible for sperm motility. Lack of motility in hypertonic media was not an absolute indicator of cell death, and unstained sperm overestimated sperm viability, which are factors to consider when sperm are being evaluated, especially relative to cryopreservation.     

The heparin-glutathione test: an alternative to the hypo-osmotic swelling test to select viable sperm for intracytoplasmic sperm injection

OBJECTIVE: To evaluate the heparin-glutathione test (HEGLUT) for the selection of viable sperm for intracytoplasmic sperm injection (ICSI). DESIGN: A prospective study. SETTING: Department of Pediatrics, Obstetrics and Gynecology, University of Valencia and Instituto Valenciano de Infertilidad. PATIENT(S): Semen samples from healthy donors and patients with infertility. INTERVENTION(S): Sperm samples were kept in culture for different periods in Ham's F-10 medium supplemented or not supplemented with heparin, reduced glutathione (GSH), or a heparin-GSH mixture. Control and heparin-GSH-treated spermatozoa were injected into hamster oocytes. The HEGLUT and ICSI were performed. MAIN OUTCOME MEASURE(S): Sperm nuclear decondensation, progressive and nonprogressive motility, and male pronucleus formation. RESULT(S): The maximum proportion of sperm nuclear decondensation (28.7%+/-2.1% versus 2.6%+/-0.5% in the control group) was reached after 60 minutes of incubation in the presence of a heparin-GSH mixture. Differences in the percentages of progressive and nonprogressive motility among treatments and times of incubation, although statistically significant, were biologically negligible. No statistically significant differences were observed in the rate of sperm head decondensation (8.2% [4/49] versus 11.1% [6/54]) and male pronucleus formation (18.4% [9/49] versus 22.2% [12/541) after the injection of control and treated spermatozoa into hamster oocytes. CONCLUSION(S): The HEGLUT may offer an alternative to the hypo-osmotic swelling test for the selection of viable sperm for ICSI.     

Effect of the phosphodiesterase inhibitor Pentoxyfylline on human sperm motility

Tyrode fluid and Tyrode fluid plus Pentoxifylline were individually added to aliquots of semen samples obtained from 6 normal men and 6 infertile patients considered to have idiopathic normogonadotropic oligoasthenozoospermia. Pentoxifylline was added to final concentrations of 0.15, 0.30 and 0.60 mM. One aliquot with no addition served as control. Samples were incubated in 37 degrees C and observed by light microscopy at 30 minutes and at 1, 2 and 4 hours after obtaining the material. At observation time, semen quality was evaluated by determining the percentages of forwardly progressive spermatozoa, slowly progressive spermatozoa, "in situ" motile spermatozoa, live and non-motile spermatozoa and dead spermatozoa. Results reported included only the first and last category. Tyrode fluid did not affect significantly the motility and the duration of activity of spermatozoa. Ejaculated human spermatozoa both from normal and asthenozoospermic men added the Pentoxifylline at 0.30 and 0.60 mM showed a longer lasting activity than those of control semen and semen added only with Tyrode fluid.     

Patients with absolutely immotile spermatozoa and intracytoplasmic sperm injection

The microinjection of completely immotile spermatozoa may impair the outcome of intracytoplasmic sperm injection (ICSI). Eleven couples underwent an initial ICSI cycle with 100% immotile freshly ejaculated spermatozoa. Two-pronuclear fertilization ensued in 18 of 145 (12.4%) successfully injected oocytes. None of these cycles resulted in a pregnancy. Nine couples underwent ICSI in subsequent cycles (n = 16). Ejaculated spermatozoa were injected in 15 cycles and testicular spermatozoa in one cycle. In 10 of the 15 cycles, motile spermatozoa were available at the time of injection. Motile testicular spermatozoa could also be injected. In the subsequent cycles, 91 of 176 (51.7%) successfully injected oocytes fertilized normally and four patients became pregnant. In the subsequent cycles where again immotile spermatozoa had to be injected no pregnancies occurred. In four subsequent cycles embryo cryopreservation was carried out. After replacement of two frozen-thawed embryos one additional pregnancy was obtained. In all, five healthy infants were born. It has been ascertained that motile spermatozoa can be detected either in repeated ejaculates or after testicular biopsy. The causes of total asthenozoospermia are variable and the problem is a sporadic rather than a permanent condition.     

