Testicular Sperm Extraction Samples Research Articles

P-027 Sperm motility and calcium ionophore treatment influence fertilization and pregnancy outcomes in TESE-ICSI

Abstract Study question Are there differences in fertilization and pregnancy rates post-TESE-ICSI between motile and immotile sperm, and do these vary in treatments with and without calcium ionophore? Summary answer Post-TESE-ICSI data reveals differences in fertilization and pregnancy rates when comparing motile vs. immotile sperm and treatments with and without calcium ionophore after TESE-ICSI. What is known already Testicular sperm extraction (TESE) followed by intracytoplasmic sperm injection (ICSI) poses a challenge for every IVF laboratory. This is particularly true in patients exhibiting azoospermia, where only a few and mostly immotile sperm are present in testicular tissue. In these cases, a calcium ionophore treatment for activation of the oocyte may help to overcome poor prognosis fertilization outcome, and thereby enhancing the likelihood to achieve a transfer leading to a successful pregnancy. Although both techniques, TESE-ICSI and activating substances are commonly applied, detailed analyses for individual counselling of couples are missing. Study design, size, duration This study analyzed 787 TESE-ICSI cycles (2015-2023) regarding patient age, number of attempts, oocyte maturity, fertilization and pregnancy rate after fresh embryo transfer. Cycles were split into four groups: (A) at least one motile sperm was injected (n = 493); (B) only immotile sperm were used (n = 294); (C) calcium ionophore (n = 474); (D) no calcium ionophore (n = 313). 17 cycles were excluded from the study because no sperm were found on the day of TESE-ICSI and oocytes were vitrified. Participants/materials, setting, methods In our IVF clinic, calcium ionophore treatments are only offered if a pre-screening of TESE samples reveals the absence of motile spermatozoa or if a previously conducted TESE-ICSI attempt resulted in a low fertilization rate (<30%) or a total fertilization failure. Statistical analyses was performed to compare the motile and immotile sperm group and the groups with or without calcium ionophore treatment. Main results and the role of chance No differences were found in all groups (A-D) regarding age of female or male patient and number of collected oocytes per group. However, our results demonstrate a difference in the mean number of ICSI attempts when comparing motile sperm and immotile sperm (2.0±1.3 vs. 2.3±1.6, p < 0.05), and when comparing attempts with calcium ionophore treatment and those without (2.4±1.6 vs. 1.8±1.8, p < 0.0005). The mean fertilization rate (2PN stage) was higher in the motile sperm group (A) compared to the immotile group (B) (57.8% vs. 28.4%, p < 0.0005) and also higher in attempts of group (D) in comparison to attempts of group C (56.8% vs. 40.2%, p < 0.0005). While fertilization failure occurred less often in group (A) (3.2%) it occurred to a higher rate in group (B) (19.4%), followed by group (C) (12.0%). Biochemical (BPR) and clinical pregnancy rates (CPR) showed a significant difference between the motile and immotile sperm group (BCR: 33.2% vs. 12.3%, p < 0.0005; CPR: 25.6% vs. 9.3%, p < 0.0005), but no differences between the two calcium ionophore groups. Limitations, reasons for caution This analysis was done retrospectively. A potential subgroup analysis of groups (A) and (B) into calcium ionophore use and of groups (C) and (D) into motile or immotile sperm will reveal the effect of calcium ionophore on success rates regardless of sperm motility. Wider implications of the findings Our data provides insight into success rates of TESE-ICSI cycles, especially if only immotile sperm were present. This will serve as a helpful guide for physicians when counseling patients about their success rates and to optimize the outcome in cases with poor prognosis by using a calcium ionophore treatment. Trial registration number not applicable

An Alternative Application of Magnetic-Activated Cell Sorting: CD45 and CD235a Based Purification of Semen and Testicular Tissue Samples.

