271 THE EFFECT OF TEMPERATURE ON HUMAN TESTICULAR TISSUE TO OPTIMIZE THE IN VITRO MATURATION OF PRE-FREEZE MOTILITY

2009 ◽  
Vol 21 (1) ◽  
pp. 233
Author(s):  
M. C. Schiewe ◽  
A. Spitz ◽  
R. E. Anderson
Keyword(s):  
Sperm Motility ◽  
Seminiferous Tubules ◽  
Type I ◽  
Obstructive Azoospermia ◽  
Percentage Increase ◽  
Testicular Sperm ◽  
Incubation Condition ◽  
Progressive Motility ◽  
Over Time

The development of intracytoplasmic sperm injection has made the use of testicular sperm a viable option for infertile men with obstructive and nonobstructive azoospermia. Over the past decade, testicular biopsies have been handled and processed using a variety of different methods. Whole biopsy pieces can be effectively cryopreserved in a 10% glycerol diluent (Schiewe et al. 1997 67, S115 abst); however, the ability to find viable, motile sperm post-thaw is improved when prefreeze motility exists. The purpose of this study was to comparatively document in vitro sperm motility enhancement over time at different temperatures, and to prove that an intermediate temperature (28 to 30°C) would optimize sperm longevity for up to 1 week. In this study, 10 men with obstructive azoospermia underwent a surgical, open testicular biopsy procedure. Each biopsy was placed in HEPES buffered-human tubal fluid (mHTF) medium supplemented with 5% human serum albumin (HSA; Irvine Sci., Santa Ana, CA, USA) and transported to the laboratory at room temperature. Each testis biopsy (TBx) was dissected into 8 equal pieces (approximately 2 × 2 × 1 mm). Five intact pieces of TBx were cryopreserved in separate cryovials for future use. The remaining TBx tissue was subdivided into 1 of 3 temperature treatment groups (24, 30, or 37°C) for extended IVM. The 30°C incubation condition was achieved by placing the dish(es) in a Styrofoam box placed on a 37°C warming plate. Each TBx was placed into a separate 100 × 35 mm Falcon dish in 150-μL droplets of mHTF under oil and shredded by needle dissection to disperse the contents of the seminiferous tubules. Reminant tissue was placed into another droplet for additional dissection, as needed. Sperm were analyzed for motility (graded as Type I = twitching, II = undulating, III = slow progression, and IV = rapid progression) at 0, 24, 96, and 144 h without replacing the IVM medium. The percentage increase in motility, compared with 0 h, was statistically contrasted across temperature treatments by chi-squared analysis. Testicular sperm motility ranged from 5 to 25% at 0 h and increased in all groups at 24 h. There was no difference in total motility at 24 h, but progressive motility was higher (P < 0.05) at 37°C compared with 24°C. Significant differences (P < 0.05) were observed in total percentage of motility/percentage of progressive motility at 96 h with treatment differences (*) being 30°C (66%*/44%*) > 24°C (42%*/14%) > 37°C (18%*/10%). This statistical trend continued at 144 h with 30°C (42%*/23%*) > 24°C (24%*/8%) > 37°C (9%*/5%). During this study, a viable pregnancy was achieved using a 30°C sample 8 days post biopsy, exceeding 2 previous healthy triplet pregnancies, which were successful using 4-day-old TBx specimens in 1996 and 2004. This study confirms that an intermediate IVM temperature of 30°C is optimal to enhance TBx cryopreservation, which in our laboratory is generally performed at 24 to 72 h of IVM. Our routine pregnancy success in applying ICSI with IVM, thawed TBx sperm discounts the concern some individuals express regarding DNA fragmentation of sperm over time.

scholarly journals Two Decades from the Introduction of Microdissection Testicular Sperm Extraction: How This Surgical Technique Has Improved the Management of NOA

2021 ◽  
Vol 10 (7) ◽  
pp. 1374
Author(s):  
Nahid Punjani ◽  
Caroline Kang ◽  
Peter N. Schlegel
Keyword(s):  
Testicular Tissue ◽  
Seminiferous Tubules ◽  
Testicular Sperm Extraction ◽  
Obstructive Azoospermia ◽  
Testicular Sperm ◽  
Sperm Retrieval ◽  
Vitro Fertilization ◽  
Surgeon Experience ◽  
Patient Optimization

