Efficacy of the second micro-testicular sperm extraction after failed first micro-testicular sperm extraction in men with nonobstructive azoospermia.

OBJECTIVE: To evaluate the efficacy of the second micro-testicular sperm extraction (TESE)in men with nonobstructive azoospermia in whom the first micro-TESE failed. DESIGN: Retrospective. SETTING: Private clinic. PATIENT(S): One hundred twenty-five men with nonobstructive azoospermia with failed previous micro-TESE. The patients were divided into 2 groups according to their surgical sperm retrieval status during the second micro-TESE. If sperm could not be found, these patients were classified as Group 1, and, if sperm was found, the patients were classified as Group 2. The 2 groups were compared for clinical parameters and pathologic findings. INTERVENTION(S): Micro-TESE. MAIN OUTCOME MEASURE(S): Surgical sperm retrieval status. RESULT(S): Sperm was recovered successfully in 23 of 125 (18.4%) men with the second micro-TESE. Testicular volume was significantly lower in Group 2 (8.2 ± 5.4 mL) than Group 1 (11.3 ± 5.3 mL). Seven of 14 (50%) patients with Klinefelter's Syndrome had sperm recovery with repeat micro-TESE. The sperm retrieval rate was significantly higher in the Leydig cell hyperplasia and tubular sclerosis groups than in the Sertoli cell only and maturation arrest groups (54.5%, 10.1%, and 18.6%, respectively). CONCLUSION(S): On the basis of our results, 18.4% of men with failed first micro-TESE had a probability of sperm retrieval with the second micro-TESE. Patients with successful sperm recovery had smaller testicular volumes than those with a failed second attempt. Severe testicular atrophy was not a contraindication for the second micro-TESE in such patients.

Microfluidic System for Rapid Isolation of Sperm From Microdissection TESE Specimens.

OBJECTIVES: To demonstrate a novel prototype microfluidic system for rapid isolation of sperm from real and simulated microdissection testicular sperm extraction samples. METHODS: The novel microfluidic system was tested using minced testicular biopsies from patients with nonobstructive azoospermia. The samples were split into 2 portions, conventional processing vs microfluidic. The embryologists were blinded to the processing protocol and searched the specimens for sperm after processing. We recorded the number of sperm found and the time to sperm identification and compared the sperm retrieval rates. RESULTS: When compared to conventional methods, samples processed through the microfluidic system were cleaner (decreased somatic cells/debris), with the average number of sperm identified per minute improving from 1.52 sperm per minute for the control and 13.5 sperm per minute with the device yielding an 8.88 fold improvement in the sperm found per minute for the device as compared to the control. Preliminary viability and morphology tests show a minimal impact on sperm processed through the microfluidic system. CONCLUSION: The presented microfluidic system can facilitate rapid and efficient isolation of sperm from microdissection testicular sperm extraction samples. A prospective clinical trial to verify these results is needed to confirm this preliminary data.

Successful pregnancy in double factor infertility: Sperm in vitro culture by modified testicular fine-needle aspiration.

INTRODUCTION: Infertility may depend up to 27% of couples on both partners. In patients with obstructive azoospermia, testicular fine-needle aspiration represents a good option to retrieve spermatozoa, in order to perform an assisted reproductive treatment. In vitro maturation of testicular spermatozoa could be the better choice of treatment in view of the increased motility, improving fertilization and pregnancy rates. CASE DESCRIPTION: A 34-year-old azoospermic man and his 33-year-old partner referred for treatment of simultaneous male and female infertility factor. The woman presented a diminished ovarian reserve, with serum follicle stimulating hormone value of 27.15 IU/L. The man underwent trans-rectal and testicular ultrasounds that detected the congenital absence of proximal vas deferens on the right side and the absence of seminal vesicle and distal vas deferens on the left side. We proposed a chance to have their own biological child. The man underwent modified testicular fine-needle aspiration using a 18-gauge butterfly needle. Sperm retrieval was successful with 0.001 × 106 spermatozoa/mL and absence of motility. Testicular sperm suspension was cultured for 24 h to identify sperm viability, achieving 10% of sperm motility. Two metaphase II oocytes were retrieved and processed with intracytoplasmic sperm injection. Clinical pregnancy with live birth was obtained. CONCLUSION: Performing modified testicular fine-needle aspiration increases successful sperm retrieval. Testicular sperm in vitro culture for 24 h proved to be a real and practical technique to increase sperm motility, in order to select mature and viable spermatozoa and improve successful intracytoplasmic sperm injection outcomes.