Magnetic activated cell sorting (MACS) is a well-known sperm selection technique, which is able to remove apoptotic spermatozoa from semen samples using the classic annexinV based method. Leukocytes and erythrocytes in semen samples or in testicular tissue processed for in vitro fertilization (IVF) could exert detrimental effects on sperm. In the current study, we rethought the aforementioned technique and used magnetic microbeads conjugated with anti-CD45/CD235a antibodies to eliminate contaminating leukocytes and erythrocytes from leukocytospermic semen samples and testicular tissue samples gained via testicular sperm extraction (TESE). With this technique, a 15.7- and a 30.8-fold reduction could be achieved in the ratio of leukocytes in semen and in the number of erythrocytes in TESE samples, respectively. Our results show that MACS is a method worth to reconsider, with more potential alternative applications. Investigations to find molecules labeling high-quality sperm population and the development of positive selection procedures based on these might be a direction of future research.

Automated rare sperm identification from low-magnification microscopy images of dissociated microsurgical testicular sperm extraction samples using deep learning

To develop a machine learning algorithm to detect rare human sperm in semen and microsurgical testicular sperm extraction (microTESE) samples using bright-field (BF) microscopy for nonobstructive azoospermia patients. Spermatozoa were collected from fertile men. Testis biopsies were collected from microTESE samples determined to be clinically negative for sperm. A convolutional neural network based on the U-Net architecture was trained using 35,761 BF image patches with fluorescent ground truth image pairs to segment sperm. The algorithm was validated using 7,663 image patches. The algorithm was tested using 7,663 image patches containing abundant sperm, as well as 7,985 image patches containing rare sperm. Invitro fertilization center and university laboratories. Normospermic and nonobstructive azoospermia patients. None. Precision (positive predictive value [PPV]), recall (sensitivity), and F1-score of detected sperm locations. For sperm-only samples, our algorithm achieved 91% PPV, 95.8% sensitivity, and 93.3% F1-score at ×10 magnification. For dissociated microTESE samples doped with an abundant quantity of sperm, our algorithm achieved 84.0% PPV, 72.7% sensitivity, and 77.9% F1-score. For dissociated microTESE samples doped with rare sperm, our algorithm achieved 84.4% PPV, 86.1% sensitivity, and 85.2% F1-score. Rare sperm can be detected in patients' testis biopsy samples for potential subsequent use in invitro fertilization-intracytoplasmic sperm injection. A machine learning algorithm can use BF images at ×10 magnification to accurately detect sperm locations using automated imaging.

O-150 Predicting the sperm retrieval of microsurgical testicular sperm extraction (mTESE) in infertile men with azoospermia on the day of TESE-ICSI