The treatment of men with non-obstructive azoospermia (NOA) has improved greatly over the past two decades. This is in part due to the discovery of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), but also significantly due to improvements in surgical sperm retrieval methods, namely the development of microdissection testicular sperm extraction (mTESE). This procedure has revolutionized the field by allowing for identification of favorable seminiferous tubules while simultaneously limiting the amount of testicular tissue removed. Improving sperm retrieval rates is imperative in this cohort of infertile men as there are a limited number of factors that are predictive of successful sperm retrieval. Currently, sperm retrieval in NOA men remains dependent on surgeon experience, preoperative patient optimization and teamwork with laboratory personnel. In this review, we discuss the evolution of surgical sperm retrieval methods, review predictors of sperm retrieval success, compare and contrast the data of conventional versus mTESE, share tips for optimizing sperm retrieval outcomes, and discuss the future of sperm retrieval in men with NOA.

scholarly journals Short-term storage of tiger salamander (Ambystoma tigrinum) spermatozoa: The effect of collection type, temperature and time

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0245047
Author(s):  
Amanda B. Gillis ◽  
Emmet L. Guy ◽  
Andrew J. Kouba ◽  
Peter J. Allen ◽  
Ruth M. Marcec-Greaves ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Hormone Treatment ◽  
Effect Of Temperature ◽  
Initial Assessment ◽  
Ambystoma Tigrinum ◽  
Tiger Salamander ◽  
Storage Duration ◽  
Spermatozoa Motility ◽  
Over Time

The aims of this project were to characterize tiger salamander (Ambystoma tigrinum) spermatozoa motility over time, when excreted as either milt or spermic urine prior to packaging into a spermatophore, and to determine the effect of temperature on sperm motility. A split-plot design was utilized to assess the motility of the two pre-spermatophore sample types at two temperatures, 0°C and 20°C (n = 10 for each treatment). Spermiation was induced through exogenous hormone treatment of luteinizing hormone releasing hormone analog in order to collect both milt and spermic urine, which were evaluated for motility, divided into two separate aliquots, and subsequently stored in either an ice-bath (0°C) or on the benchtop (20°C). The decay rate of sperm motility was assessed by reevaluating subsamples at 0.5, 1, 2, 3, 5, 7, and 24 hours following the initial assessment. Results showed that sperm stored at 0°C had significantly higher progressive, non-progressive, and total motility for both sperm collection types over time. An interaction was found between collection type and time, with milt exhibiting lower initial motility that was more sustainable over time, compared to spermic urine. For both milt and spermic urine, motility decreased rapidly with storage duration, indicating samples should be used as soon as possible to maximize motility for in-vitro fertilization and cryopreservation. This is the first study to describe the differences in sperm motility between milt and spermic urine from an internally fertilizing caudate and demonstrates the benefits of near freezing temperatures on sperm longevity.

Fertilizing capacity of epididymal and testicular sperm using intracytoplasmic sperm injection (ICSI)

1995 ◽  
Vol 7 (2) ◽  
pp. 281 ◽  
Author(s):  
SJ Silber ◽  
P Devroey ◽  
H Tournaye ◽  
Steirteghem AC Van
Keyword(s):  
Pregnancy Rate ◽  
Intracytoplasmic Sperm Injection ◽  
Pregnancy Rates ◽  
Obstructive Azoospermia ◽  
Motile Sperm ◽  
Epididymal Sperm ◽  
Testicular Sperm ◽  
Cleavage Rate

For men with uncorrectable obstructive azoospermia, their only hope of fathering a child is microsurgical epididymal sperm aspiration (MESA) combined with in vitro fertilization (IVF). In 1988, proximal epididymal sperm were demonstrated to have better motility than senescent sperm in the distal epididymis, and it was thought that retrieval of motile sperm from the proximal epididymis would yield reliable fertilization and pregnancy rates after conventional IVF. However, the results to date have been poor, and although a minority of patients achieved good fertilization rates with IVF, the vast majority (81%) had consistently poor or no fertilization and the pregnancy rate averaged only 9%. Recently, intracytoplasmic sperm injection (ICSI) has been successfully used to achieve fertilization and pregnancies for patients with extreme oligoasthenozoospermia. ICSI has therefore been applied to cases of obstructive azoospermia and, in this report, 67 MESA-IVF cases are compared with 72 MESA-ICSI cases. The principle that motile sperm from the proximal segments of the epididymis should be used for ICSI was followed, although in the most severe cases in which there was an absence of the epididymis (or absence of sperm in the epididymis), testicular sperm were obtained from macerated testicular biopsies. These sperm only exhibited a weak, twitching motion. In 72 consecutive MESA cases, ICSI resulted in fertilization and normal embryos for transfer in 90% of the cases, with an overall fertilization rate of 46%, a cleavage rate of 68%, and ongoing or delivered pregnancy rates of 46% per transfer and 42% per cycle. The pregnancy and take-home baby rates increased from 9% and 4.5% with IVF to 53% and 42% with ICSI. There were no differences between the results for fresh epididymal, frozen epididymal or testicular sperm, and the number of eggs collected did not affect the outcome. The results were also unaffected by the aetiology of the obstruction such as congenital absence of the vas deferens or failed vasoepididymostomy. The only significant factor which affected the pregnancy rate was female age. It is concluded that although complex mechanisms involving epididymal transport may be beneficial for conventional fertilization of human oocytes (in vivo or in vitro), none of these mechanisms are required for fertilization after ICSI. Given the excellent results with epididymal and testicular sperm, ICSI is obligatory for all future MESA patients. Finally, the use of ICSI with testicular sperm from men with non-obstructive azoospermia is also discussed.