Effectiveness of urethral catheterization under ultrasound guidance for semen collection from Asiatic black bears (Ursus thibetanus).

The Asiatic black bear (ABB; Ursus thibetanus ussuricus) is a globally endangered species, and measures to help increase their population are necessary. For the successful restoration of this species, artificial breeding as well as conservation translocation are considered important. The aims of the present study were to evaluate the feasibility and effectiveness of urethral catheterization (UC), which is effectively used in feline species, for semen collection from ABBs and establish the optimal protocol for semen collection via this technique. Seven clinically healthy, adult male ABBs (age, 6-13 years; weight, 130-180 kg) housed at the Species Restoration Technology Institute, Korea were included in this study. All study procedures were performed during the breeding season (June to August) over 3 consecutive years. Semen samples were collected once or three times from all bears by ultrasound-guided UC or electroejaculation (EE) under general anesthesia, and their characteristics, including sperm motility, were evaluated. The day of semen collection was defined as Day 0. The semen collected by the UC method was stored at 4 °C, and sperm motility was evaluated at the same time every day for 16 days. The successful collection rates for the UC and EE methods were 92.3% and 53.8%, respectively. The sperm concentration (4718.9 ±â€¯1526.1 vs. 185.0 ±â€¯34.2 × 106/ml), total sperm count (1196.6 ±â€¯955.5 vs. 100.9 ±â€¯70.0 × 106), sperm motility score (4.39 ±â€¯0.78 vs. 3.00 ±â€¯1.73), viability (98.2 ±â€¯2.3 vs. 82.7 ±â€¯19.6), and the proportion of spermatozoa with intact acrosomes (92.2% ±â€¯9.3% vs. 75.6% ±â€¯10.6%) were higher with the UC method than with the EE method, whereas the proportion of spermatozoa with an abnormal morphology (23.1% ±â€¯4.6% vs. 45.6% ±â€¯19.5%) was lower with the former than with the latter. Over the course of cool storage, there was an overall decrease in the total motility, progressive motility, and viability, although viability was >50% until Day 10. These findings suggest that ultrasound-guided UC is a useful and feasible tool for the collection of high-quality semen from ABBs. The collected semen remains viable for up to 10 days, with high sperm motility maintained for up to 7 days, when stored at 4 °C.

Modeling of behavioral responses for successful selection of easy-to-train rams for semen collection with an artificial vagina.

The aim of this study was to analyze the reproductive behavioral responses in Australian Merino rams, to identify those related to a faster training for semen collection with an artificial vagina. Eight Australian Merino rams, aged 1.5 years and with no prior sexual experience, were randomly selected from an extensively grazed flock. One immobilized ewe with no hormone stimulation was used for rams to sexually interact and mount. The frequencies of approaching, sniffing, flehmen, pushing, pawing with chin resting, and tongue flicking were recorded during eight training and three post-training assessments periods. In addition, the duration of sniffing and flehmen responses, as well as the time from when the ram started to approach the ewe until the mount with ejaculation (completed mount) were recorded. Descriptive, correlation, and modeling analyses were performed. Amongst the rams, four mounted the ewe and ejaculated for the first time during the training phase, and three mounted and ejaculated for the first time after the training phase. The remaining ram mounted the ewe and ejaculated for the first time during the post-training evaluation in the following year. A great variability in the behavior repertoire was observed among rams. The correlation analysis indicated that the completed mount was associated with the behaviors during the approaching response. The expression of the sniffing response decreased between the training phase and post-training evaluation, while the responses of pushing the ewe and tongue flicking ceased to occur. Pawing the side of the ewe with the chin resting on the back of the ewe and flehmen responses, however, continued between the training and post-training phases. This led to a decrease in the time from when the ram started to approach the ewe until the completed mount. It is concluded that the responses of approaching the ewe, pawing the side of the ewe with chin resting on the ewe, and sniffing of the ewe (the latter occurring only during the training phase) are behavioral indicators that could be used for selection of easy-to-train rams for purposes of semen collection with an artificial vagina.