Abstract Study question Is the sperm retrieval rate of a small, pre-processed sample (PPS) of each TESE-biopsy representative for the sperm outcome on the day of ICSI? Summary answer The analysis of a PPS reliably reflects the probability of finding comparable numbers of sperm at time of TESE-ICSI. What is known already Azoospermia is defined as a condition where no spermatozoa are found in the ejaculate and is diagnosed in up to 15% among infertile men and in 11% of all patients attending our centre. The combination of testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI) has become the standard treatment of azoospermic patients. However, no validated standard procedure has been identified to predict the exact sperm outcome of the cryopreserved TESE samples prior to TESE-ICSI so far. For optimal management of TESE-biopsies and the respective ICSI treatment, we developed a stepwise approach for the analysis of tissue samples. Study design, size, duration We retrospectively analysed the outcome of 872 microsurgically retrieved testicular biopsies of 198 patients of legal age who had a TESE-ICSI at our department between 2009 and 2019. From all 872 mTESE biopsies the number of sperm extracted from a small, pre-processed sample (PPS) before freezing procedure were known. The PPS was then compared to the number of sperm retrieved from the corresponding thawed specimen on the day of TESE-ICSI. Participants/materials, setting, methods During micro-TESE eight samples per testis are retrieved, then 1/10 of each biopsy is removed, digested with collagenase and screened for spermatozoa (pre-processed sample, PPS). If less than 100 spermatozoa are detected the absolute sperm number is recorded, otherwise the result is displayed as the maximum value of 100 sperm. On the day of ICSI, one or more TESE biopsies are thawed and processed for TESE-ICSI; the absolute sperm number is counted again. Main results and the role of chance Comparing the sperm yield of 872 TESE samples at time of ICSI to its respective PPS showed a similar sperm outcome with a minor deviation of ± 5 spermatozoa in 73.6% of all biopsies. However, 12.9% of the specimen had less and 13.4% had more spermatozoa. A negative sperm retrieval in the initial PPS was confirmed in 93.1% (268/288). PPS with 1-4 spermatozoa had a 27.2% (43/158) risk of complete absence of sperm on the day of ICSI, yet sperm detection (≥1 sperm) was positive in 72.8% (115/158) of the biopsies. With initially ≥5 spermatozoa present in the PPS, only 0.9% (4/426) of the biopsies had no sperm on the day of ICSI, vice versa 99.1% (422/426) were spermatozoa positive. A significant (p = 0.01) and strong (rs = 0.926) correlation of the sperm retrieval rates of the PPS and the ICSI sample was found meaning that the PPS reflects very well the sperm retrieval rate of the cryopreserved mTESE biopsy thawed at time of TESE-ICSI. However, if ≤ 4 sperm are found in the PPS, there is a relevant risk for a negative sperm retrieval on the day of ICSI and the couple should be carefully advised before start of treatment. Limitations, reasons for caution This analysis focussed on sperm prediction in cases of severe male factor infertility and therefore the sperm yield on the day of ICSI was chosen as primary outcome. The reproductive competence of the retrieved sperm in terms of pregnancy and birth rates should be subject to further investigation. Wider implications of the findings Treatment options for azoospermic patients are mostly related to the ability to find sperm on the day of ICSI. However, validated standards for sperm processing are missing. Therefore, a PPS seems to be a good option for prediction of sperm retrieval and improves counselling of the patients prior to TESE-ICSI. Trial registration number not applicable

Testicular sperm extraction vs. ejaculated sperm use for nonazoospermic male factor infertility

To study the potential benefit of testicular sperm compared with ejaculated sperm for men with oligospermia. After exemption from institutional review board approval, we performed a retrospective cohort study using the Mayo Clinic Assisted Reproductive Technology database. Single academic center. Couples with nonazoospermic male factor infertility (total motile sperm <25 million per ejaculate) undergoing intracytoplasmic sperm injection with sperm obtained by testicular sperm extraction (TESE) or ejaculated sperm between 2016 and2019. Invitro fertilization, Intracytoplasmic sperm injection, TESE. The primary outcome was live birth rate. The secondary outcomes were fertilization rate, blastulation rate, pregnancy rate, and miscarriage rate. Subjects in the two groups were similar in age, body mass index, and ovarian reserve. Baseline sperm parameters were similar in the two groups: total motile sperm (5.4 in the ejaculate sperm group vs. 3.6 million motile per ejaculate), except that baseline motility was higher in the group that used ejaculated sperm (40% vs. 29%). The total number of mature oocytes retrieved was similar in the two groups, but the use of TESE was associated with a 20% decrease in fertilization (60.0% vs. 80.6%) and half the number of blastocyst embryos (two vs. four) compared with ejaculated sperm. Compared with ejaculated sperm, use of TESE did not improve the miscarriage rate (11% vs. 9%) or the live birth rate (50.0% vs. 31.3%). Patients with male factor infertility and oligozoospermia did not have improved ICSI outcomes with the use of TESE samples compared with ejaculated sperm.