Prolonged survival of human spermatozoa when co-incubated with epididymal cell cultures

Human epididymal tissue was recovered from 11 patients undergoing orchidectomy without anti-androgen treatment. Everted epithelial fragments from the caput and corpus epididymis of six patients were successfully cultured in a modified RPMI 1640 medium supplemented with HEPES and androgens for up to 110 days (mean 56 +/- 28) in 5% CO(2) in air at 37 degrees C. Epithelial cells from human oviduct and non-reproductive tract cells (breast epithelial cells, fibroblasts) were also cultured for comparison. The proportion of epididymal epithelial cells in primary cultures assessed by immunofluorescent localization using a cytokeratin monoclonal antibody was shown to be >70% for the first 6-8 weeks of culture. Light and electron microscopy indicated that epithelial cells maintained polarity and some normal morphology during the culture period. Washed epididymal or ejaculated spermatozoa prepared by a 'swim-up' procedure were co-incubated (i) directly with epididymal cells in culture wells, (ii) in 12 mm Millicell inserts within culture wells, thereby preventing contact of spermatozoa with culture cells; and (iii) in culture medium alone. A significant proportion of spermatozoa in direct contact with culture cells or in Millicell inserts were viable after 6 days of co-incubation (30-45%) and exhibited progressive motility, while all spermatozoa in medium alone were non-motile by 3 days. Using computer-assisted sperm analysis it was shown that the progressive motility of viable spermatozoa decreased gradually for the first 5 days in culture and then remained constant (approximately 30 microm/s, average path velocity). After 12 days of co-incubation, 15 +/- 4% of spermatozoa in direct contact with epithelial cells remained motile; in one experiment, a few spermatozoa (<1%) were motile at 17 days. Light and electron microscope observations indicated that prolonged sperm survival was associated with close apposition of spermatozoa (by equatorial segment) to the apical membrane of epithelial cells. Oviductal epithelial cells were also beneficial for sperm survival, but other cell types had no effect.     

A new method for freezing testicular biopsy sperm: three pregnancies with sperm extracted from cryopreserved sections of seminiferous tubule

OBJECTIVE: To determine whether spermatozoa, located in the seminiferous tubules obtained by needle puncture testicular biopsy, could be cryopreserved successfully within the tubules and subsequently be used for in oocyte fertilization via intracytoplasmic sperm injection (ICSI) after the spermatozoa were removed from the thawed tubules. DESIGN: Clinical case series. SETTING: Private IVF unit. PATIENT(S): Six azoospermic patients (four obstructive, two maturation arrest). MAIN OUTCOME MEASURE(S): Survival rate of thawed spermatozoa, fertilization rate of oocytes after ICSI with spermatozoa extracted from thawed tubules and pregnancies. RESULT(S): All six patients had adequate motile spermatozoa extracted from the thawed tubule sections, and all patients achieved fertilization via ICSI with their partner's eggs. The fertilization rate was 46%, compared with 56% obtained in other previous patient cycles using fresh testicular spermatozoa. Three pregnancies resulted from five ETs. CONCLUSION(S): Cryopreservation and subsequent thawing of seminiferous tubules proved to be a simple and successful method for storage of testicular spermatozoa, reducing the need for repetitive testicular biopsies and increasing the likelihood of sperm availability on the day of oocyte retrieval.     