Using microscope for onco-testicular sperm extraction for bilateral testis tumors.

OBJECTIVE: To demonstrate a step-by-step approach to the use of the operating microscope for onco-testicular sperm extraction. DESIGN: Video presentation. SETTING: University hospital. PATIENT(S): A 34-year-old man (status post right orchiectomy at another institution for pT3 pure seminoma with negative preoperative tumor markers) was referred for contralateral orchiectomy for multifocal left testis mass and fertility preservation. A postoperative semen analysis for attempted cryopreservation of ejaculated semen identified azoospermia. INTERVENTION(S): Left radical orchiectomy, left microsurgical onco-testicular sperm extraction (TESE). MAIN OUTCOME MEASURE(S): Intraoperative technique with commentary highlighting tips for successful fertility preservation via microsurgical onco-TESE. Discussion of alternatives. RESULT(S): This video provides a step-by-step guide to microsurgical onco-TESE coordinated with radical orchiectomy for testis cancer as a means of fertility preservation in an azoospermic patient. Preoperative imaging with scrotal ultrasound can serve as a useful guide for targeting microdissection to areas of normal testicular parenchyma for extraction of seminiferous tubules likely to host normal spermatogenesis. This patient had successful recovery and cryopreservation of abundant testicular sperm following targeted ex-vivo testicular microdissection. CONCLUSION(S): Microsurgical onco-TESE may be offered to azoospermic patients when undergoing orchiectomy for testis cancer. Use of preoperative imaging and the surgical microscope guide surgical dissection and optimize sperm recovery.

Evaluation of Prognostic Factors and Determinants in Surgical Sperm Retrieval Procedures in Azoospermic Patients.

INTRODUCTION: Main categorisation of azospermic is in two grups: nonobstructive azoospermia (NOA) and obstructive azoospermia (OA). We had evaluation of prognostic factors and determinants in sperm retrieval procedures in azoospermic patients. METHODS: Retrospective evaluation observed 21 selected patients with NOA and OA azoospermia, after that complete history, physical examination with ultarsound volume of testis and hormonal profile. Hormonal profile included: follicle stimulating hormone (FSH), luteinizing hormone (LH), testosteron (T) and prolactin (P) serum levels. Also karyotype and Y-deletion analysis were done and analyzed. RESULTS: 9 OA patients (42,9%) were undergone for TESE operation and 12 NOA patients (57,1%) for Micro-TESE operation. All TESE procedures were positive (100%). Micro-TESE in 12 selected NOA patients, 5 patients (41,6%) were positive and 7 patients (58,4%) negative. Patients testicular size, serum FSH and testosterone level showed correlation in success of sperm retrieval procedures. CONCLUSION: TESE is elected procedure for obstructive azospermia (OA). Micro-TESE is appropriate sperm retrieval procedurec for patients with non-obstructive azoospermia (NOA) and correlate with high FSH and small volume of testis.

Comparison of three methods of penile vibratory stimulation for semen retrieval in men with spinal cord injury.

STUDY DESIGN: Prospective, three-way crossover design. OBJECTIVE: Penile vibratory stimulation (PVS) is recommended as the first line of treatment for semen retrieval in anejaculatory men with spinal cord injury (SCI). This study compared ejaculatory success rates and patient preference for three methods of PVS within the same group of men with SCI. SETTING: Major medical university. METHODS: Fifteen men with SCI each received three methods of PVS. Method 1 (M1): applying one FertiCare Personal device to the dorsum or frenulum of the glans penis; Method 2 (M2): 'sandwiching' the glans penis between two FertiCare devices; Method 3 (M3): sandwiching the glans penis between the two vibrating surfaces of the Viberect-X3 device. The order of M1, M2 and M3 was varied to control for sequencing effects. Following each PVS trial, subjects rated their experience on a questionnaire with scaled responses. RESULTS: Ejaculation success rates were high for each method; however, ejaculation latency was significantly longer with M3 compared with M1 or M2. In survey questions about patient preference, there were no significant differences between M1 and M2. In contrast, M3 was rated lower than M1 and M2 in patient preference. Semen collection may be more difficult with the Viberect device. CONCLUSIONS: On the basis of these findings, we recommend attempting PVS with one FertiCare device. If that fails, use two FertiCare devices. Although the Viberect-X3 was preferred less by patients, it had similar efficacy as the Ferticare vibrator(s) and may be suitable for home use by some patients.