The role of nitric oxide on spermatogenesis in infertile men with azoospermia

Objectives: The underlying pathophysiological mechanisms of azoospermia is still unclear. The aim of the study was to evaluate nitric oxide synthase (NOS) isoforms and free radical release in testicular sperm extraction (TESE) in infertile men with azoospermia. Materials and methods: The study included 40 men (mean age: 37.2±2 years; range 25 to 55 years) with azoospermia which were divided into two groups: spermatozoa-present (n=20) and spermatozoa-absent (n=20). Testicular samples were examined morphologically, immunohistochemically, and biochemically. The TESE samples were examined according to number of mast cells stained with toluidine blue; immunohistochemically with three types of NOS isoforms, and free radicals were measured with chemiluminescence method, respectively. Results: Endothelial NOS (eNOS) reaction in spermatozoa-present group was considerably higher than spermatozoa-absent group (p&lt;0.001). Compared to the spermatozoa-present group, inducible NOS (iNOS) reaction was higher than the spermatozoa-absent group (p&lt;0.05). Neuronal NOS (nNOS) reaction was only prominent in Leydig cells in both groups. Mast cells increased (p&lt;0.05) in the interstitial area surrounding seminiferous tubules in spermatozoa-absent samples. Superoxide radical generation in spermatozoa-present samples was significantly lower (p=0.0003). The peroxynitrite ratio in spermatozoa-absent samples was significantly higher (p=0.0038). Conclusion: These results suggest that eNOS, iNOS, and mast cells play an important role in spermatogenesis process in azoospermic men.

Cross flow coupled with inertial focusing for separation of human sperm cells from semen and simulated TESE samples.

A triplet spiral channel coupled with cross-flow filtration has been designed and fabricated in an effort to separate sperm cells from either semen or simulated testicular sperm extraction (TESE) samples. This device separates a fraction of cells from the sample by taking advantage of inertial focusing combined with hydrodynamic filtration in multiple micro-slits. Compared to the conventional swim-up technique, the proposed microfluidic device is capable of efficiently separating sperm cells without any tedious semen sample processing and centrifugation steps with a lower level of reactive oxygen species and DNA fragmentation. The device processing capability on the simulated TESE samples confirmed its proficiency in retrieving sperm cells from the samples with an approximate yield of 76%. Conclusively, the introduced microfluidic device can pave the path to proficiently separate sperm cells in assisted reproductive treatment cycles.

A preliminary study of sperm identification in microdissection testicular sperm extraction samples with deep convolutional neural networks.

Sperm identification and selection is an essential task when processing human testicular samples for in vitro fertilization. Locating and identifying sperm cell(s) in human testicular biopsy samples is labor intensive and time consuming. We developed a new computer-aided sperm analysis (CASA) system, which utilizes deep learning for near human-level performance on testicular sperm extraction (TESE), trained on a custom dataset. The system automates the identification of sperm in testicular biopsy samples. A dataset of 702 de-identified images from testicular biopsy samples of 30 patients was collected. Each image was normalized and passed through glare filters and diffraction correction. The data were split 80%, 10%, and 10% into training, validation, and test sets, respectively. Then, a deep object detection network, composed of a feature extraction network and object detection network, was trained on this dataset. The model was benchmarked against embryologists' performance on the detection task. Our deep learning CASA system achieved a mean average precision (mAP) of 0.741, with an average recall (AR) of 0.376 on our dataset. Our proposed method can work in real time; its speed is effectively limited only by the imaging speed of the microscope. Our results indicate that deep learning-based technologies can improve the efficiency of finding sperm in testicular biopsy samples.

Sperm chromosome abnormalities in patients with normal karyotype and in translocation carriers: clinical relevance for assisted reproductive technology