Serum levels of tumor necrosis factor in squamous cell carcinoma of the head and neck

Two experiments were conducted to study the effect(s) of Borrelia burgdorferi and its metabolites (toxicants?) on canine spermatozoa, using B burgdorferi type strain B-31 and ejaculates from 5 sexually mature dogs. In Experiment 1 the spirochetes were cocultured with semen and incubated under various conditions, and in Experiment 2 the spirochetes were sonicated to release the metabolites/toxicants. The sonicate was then cultured with the semen. The parameters investigated were kinematics and percentage of sperm motility, morphology, and sperm response to the hypoosmotic swelling test and acrosome reaction. There were no visible physical interaction between either dead or motile spirochetes and viable or dead spermatozoa. Neither the spirochetes per se nor their metabolites/toxicants had any significant adverse effect on the functional and morphological characteristics of the canine spermatozoa. It is possible that the exposure times for incubation were not long enough for metabolites or toxicants in the sonicate to significantly affect sperm characteristics. Some investigators have reported that B burgdorferi contain biologically active LPS-like endotoxins. It is also likely that storage denatures B burgdorferi metabolites and other intracellular products in the sonicate and, thus, negates any effects the medium or the sonicate might have on the spermatozoa. The apparent lack of effect suggests that either peripheral metabolism or action on other organ(s) may be required for deleterious action of the spirochetes and/or their toxicants on spermatozoa. It was concluded that B burgdorferi and/or metabolites/toxicants do not have any significant deleterious effects on the functional and morphological characteristics of the postspermatogenic spermatozoa. Interaction of the spirochetes with in vivo conditions may be needed to adversely affect spermatozoa.     

Persistent spermatozoa after vasectomy: a survey of British urologists

OBJECTIVES: To determine the rate of, and main indications for, repeat vasectomy in our department, and to assist in policy-making procedures by determining how urologists in England and Wales manage those men who show small but persistent quantities of motile or non-motile spermatozoa in their ejaculate after vasectomy. SUBJECTS AND METHODS: A retrospective review of all of the vasectomies and repeat vasectomies performed by the Urology Department at Southmead Hospital during a 14-month period was undertaken to determine the rate of and indications for repeat vasectomy. Subsequently, every consultant urologist in England and Wales was canvassed with a questionnaire to determine whether they repeated vasectomy in the presence of persistent motile or non-motile sperms and if so, after what time interval. Any experience of pregnancies arising from these groups was also assessed, and any relevant comments invited. RESULTS: The local review revealed that 5% of all vasectomies were repeated within 6-36 months. Of these, 87% were performed because of persistent sperms in post-vasectomy semen samples, the majority of which showed sperm concentrations of one in 50 to one in 100 high-power fields. A response of 56% was obtained to the questionnaire and of those responding, 23% never repeated a vasectomy where there were presistent non-motile sperms, but almost all urologists would eventually repeat vasectomy where motile sperms were present. The median interval between the first and second vasectomies was 6 months and 12 months for motile and non-motile sperm, respectively. Apart from those cases already published, there was little experience of pregnancy arising from men with persistently few motile or non-motile sperms. CONCLUSIONS: The risk of pregnancy occurring in the presence of non-motile sperms was estimated to be less than the established risk of late recanalization, and this survey provides both logical and medico-legal support for issuing a 'special clearance' to men with few persistent non-motile sperm after vasectomy, providing the risks of pregnancy are properly discussed and documented. For motile sperm, however, there appears to be a stronger precedent for repeating the vasectomy. The technique used for post-vasectomy semen analysis was also an important consideration when determining any policy regarding such cases.     

Three-dimensional low dose gadolinium-enhanced peripheral MR venography

Cases with absolute immotile sperm syndrome are rare, and include the genetic defect of immotile cilia syndrome with the absence of dynein arms in the flagellum. We attempted to increase the percentage of viable spermatozoa to improve the outcome of intracytoplasmic sperm injection (ICSI). Three couples in whom repeated analysis of the male partners indicated 100% sperm immotility underwent an in-vitro fertilization (IVF) procedure in which ICSI was performed. On their first ICSI cycle the males produced a single ejaculation while in their successive ICSI cycles they were requested to repeatedly ejaculate (two to four times) and only the last ejaculation was used. The eosin-Y test was performed on each used sample. Following their first treatment, one couple had one repeated treatment cycle, another had two and the third couple had three repeated treatment cycles. The mean percentages of viable spermatozoa were 41+/-7.4 and 71+/-6.9% in the first and repeated cycles respectively (P < 0.01; t-test). Of the 39 oocytes injected in the first ICSI cycles only one (3%) was normally fertilized (2PN) compared with 41 (48%) of the 85 oocytes injected in the repeated ICSI cycles. One (3%) embryo in the first and 35 (41%) embryos in the repeated ICSI cycles respectively were obtained (P < 0.001), enabling their replacement into the uterine cavity in all the repeated cycles. One woman (in a repeated cycle) conceived a twin pregnancy and delivered two healthy babies. The use of spermatozoa from repeated ejaculation is recommended in men with absolutely immotile spermatozoa so as to obtain significantly better viability and fertilizing capacity.     