In-vitro maturation (IVM) of fresh and frozen-thawed testicular sperm in non-obstructive azoospermia (NOA) improves ICSI outcomes

2002 ◽  
Vol 78 ◽  
pp. S62-S63
Author(s):  
N Khotari ◽  
Pramila Dandekar ◽  
Stephen Dickstein ◽  
Diana Wong ◽  
Bin Wu ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Obstructive Azoospermia ◽  
Testicular Sperm ◽  
Icsi Outcomes

Development of geniculocortical axon arbors in a primate

1990 ◽  
Vol 5 (3) ◽  
pp. 291-309 ◽  
Author(s):  
S. L. Florence ◽  
V. A. Casagrande
Keyword(s):  
White Matter ◽  
Visual Experience ◽  
Striate Cortex ◽  
Growth Cones ◽  
Type I ◽  
Type Ii ◽  
Distinctive Features ◽  
Over Time

AbstractThe main objective of the present study was to describe the postnatal development of magnocellular and parvocellular LGN axons within the primate striate cortex. For this purpose, we bulk labeled axons in neonatal prosimians (galagos) in vivo or in vitro at regular intervals from birth (PO) to 12 weeks after birth by injecting horseradish peroxidase (HRP) into white matter anterior to the striate cortex. Filled axons within layer IV were reconstructed, quantitatively analyzed, and compared to a population of adult axons described previously (Florence & Casagrande, 1987).Our results show that although axons are morphologically immature at birth, they are restricted to the upper (IVα) and lower (IVβ) tiers of layer IV of the striate cortex as in adults. In adults, we referred to the presumed magnocellular LGN axons terminating in IVα as type I and the presumed parvocellular axons terminating in IVβ as type II. We used the same convention for developing axons.From birth to 3 weeks postnatal, type I and II axon classes are more variable in appearance than adult counterparts, and are not morphologically class distinct. As axons mature, parent axon shafts increase in caliber, arbors become smaller and more radial, and other immature features (e.g. spikes, protrusions, growth cones) are less evident. Both arbor classes mature slowly and some still exhibit immature features (e.g. growth cones) as late as 12 weeks postnatally. Although arbors do not show class-distinctive features until late in development, each class does show some unique maturational trends. Type I arbors are only slightly larger than adult counterparts at birth, whereas type II arbors are dramatically larger. Type I arbors increase in branch complexity with age, whereas type II arbors simply show a shift in complexity toward the center of the arbor with decreasing size over time. These growth trends suggest that magnocellular and parvocellular pathways to cortex could be differentially vulnerable to the manipulation of postnatal visual experience.

scholarly journals Vitamin C Ameliorates Tetrahydrocannabinol-Induced Spermatotoxicity In-Vitro

2020 ◽  
Author(s):  
Abdullateef Isiaka Alagbonsi ◽  
Luqman Aribidesi Olayaki
Keyword(s):  
Vitamin C ◽  
Sperm Motility ◽  
Cannabinoid Receptors ◽  
Group V ◽  
Progressive Motility ◽  
Straight Line ◽  
Lateral Head

Abstract Background: We investigated the in-vitro effects of vitamin C on delta-9-tetrahydrocannabinol (THC)-induced reduction in sperm motility and kinematics. Methods: Rats semen were randomly divided into 5 groups. Groups I-III received placebo, THC (1 mM), and vitamin C (5 mM) respectively. Groups IV and V received THC and vitamin C, but group V was additionally pre-treated with cannabinoid receptors’ blockers (CBs-) 1 (SR141716) and 2 (AM-630). Results: The sperm progressive motility, average path velocity (VAP), curvilinear velocity (VCL), straight-line velocity (VSL), amplitude of lateral head (ALH) and beat cross frequency (BCF) were reduced by THC but increased by vitamin C when compared to control. Vitamin C inhibited the THC-induced reduction in these parameters in the absence of CBs- 1 and 2, and even caused additional increases in progressive motility, VAP and VCL above the control levels with CBs-. Conclusion: In conclusion, vitamin C ameliorates THC-induced reduction in sperm motility in-vitro by modulation of sperm kinematics.