Research questionWhat is the proportion of chromosomally abnormal spermatozoa in men with a history of reproductive failure, including patients with normal karyotype and carriers of translocations? Should this analysis be included in a clinical setting to define the best treatment options for infertile couples? DesignAneuploidy for chromosomes XY, 13, 15, 16, 17, 18, 21, 22 was tested by fluorescent in-situ hybridization (FISH) in 1665 samples from couples with normal karyotype having had at least three previous IVF failures, miscarriages, or both (group-A). A FISH test was also carried out in 76 samples from carriers of translocations (group B) to detect the proportion of spermatozoa with unbalanced rearrangement. ResultsIn group A, the lowest incidence of aneuploid sperm cells was found in men with normozoospermia (1.3%, range 0.09–6.31%) compared with men with moderate oligoasthenoteratozoospermia (2.1%, range 0.41–16.6%, P < 0.001), severe oligoasthenoteratozoospermia (4.7%, range 0.53–30.77, P < 0.001), microepididymal sperm aspiration (3.1%, range 1.19–24.24, P < 0.001) and testicular sperm extraction samples (5.8%, range 1.54–33.3, P < 0.001). In group B, the proportion of spermatozoa with unbalanced rearrangement was significantly higher in reciprocal (63%, range 10.0–87.6%) than in Robertsonian translocations (16%, range 4.3–51.0%, P < 0.001). ConclusionsPatients with poor prognosis of term pregnancy tend to generate high proportions of chromosomally abnormal spermatozoa, especially in severe male factor cases. Corresponding frequencies occur at wide ranges; therefore, the FISH test is needed to assess the proportion of spermatozoa with altered chromosome condition. A flowchart, which included the FISH test, was designed to assist clinicians guide couples with poor prognosis of pregnancy, on the most indicated treatment options.

Microfluidic System for Rapid Isolation of Sperm From Microdissection TESE Specimens

To demonstrate a novel prototype microfluidic system for rapid isolation of sperm from real and simulated microdissection testicular sperm extraction samples. 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. 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. 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.

Testicular human spermatozoa cryopreservation correlation between sperm head vacuoles, DNA fragmentation and mitochondrial membrane potential

Abstract Background There are difficulties associated with testicular sperm freezing. Different methods of sperm cryopreservation developed. Not enough detailed studies about the real efficacy of these techniques exist. For sperm morphologic assessment, motile sperm organelle morphology examination (MSOME) is able to identify not only conventional morphological sperm alterations but also sperm head vacuoles. Objective To assess the effect of cryopreservation on testicular spermatozoa vacuoles by MSOME and its correlation to DNA fragmentation and mitochondrial membrane potential. Materials and methods After preparation, testicular sperm extraction samples of 15 azoospermic men, aged 20–40 years old were divided into three groups. Group 1 was assessed freshly. Group 2 was cryopreserved with vitrification method and Group 3 with cooling in liquid nitrogen vapor using droplet. Pre and post warming assessment in terms of spermatozoa head vacuoles by MSOME, DNA fragmentation, and mitochondrial membrane potential were performed. Results The number of spermatozoa with no vacuoles significantly decreased after two cryopreservation techniques (P 0.05). DNA fragmentation and mitochondrial membrane potential increased after cryopreservation (P Conclusion Cryopreservation affects spermatozoa vacuolization, DNA structure, and mitochondrial membrane potential. Using MSOME in the selection of post-thaw morphologically normal testicular spermatozoa for ICSI procedure will be of particular value.

Infectious, inflammatory and 'autoimmune' male factor infertility: how do rodent models inform clinical practice?