Possible mechanisms involved in apoptosis of colon tumor cell lines induced by deoxycholic acid, short-chain fatty acids, and their mixtures

OBJECTIVE: To study the effects of cryopreservation on the quality of human testicular spermatozoa and the efficiency of intracytoplasmic sperm injection (ICSI) with frozen-thawed testicular sperm into metaphase II oocytes in vitro-matured from the germinal-vesicle stage oocyte. DESIGN: Preclinical freezing study on supernumarary testicular spermatozoa after ICSI. SETTING: Tertiary IVF center coupled with an institutional research environment. PATIENT(S): Twenty-nine patients undergoing excisional testicular biopsy for ICSI. INTERVENTION(S): Isolated testicular spermatozoa were cryopreserved and thawed; frozen-thawed motile testicular spermatozoa were microinjected. MAIN OUTCOME MEASURE(S): Prefreezing and post-thawing motility and viability, survival rate, fertilization rate, cleavage rate, and embryo quality after ICSI. RESULT(S): Mean percentage motility decreased from 21% before freezing to 6% after thawing. Vitality was impaired to a similar extent, decreasing from 68% to 22% (32% recovery rate). Injection of frozen-thawed testicular spermatozoa into in vitro-matured oocytes resulted in a fertilization rate of 50.9%. Cleavage rate was severely impaired. Half of the fertilized oocytes became arrested in the one-cell stage. CONCLUSION(S): Despite the low quality of the fresh testicular spermatozoa, a high percentage of prepared testicular sperm fractions showed survival and motility after the freezing and thawing process. Injection of frozen-thawed testicular sperm into matured oocytes resulted in fertilization rates comparable with these with fresh testicular sperm, but cleavage rates were severely impaired, which might be due to source of oocytes used for ICSI.     

Zona pellucida as physiological trigger for the induction of acrosome reaction

To evaluate the kinetics of acrosome reaction, sperm samples from four fertile donors were prepared by swim-up and incubated with solutions of human zonae containing 0.1, 0.15, 0.3, 0.5 and 1.0 zonae microliter-1. After 20, 40 and 60 min of incubation at 37 degrees C, aliquots were taken for evaluation of the acrosomal status. The results showed a distinct time- and dose dependence of the acrosome reaction induced by solubilized zona proteins. After 60 min of incubation in 1.0 zonae microliter-1, about 80% of the spermatozoa showed signs of acrosomal loss; about 40% were completely acrosome-reacted. In addition, zona-bound sperm showed the same ratios of acrosome-reacted spermatozoa in control experiments. The velocity of acrosome reaction was calculated by means of a double-reciprocal plot being 2.0-2.5% min-1 for completely reacted spermatozoa and those showing signs of acrosome reaction. However, both subgroups differed considerably in their constants of equilibrium (K = 2.0 ZP microliter-1 and K = 0.2 ZP microliter-1, respectively). In nonreacted and partly reacted spermatozoa results might indicate a disturbed course of acrosome reaction or possibly the existence of different subpopulations in respect of sperm competition.     