Multiple needle-pass percutaneous testicular sperm aspiration as first-line treatment in azoospermic men.

Percutaneous testicular sperm aspiration (TESA) has been known for decades as a simple, minimally invasive approach to sperm retrieval in azoospermic men. Because of lower reported sperm retrieval rates (SRR) when compared with microdissection testicular sperm extraction (mTESE), many centers now use mTESE as the first choice for retrieving spermatozoa in nonobstructive azoospermia (NOA). Objectives of this study were to evaluate the outcome and safety of TESA and mTESE in the treatment of azoospermia and to investigate the usefulness of a prognostic TESA to individualize protocols for couples and limit the use of invasive testicular procedures. IRB approval was obtained to retrospectively evaluate 208 patients undergoing multiple needle-pass TESA between 1999 and 2014. Prognostic TESA was performed on 125 men with NOA and 82 with obstructive azoospermia (OA). Nine NOA men and 31 OA men with previously demonstrated spermatozoa had a subsequent therapeutic TESA while nine NOA men with a failed TESA proceeded to mTESE. Main outcome measures were complication rates and SRR. SRR of the prognostic TESA was 30% (38/125) for NOA men and 100% (82/82) for OA men. Eight/nine NOA men and 31/31 OA men had spermatozoa found for intracytoplasmic sperm injection in a subsequent therapeutic TESA. In nine NOA men in whom a TESA produced no spermatozoa, only one had spermatozoa found with mTESE. Overall complication rates of TESA and mTESE were 3% (7/267) and 21% (3/14), respectively. TESA provides reasonable SRR and is a safe procedure. Successful prognostic TESA indicates future success with therapeutic TESA. Men with a failed TESA have a limited chance of sperm retrieval using mTESE. Approaching azoospermic men with an initial prognostic TESA followed by either therapeutic TESA and/or mTESE is an efficient algorithm in the management of azoospermia and limits the use of more invasive procedures.

Semen collection methods affect the bacterial composition of post-thawed semen of silver barb (Barbodes gonionotus).

Biosafety issue associated with the risk of pathogenic contamination of cryopreserved semen is a common concern because of associated declines in sperm quality, storage period and disease transmission. This study was conducted to evaluate the effects of methods of semen collection on sperm quality and bacterial composition of post-thawed semen of silver barb (Barbodes gonionotus). Semen collection methods consisted of four treatments: (1) hand-stripping of abdomen without rinsing of urogenital area with water, (2) hand-stripping of abdomen after rinsing of urogenital area with water, (3) catheterization without rinsing of urogenital area with water and (4) catheterization after rinsing of urogenital area with water. Semen diluted with calcium-free Hank's balanced salt solution containing 10% dimethylsulfoxide (DMSO) was frozen at a freezing rate of -8°Cmin(-1) before plunging in liquid nitrogen. Post-thawed semen collected by catheterization after rinsing urogenital area had the lowest bacterial number, about 2-log reduction of total heterotrophic, Gram negative and pseudomonad bacteria, compared with the other three collection treatments. However, percentages of motile and viable sperm were not significantly (P>0.05) different among treatments. This method eliminated Flavobacterium aquatile, Bacillus megaterium, Kocuria varians, Staphylococcus haemolyticus and Aeromonas media in cryopreserved semen. This is the first report demonstrating the effects of semen collection methods on bacteriological quality of frozen-thawed fish semen.

Separation efficiency of a microfluidic sperm sorter to minimize sperm DNA damage.