BACKGROUNDInfection and inflammation of the reproductive tract are significant causes of male factor infertility. Ascending infections caused by sexually transmitted bacteria or urinary tract pathogens represent the most frequent aetiology of epididymo-orchitis, but viral, haematogenous dissemination is also a contributory factor. Limitations in adequate diagnosis and therapy reflect an obvious need for further understanding of human epididymal and testicular immunopathologies and their contribution to infertility. A major obstacle for advancing our knowledge is the limited access to suitable tissue samples. Similarly, the key events in the inflammatory or autoimmune pathologies affecting human male fertility are poorly amenable to close examination. Moreover, the disease processes generally have occurred long before the patient attends the clinic for fertility assessment. In this regard, data obtained from experimental animal models and respective comparative analyses have shown promise to overcome these restrictions in humans.OBJECTIVE AND RATIONALEThis narrative review will focus on male fertility disturbances caused by infection and inflammation, and the usefulness of the most frequently applied animal models to study these conditions.SEARCH METHODSAn extensive search in Medline database was performed without restrictions until January 2018 using the following search terms: ‘infection’ and/or ‘inflammation’ and ‘testis’ and/or ‘epididymis’, ‘infection’ and/or ‘inflammation’ and ‘male genital tract’, ‘male infertility’, ‘orchitis’, ‘epididymitis’, ‘experimental autoimmune’ and ‘orchitis’ or ‘epididymitis’ or ‘epididymo-orchitis’, antisperm antibodies’, ‘vasectomy’. In addition to that, reference lists of primary and review articles were reviewed for additional publications independently by each author. Selected articles were verified by each two separate authors and discrepancies discussed within the team.OUTCOMESThere is clear evidence that models mimicking testicular and/or epididymal inflammation and infection have been instructive in a better understanding of the mechanisms of disease initiation and progression. In this regard, rodent models of acute bacterial epididymitis best reflect the clinical situation in terms of mimicking the infection pathway, pathogens selected and the damage, such as fibrotic transformation, observed. Similarly, animal models of acute testicular and epididymal inflammation using lipopolysaccharides show impairment of reproduction, endocrine function and histological tissue architecture, also seen in men. Autoimmune responses can be studied in models of experimental autoimmune orchitis (EAO) and vasectomy. In particular, the early stages of EAO development showing inflammatory responses in the form of peritubular lymphocytic infiltrates, thickening of the lamina propria of affected tubules, production of autoantibodies against testicular antigens or secretion of pro-inflammatory mediators, replicate observations in testicular sperm extraction samples of patients with ‘mixed atrophy’ of spermatogenesis. Vasectomy, in the form of sperm antibodies and chronic inflammation, can also be studied in animal models, providing valuable insights into the human response.WIDER IMPLICATIONSThis is the first comprehensive review of rodent models of both infectious and autoimmune disease of testis/epididymis, and their clinical implications, i.e. their importance in understanding male infertility related to infectious and non-infectious/autoimmune disease of the reproductive organs.

Improvement of motility after culture of testicular spermatozoa: the effects of incubation timing and temperature.

BackgroundSperm motility is the reliable parameter that roles in success of intracytoplasmic sperm injection (ICSI), especially in azoospermia. Selection of appropriate culture duration, temperature and media for enhancing the sperm motility is an important issue in assisted reproduction program. The aim was to evaluate the sperm motion characteristics after culturing of testicular sperm extraction (TESE) samples at different temperatures and time intervals.MethodsIn this prospective study, 27 TESE samples were collected from young azoospermic patients. The samples were cultured in Ham’s F10+20% HAS, at different temperatures (incubation at 37 vs. 25 °C) and sperm total motility was assessed at different time intervals of 0, 24, 48 and 72 h post testicular biopsy.ResultsIn vitro culture at 25 °C changed sperm motility from 13% immediately after biopsy to 76% at 24 h, 43% at 48 h and 15% at 72 h. At 37 °C, the sperm motion feature was changed to 67% at 24 h, 38.40% at 48 h and 12.03% at 72 h. Sperm motility change at 24 h was incremental in both conditions of culturing, but significant at 25 °C (P≤0.05).ConclusionsThe ideal in vitro culture for testicular spermatozoa was at 25 °C after 1 day of culture, which optimized the sperm motility in azoospermic TESE samples.

Is ex vivo microdissection testicular sperm extraction indicated for infertile men undergoing radical orchiectomy for testicular cancer? Case report and literature review

To report a case of an infertile man with nonobstructive azoospermia who underwent simultaneous radical orchiectomy for testicular cancer and testicular sperm extraction (TESE) for preservation of fertility. Case report and literature review. University teaching hospital. A couple being treated for infertility. Radical orchiectomy with simultaneous TESE. Sperm retrieval, histologic evaluation of archived testicular pathology slides. We retrieved 20 spermatozoa from the multiple random TESE samples obtained at radical orchiectomy. Histologic evaluation of the archived testicular pathology slides revealed that the testis contained several foci of active spermatogenesis, suggesting that a significantly greater number of spermatozoa would likely have been retrieved had a microdissection TESE been performed instead of the multiple TESEs. We propose that microdissection TESE should be considered the preferred sperm retrieval technique at the time of radical orchiectomy in men with coexistent nonobstructive azoospermia and testicular cancer.