Effects of alprostadil and prazosin on motility, viability and membrane integrity of human sperm

PURPOSE: We evaluated the effects of alprostadil, prazosin hydrochloride, and alprostadil/prazosin hydrochloride, agents used in the clinical treatment of male erectile dysfunction, on the motility, viability and membrane integrity of human sperm. MATERIALS AND METHODS: Ten healthy volunteers provided semen samples that were incubated with 0.4 mg./ml. alprostadil, 0.1 and 0.2 mg./ml. prazosin hydrochloride and 0.4 mg./ml. alprostadil plus 0.1 mg./ml. prazosin hydrochloride for 2 hours. Control incubations included polyethylene glycol 1450, the formulation vehicle for the clinical use of alprostadil and prazosin, and Ham's F-10 buffer. Serial evaluations of percent sperm motility, percent viability, membrane function (by hypo-osmotic swelling test) and several computer generated measurements of sperm motion, including straight line velocity, curvilinear velocity, linearity and amplitude of lateral head displacement, were made. RESULTS: None of the agents had a significant impact on the percentage of motile or viable sperm or on sperm membrane function. Incubation with 0.2 mg./ml. prazosin reduced straight line velocity and curvilinear velocity significantly compared with the other agents. These changes were most likely a direct result of the viscosity of the 0.2 mg./ml. prazosin solution and not a cellular or metabolic effect on the sperm. CONCLUSIONS: Alprostadil and prazosin hydrochloride at doses used in transurethral therapy for erectile dysfunction have no effect on the motility, viability and membrane integrity of human sperm.     

Y chromosome deletions in azoospermic and severely oligozoospermic men undergoing intracytoplasmic sperm injection after testicular sperm extraction

Y chromosome deletions encompassing the AZFc region have been reported in 13% of azoospermic men and 7% of severely oligozoospermic men. We examined the impact of these Y deletions on the severity of testicular defects in 51 azoospermic men undergoing intracytoplasmic sperm injection (ICSI) after testicular sperm extraction (TESE) and 30 men with severe oligozoospermia undergoing ICSI after ejaculation of spermatozoa. In addition, five azoospermic patients shown previously to have Y chromosome deletions underwent histological evaluation of their previously obtained testis biopsy specimens. A further 27 azoospermic men underwent TESE-ICSI, but not Y chromosome DNA testing. Ten of 51 azoospermic men (20%) who underwent TESE-ICSI and Y-DNA testing were found to be deleted for portions of the Y chromosome AZFc region. Of these 10, five had spermatozoa retrievable from the testis, and in two cases the wives became pregnant. Of the 41 azoospermic men with no Y chromosome deletion, 22 (54%) had spermatozoa retrievable from the testis, and in 12 cases (29%) the wives became pregnant. Four of 30 (13%) severely oligozoospermic patients were found to be deleted for AZFc and in three (75%) of these pregnancy was achieved. The other 26 severely oligozoospermic couples who had no AZFc deletions underwent ICSI, and 12 (46%) have an ongoing or delivered pregnancy. The embryo implantation rate was not significantly different for azoospermic (22%), oligozoospermic (16%), Y-deleted (14%) or Y-intact (18%) men. Of the total of 19 infertile men who had Y chromosome deletions, 14 had deletions within Y chromosome intervals 6D-6F, in the AZFc region. Twelve of those 14 had some spermatozoa (however few in number) in the ejaculate or testis. Five of the Y-deleted men had deletions that extended more proximally on the Y chromosome, and in none of these could any spermatozoa be observed in either ejaculate or testis. These results support the concept that, in azoospermic or oligozoospermic men with Y chromosome deletions limited to intervals 6D-6F (AZFc), there are generally very small numbers of testicular or ejaculated spermatozoa. Larger Y deletions, including and extending beyond the AZFc region and encompassing more Y genes, tend to be associated with a total absence of testicular spermatozoa. In those cases where spermatozoa were retrieved, the presence of Y deletions had no obvious impact on fertilization or pregnancy rate.     