OBJECTIVE: To evaluate whether microfluidic sperm sorters (MFSSs) allow effective recovery of sorted motile sperm without DNA damage compared with the centrifugation and swim-up procedure. DESIGN: Experimental laboratory study. All participants completed questionnaires regarding previous and/or current diseases, surgery, reproductive experiences, lifestyle factors, and date of the preceding ejaculation. SETTING: University research laboratory. PATIENT(S): Male volunteers were recruited without setting conditions. Semen samples from healthy volunteers (n = 37) were collected in sterile containers by masturbation. INTERVENTION(S): Flow cytometric measurement and sperm chromatin structure assay analysis of DNA damage after sperm preparation using MFSS and the centrifugation and swim-up procedure. MAIN OUTCOME MEASURE(S): Efficacy and efficiency of sperm preparation, correlation between sperm DNA fragmentation index (DFI) and semen parameters, and relationship between basic characteristics and DFI after the centrifugation and swim-up procedure. RESULT(S): Final sperm concentration and motility were significantly different between the centrifugation and swim-up procedure and MFSS sperm preparations. A significantly lower sperm DNA fragmentation rate was detected with MFSS compared with the centrifugation and swim-up procedure use. No correlation was observed between DFI and smoking or drinking, but significant correlations were observed between DFI and medication use and sexual abstinence duration. CONCLUSION(S): MFSSs can be used to efficiently and reliably prepare sperm compared with the centrifugation and swim-up procedure. Further research on the clinical use of MFSSs is required to evaluate the safety and usefulness of this device.

Current and evolving uses of optical coherence tomography in the genitourinary tract.

Optical coherence tomography is an emerging imaging modality that provides high-resolution, real-time, cross-sectional visualization of urologic tissue with promising results. Early studies have demonstrated detailed, accurate histologic information of tissues sampled. Optical coherence tomography (OCT) has also been applied in evaluating malignancy of the bladder, prostate, and kidney. In the bladder, it can assist in the identification, biopsy, and intraoperative resection of lesions suspicious for bladder cancer. Intraoperative use of OCT during radical prostatectomy can improve visualization of the neurovascular bundle and surgical margins. Several small, ex vivo studies have also shown promising results in the ability of OCT to demonstrate histopathologic alterations to renal morphology such as in renal ischemia and malignancy. In men with non-obstructive azoospermia, OCT has also been used in improving sperm retrieval rates by assisting in the identification of tubules with isolated foci of spermatogenesis. Common limitations of OCT include limited depth of penetration and limited number of current clinical studies.

In vivo serial sampling of epididymal sperm in mice.

This study was undertaken to refine the techniques of in vivo collection of sperm in the mouse. The principal objective was to offer a viable, safe and reliable method for serial collection of in vivo epididimary sperm through the direct puncture of the epididymis. Six C57Bl/6J males were subjected to the whole experiment. First we obtain a sperm sample of the right epididymis, and perform a vasectomy on the left side. This sample was used in an in vitro fertilization (IVF) experiment while the males were individually housed for 10 days to let them recover from the surgery, and then their fertility was tested with natural matings until we obtained a litter of each one. After that, the animals were subjected another time to the same process (sampling, recover and natural mating). The results of these experiments were a fertilization average value of 56.7%, and that all the males had a litter in the first month after the natural matings. This study documented the feasibility of the epididimary puncture technique to in vivo serial sampling of sperm in the mouse.

Optimizing TESE-ICSI by laser-assisted selection of immotile spermatozoa and polarization microscopy for selection of oocytes.

For most azoospermic men testicular sperm extraction (TESE) is the only treatment, however it presents challenges for the ART laboratory, as the retrieval of motile spermatozoa is difficult. In the absence of sperm movement no unequivocal distinction can be made between either dead or immotile, but vital spermatozoa. However, a single laser shot directed to the tip of the tail allows recognition of viability because the flagellum coils at the area of impact. To rank the quality and the maturity of oocytes, polarization microscopy can be used. The zona score and the visualization of the meiotic spindle correlate with implantation and pregnancy rates. We compared 65 TESE-ICSI cycles of the years 2007 and 2008 (Group 1, G1) with 58 TESE-ICSI cycles of the years 2009 and 2010 (Group 2, G2). Testicular spermatozoa were injected according to motility and morphology into selected oocytes. In G1 both, oocyte and spermatozoa were rated using light microscopy only, whereas in G2 the laser was used for sperm selection and the oocytes were rated by light and polarization microscopy. In G2 we enhanced our fertilization rate (FR) significantly in comparison to G1 (G1 42.1% vs. G2 52.7%, p < 0.001). The fertilization rate with immotile, but vital spermatozoa improved significantly when applying laser-based selection (p = 0.006). The laser selection of immotile spermatozoa and the use of polarization microscopy can enhance the FR of TESE-ICSI. No negative effect of the laser was seen on birth rates. The FR with immotile, but vital spermatozoa clearly benefits from laser selection and is a non-hazardous and safe method for the selection of viable but immotile sperm. To our knowledge this is the first report using new technology creating novel endpoints for the analysis of spermatozoa and oocytes in TESE-ICSI.