In vitro production of haploid cells after coculture of CD49f+ with Sertoli cells from testicular sperm extraction in nonobstructive azoospermic patients

To isolate CD49f+ cells from testicular sperm extraction (TESE) samples of azoospermic patients and induce meiosis by coculturing these cells with Sertoli cells. Prospective analysis. Research center. Obstructive azoospermic (OA) and nonobstructive azoospermic (NOA) patients. TESE, with enzymatic dissociation of samples to obtain a cell suspension, which was cultured for 4 days with 4 ng/mL GDNF. The CD49f+ cells were sorted using fluorescence-activated cell sorting (FACS) as a marker to identify spermatogonial stem cells (SSCs), which were cocultured with Sertoli cells expressing red fluorescent protein (RFP) in knockout serum replacement (KSR) media with addition of 1,000 IU/mL of follicle-stimulating hormone (FSH), 1 μM testosterone, 40 ng/mL of GDNF, and 2 μM retinoic acid (RA) for 15 days in culture at 37°C and 5% CO(2) to induce meiotic progression. Cells were collected and analyzed by immunofluorescence for meiosis progression with specific markers SCP3 and CREST, and they were confirmed by fluorescence in situ hybridization (FISH). Isolation of CD49f+ cells and coculture with Sertoli cells, meiosis progression invitro, assessment of SSCs and meiotic markers real-time polymerase chain reaction (RT-PCR), immunohistochemical analysis, and FISH. The CD49f+ isolated from the of total cell count in the TESE samples of azoospermic patients varied from 5.45% in OA to 2.36% in NOA. Sertoli cells were obtained from the same TESE samples, and established protocols were used to characterize them as positive for SCF, rGDNF, WT1, GATA-4, and vimentin, with the presence of tight junctions and lipid droplets shown by oil red staining. After isolation, the CD49f+ cells were cocultured with RFP Sertoli cells in a 15-day time-course experiment. Positive immunostaining for meiosis markers SCP3 and CREST on days 3 to 5 was noted in the samples obtained from one NOA patient. AFISH analysis for chromosomes 13, 18, 21, X, and Y confirmed the presence of haploid cells on day 5 of the coculture. Invitro coculture of SSCs from TESE samples of NOA patients along with Sertoli cells promoted meiosis induction and resulted in haploid cell generation. These results improve the existing protocols to generate spermatogenesis invitro and open new avenues for clinical translation in azoospermic patients.

Are testicular sperm in epididymal necrospermia (EN) normal?

OBJECTIVE: Evaluate the viability of testicular sperm in patients with idiopathic EN. DESIGN: Prospective clinical analysis. MATERIALS AND METHODS: Semen and testicular samples from three men with consistent absolute asthenospermia (0% motility) and between 70 to 100% necrospermia in the ejaculate were studied under electron microscopy (EM) after ruling out bacterial infections. The female partners of all three patients underwent controlled ovarian hyperstimulation (COH) for intracytoplasmic sperm injection (ICSI) with testicular sperm extraction (TESE) that was undertaken at the time of follicular aspiration. Scrotal exploration was performed under local anesthesia through a small incision and the samples obtained were were placed in modified human tubal fluid (HTF) culture media. The seminiferous tubules were dispersed with the aid of fine needles under an inverted microscope. All semen and testicular samples were studied under phase-contrast microscopy at X400. After dilution and centrifugation the pellets were fixed with 3% glutaraldehyde, post fixed in 1.3% osmium tetroxide and embedded in Epon-Araldite for electron microscopy scanning. RESULTS: Motile spermatozoa were harvested from TESE samples in all patients and five ICSI cycles were performed. Fertilization rate was 64% (9/14) (Patient 1), 83% (5/6) (Patient 2) and 79% (15/19) (Patient 3) in MII oocytes. We obtained two pregnancies in the same patient (Patient 3): an early abortion in the first attempt and a term twin pregnancy after transfer of three cryopreserved embryos. EM studies of ejaculated spermatozoa showed no evidence of flagellar abnormalities and autolysis of all tails and great part of the spermatic heads in two patients; and axonemal absence in all tails studied in one patient, suggesting a post-necrotic phenomenon. EM studies of testicular sperm showed normal flagellar configuration and the presence of axonemes without degenerative signs. CONCLUSIONS: It has been proposed that in EN spermatozoa are produced normally in the testis and that death of the sperm cells occurs during transit through the epididymus, either during storage or when contacting seminal fluid. Our patients had testicular spermatozoa with normal fertilization potential. The EM findings coupled with the fact that a normal twin birth was achieved through cryopreserved embryo transfer, suggests that testicular spermatozoa from patients with severe necrospermia in the ejaculate are, indeed, normal.