Pregnancy following intracytoplasmic sperm injection from an HIV-1-seropositive man

The first pregnancy achieved in a seronegative woman following in-vitro fecundation through intracytoplasmic sperm (ICSI) injection from a man with autoimmune deficiency syndrome (AIDS; HIV-1 carrier) is reported. The semen was prepared by PureSperm and swim-up techniques. Some of the motile spermatozoa obtained were used to detect the presence of HIV-1 using the polymerase chain reaction technique. HIV-1 in DNA or RNA form was not detected using this technique. The remaining spermatozoa were frozen. Ovarian stimulation in the woman was performed with long-protocol analogues and gonadotrophins. Thirteen mature oocytes were recovered, into which the thawed spermatozoa were microinjected. Nine embryos were obtained. Four were frozen, four transferred and one discarded. The woman became pregnant. Analyses for HIV-1 in the woman, performed in the first and third months of pregnancy, gave negative results. This case provides further experience with washed semen of sufficient quality for performing artificial insemination in HIV-1-serodiscordant couples (101 inseminations, 31 pregnancies, 28 deliveries, 37 babies, all healthy). In women with obstructed Fallopian tubes, or when the semen is not of sufficient quality for artificial insemination techniques to be performed, ICSI can be carried out using frozen, HIV-1-free semen.     

Testicular sperm retrieval by percutaneous fine needle sperm aspiration compared with testicular sperm extraction by open biopsy in men with non-obstructive azoospermia

The efficiency of testicular sperm retrieval by testicular fine needle aspiration (TEFNA) was compared with open biopsy and testicular sperm extraction (TESE), in 37 rigorously selected patients with non-obstructive azoospermia. All patients underwent TEFNA and TESE consecutively. Thus, each patient served as his own control. The case was regarded as successful if at least one testicular spermatozoon was found allowing intracytoplasmic sperm injection (ICSI) of at least one oocyte. The mean age of the male patients was 32.7 years (range 24-47). Whereas by TEFNA spermatozoa enabling performance of ICSI were found in only four patients out of 37 (11%), open biopsy and TESE yielded spermatozoa in 16 cases (43%). The negative predictive value of high serum follicle stimulating hormone (FSH) concentrations (> or =10 IU/l) (predicting failure to find spermatozoa for ICSI) was low (38.4%). The positive predictive value (predicting the chance to find spermatozoa for ICSI) of normal-sized testicle was not different from that of small-sized (<15 ml) testicle (50%). Complications included one case of testicular bleeding following fine needle aspiration, treated locally, and two cases of extratunical haematomata following TESE requiring no intervention. In patients with non-obstructive azoospermia, TEFNA has a significantly lower yield compared to TESE. Performance of ICSI with testicular sperm in these cases resulted in satisfactory fertilization and high embryo transfer rates. The implantation and pregnancy rates per embryo transfer were 13 and 29% respectively. Neither serum FSH values nor testicular size were predictive of the chances to find spermatozoa for ICSI. Some complications may occur even following TEFNA.     

Physical activity in usual occupation and risk of breast cancer (United States)

OBJECTIVE: To identify whether the cause, site of ductal obstruction, and characteristics of fluid aspirates are associated with the cryosurvival and fertility after thawing of sperm obtained during reconstruction of the excurrent ducts with microsurgical epididymal sperm aspiration, vasal sperm aspiration, or both. DESIGN: Prospective study. SETTING: Andrology center at a tertiary care institution. PATIENT(S): Men undergoing reconstruction of the excurrent duct and sperm aspiration (n = 42) or microsurgical epididymal sperm aspiration (n = 11). INTERVENTION(S): Sperm were tested for an association with the cause and site of obstruction. Fertilization and pregnancy rates after sperm aspiration and intracytoplasmic sperm injection (ICSI) were evaluated for fresh and frozen aspirates. MAIN OUTCOME MEASURE(S): Motile sperm count and percentage motility after thawing. RESULT(S): The motile sperm count before freezing was significantly higher in the caput epididymis than in the corpus. The motile sperm count before freezing was related inversely to the distance from the caput where the sperm were aspirated. Sperm from clear and opaque fluid aspirates had better percent motility than those from cloudy and creamy fluid aspirates. High fertilization and pregnancy rates were achieved using both fresh and frozen epididymal sperm. CONCLUSION(S): None of the factors studied was associated with cryosurvival of aspirated epididymal or vasal spermatozoa. Because motility is low after thawing, these specimens are best used with ICSI.