Efecto de la cámara de recuento utilizada sobre los parámetros espermáticos, analizados con el ISASv1® / Effect of counting chamber on seminal parameters, analyzing with the ISASv1®

Introducción: El uso de las cámaras de recuento condiciona los valores de concentración y movilidad espermática debido a su tipo de carga (capilaridad o desplazamiento de la gota) y a la altura que presenten. Recientemente, se han introducido nuevas cámaras, tanto desechables (ISAS®D4C) como reusables (Spermtrack®) con diversas alturas en cada caso. Objetivo: El objetivo del presente trabajo es validar su uso, analizando diversos parámetros espermáticos con un sistema CASA, el ISASv1®. Material y método: Se analizaron muestras de donantes voluntarios (10 para las desechables y 5 para las reusables), analizando el efecto del tipo de cámara, de la altura, del tiempo de carga y del tiempo de análisis. Resultados: Los resultados obtenidos no mostraron diferencias significativas con el tiempo de carga para las cámaras reusables ni con el tiempo de análisis en ninguna de las cámaras. Discusión: La cámara reusable de 10 μm y las desechables de 10, 16 y 20 μm no mostraron diferencias entre sí, mientras que la reusable de 20 μm presentó resultados significativamente diferentes con respecto a las demás, por lo que su uso (siguiendo la recomendación de la Organización Mundial de la Salud) debe hacerse teniendo en cuenta este hecho. Conclusión: Las nuevas cámaras ofrecen un resultado muy homogéneo, con independencia del tiempo de carga o de análisis (AU)

Introduction: The use of counting chambers conditions the sperm concentration and motility values by their type of filling (capillary or drop displacement) and chamber height. We have recently introduced new chambers, both disposable (ISAS®D4C) and reusable (Spermtrack®), with different heights in each case. Objective: This study has aimed to validate its use by analyzing different sperm parameters with a CASA system, that is, the ISASv1®. Material and method: Samples from volunteer donors (10 for the disposable and 5 for reusable), analyzing the effect of chamber type, height, filling and analysis time, were used. Results: The results obtained showed no differences within the filling time for the reusable chambers, or the analysis time in any cases. Discussion: The 10 microns reusable chamber and disposable, 10, 16 and 20 microns showed no differences between them. However, the reusable 20 microns showed significantly different results, so its use (as recommended by WHO) should be taking this into account. Conclusion: The new chambers offer very consistent results, independent of filling or time of analysis (AU)

An ultrasonically actuated silicon-microprobe-based testicular tubule assay.

Microdissection testicular sperm extraction (TESE) is an invasive surgical procedure in which sparsely located healthy larger diameter tubules carrying viable spermatazoa are identified by visual examination of the seminiferous tubules of the infertile testis under a microscope, and biopsies of regions of interest are performed. In this paper, we report on microfabricated silicon microprobes integrated with an ultrasonic horn actuator and strain gauges for microdissection probe-TESE (MP-TESE) surgery. The microprobes, with axial-force-sensitive polysilicon strain gauges, have high force sensitivity (-0.4 V/N). The probes were used to detect the boundaries between seminiferous tubules, thus enabling identification of individual tubule diameters. Insertion experiments were performed on rat testis tissue, and by monitoring the tubule puncture in the recorded force, we were able to estimate the average diameter approximately 41.2 +/- 1.6 microm of the sperm-carrying tubules in samples. We have also demonstrated the ability to sense the existence of larger tubules embedded in a mess of thinner tubules, using an analytically calculated expression for the distribution of sizes measured by the microprobe. This information is important in MP-TESE to distinguish tubules with and without fertile sperm, potentially eliminating the large incision currently required for optical spermatazoa localization.