The value of repeat testicular sperm retrieval in azoospermic men

To determine the predictive value of a previous testicular biopsy to the chance of sperm retrieval in the next testicular sperm extraction (TESE) procedure, we retrospectively analyzed the outcome of past sperm collection procedures and histopathology diagnoses of patients with nonobstructive azoospermia. Repeated TESE ensured a high recovery rate (96%) when the first recovery procedure had been successful and when hypospermatogenesis was diagnosed (77%); when no spermatozoa were found on the first attempt, a repeat TESE procedure was successful in one-third of the patients.

Sperm head's birefringence: a new criterion for sperm selection

To investigate the characteristics of birefringence in human sperm heads and apply polarization microscopy for sperm selection at intracytoplasmic sperm injection (ICSI). Prospective randomized study. Reproductive Medicine Unit, Società Italiana Studi Medicina della Riproduzione, Bologna, Italy. A total of 112 male patients had birefringent sperm selected for ICSI (study group). The clinical outcome was compared with that obtained in 119 couples who underwent a conventional ICSI cycle (control group). The proportion of birefringent spermatozoa was evaluated before and after treatment in relation to the sperm sample quality. Embryo development and clinical outcome in the study group were compared with those in the controls. Proportion of birefringent sperm heads, rates of fertilization, cleavage, pregnancy, implantation, and ongoing implantation. The proportion of birefringent spermatozoa was significantly higher in normospermic samples when compared with oligoasthenoteratospermic samples with no progressive motility and testicular sperm extraction samples. Although fertilization and cleavage rates did not differ between the study and control groups, in the most severe male factor condition (oligoasthenoteratospermic with no progressive motility and testicular sperm extraction), the rates of clinical pregnancy, ongoing pregnancy, and implantation were significantly higher in the study group versus the controls. The analysis of birefringence in the sperm head could represent both a diagnostic tool and a novel method for sperm selection.

A New Sperm Preparation Method for Testicular Sperm Extraction - Intracytoplasmic Sperm Injection (TESE-ICSI) Cycles: Simple, Effective and Rapid Method

The aim of this study was to recover spermatozoa easily from fresh or frozen-thawed testicular sperm extraction (TESE) samples. We simply minced and washed the TESE samples in medium. Spermatozoa were recovered and washed in the bottom of a PVP droplet with an injection pipette and injected into oocytes. In all cycles 100% of spermatozoa (motile and/or immotile) could be recovered. Injected oocytes were fertilized (100% per cycle and in 41.9% of oocytes) and fertilized oocytes were cleaved (100% per cycle; 38.5% of injected oocytes; 91.8% of fertilized oocytes) after ICSI with fresh or frozen-thawed TESE spermatozoa. The rates of progression to the embryo stage (2-8 cell) and blastocyst formation and their quality were similar in both the fresh sample group and the frozen-thawed sample group. Both of embryo transfer (73.5%) and embryo-blastocyst transfer (26.5%) were performed successfully. Thirty-four cycles of ICSI with testicular spermatozoa resulted in 23.5% pregnancy rates (fresh sample group, 22.2%; frozen-thawed sample group, 24%) with both embryo transfer (8%) and embryo-blastocyst transfer (66.7%). This new sperm preparation method is very simple, easy, effective and rapid for recovering spermatozoa from TESE samples for ICSI.