scholarly journals TRIM71 Deficiency Causes Germ Cell Loss During Mouse Embryogenesis and Is Associated With Human Male Infertility

2021 ◽  
Vol 9 ◽  
Author(s):  
Lucia A. Torres-Fernández ◽  
Jana Emich ◽  
Yasmine Port ◽  
Sibylle Mitschka ◽  
Marius Wöste ◽  
...  
Keyword(s):  
Male Infertility ◽  
Germ Cell ◽  
Cell Line ◽  
Rna Binding ◽  
Seminiferous Tubules ◽  
Obstructive Azoospermia ◽  
Sequencing Analysis ◽  
Mouse Embryogenesis ◽  
Human Male

Mutations affecting the germline can result in infertility or the generation of germ cell tumors (GCT), highlighting the need to identify and characterize the genes controlling germ cell development. The RNA-binding protein and E3 ubiquitin ligase TRIM71 is essential for embryogenesis, and its expression has been reported in GCT and adult mouse testes. To investigate the role of TRIM71 in mammalian germ cell embryonic development, we generated a germline-specific conditional Trim71 knockout mouse (cKO) using the early primordial germ cell (PGC) marker Nanos3 as a Cre-recombinase driver. cKO mice are infertile, with male mice displaying a Sertoli cell-only (SCO) phenotype which in humans is defined as a specific subtype of non-obstructive azoospermia characterized by the absence of germ cells in the seminiferous tubules. Infertility in male Trim71 cKO mice originates during embryogenesis, as the SCO phenotype was already apparent in neonatal mice. The in vitro differentiation of mouse embryonic stem cells (ESCs) into PGC-like cells (PGCLCs) revealed reduced numbers of PGCLCs in Trim71-deficient cells. Furthermore, TCam-2 cells, a human GCT-derived seminoma cell line which was used as an in vitro model for PGCs, showed proliferation defects upon TRIM71 knockdown. Additionally, in vitro growth competition assays, as well as proliferation assays with wild type and CRISPR/Cas9-generated TRIM71 mutant NCCIT cells showed that TRIM71 also promotes proliferation in this malignant GCT-derived non-seminoma cell line. Importantly, the PGC-specific markers BLIMP1 and NANOS3 were consistently downregulated in Trim71 KO PGCLCs, TRIM71 knockdown TCam-2 cells and TRIM71 mutant NCCIT cells. These data collectively support a role for TRIM71 in PGC development. Last, via exome sequencing analysis, we identified several TRIM71 variants in a cohort of infertile men, including a loss-of-function variant in a patient with an SCO phenotype. Altogether, our work reveals for the first time an association of TRIM71 deficiency with human male infertility, and uncovers further developmental roles for TRIM71 in the germline during mouse embryogenesis.

scholarly journals Effect of three commercial extenders on sperm motility and fertility in liquid ram semen stored at 15 °C or 5 °C

2019 ◽  
Vol 67 (3) ◽  
pp. 430-444
Author(s):  
Ander Arando ◽  
Juan Vicente Delgado ◽  
José Manuel León ◽  
Sergio Nogales ◽  
Francisco Javier Navas-González ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Soybean Lecithin ◽  
Egg Yolk ◽  
Kinematic Parameters ◽  
Progressive Motility ◽  
Head Displacement ◽  
Liquid Storage ◽  
Lateral Head

The effect of different extenders on sperm motility and fertility was evaluated during liquid storage of ram semen at 5 °C and 15 °C. The semen was collected, pooled and diluted in three commercial extenders: Inra 96® (INRA) based on skimmed milk, Biladyl® A fraction (BIL) based on egg yolk, and Ovixcell® (OVIX) based on soybean lecithin. Then, sperm motility was evaluated at 0, 6, 24, 48, 72 and 96 h. In order to evaluate fertility, samples stored at 15 °C were used after dilution in INRA and OVIX. Results showed that progressive motility was significantly higher up to 72 h of storage in sperm samples maintained at 5 °C in comparison with 15 °C, similarly for each tested diluent. When samples were stored at 5 °C in OVIX, kinematic parameters such as velocity (except curvilinear velocity, VCL), trajectory [linearity (LIN), straightness (STR), wobble (WOB)], amplitude of lateral head displacement (ALH) and beat/cross frequency (BCF) were higher than in INRA and BIL. No significant differences in pregnancy rate were detected between INRA (62.6%) and OVIX (58.9%). In conclusion, liquid storage at 5 °C with OVIX extender is an interesting option since non-animal components are used, and this extender offers similar in vitro and in vivo efficacy as other extenders containing animal components.

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