Assessment of binder of sperm protein 1 (BSP1) and heparin effects on in vitro capacitation and fertilization of bovine ejaculated and epididymal sperm

Zygote ◽  
2020 ◽  
Vol 28 (6) ◽  
pp. 489-494
Author(s):  
Paula Rodríguez-Villamil ◽  
Daiane Mentz ◽  
Felipe Ledur Ongaratto ◽  
Luis Henrique Aguiar ◽  
Jose Luiz Rodrigues ◽  
...  
Keyword(s):  
Control Group ◽  
Sperm Capacitation ◽  
Epididymal Sperm ◽  
Sperm Protein ◽  
Fertilization Rates ◽  
Bovine Sperm ◽  
Development Rates ◽  
Developmental Rates

SummaryThe present study evaluated the effect of binder of sperm protein 1 (BSP1) and/or heparin on in vitro bovine capacitation and fertilization rates using epididymal and ejaculated bovine sperm. Frozen–thawed sperm were selected and used in the following treatments. Control group: Fert-TALP medium without heparin; heparin (HEP) group: Fert-TALP with heparin (10 UI/ml); BSP1 group: Fert-TALP medium with BSP1 (10 µg/ml for ejaculated sperm; 40 µg/ml for epididymal sperm); HEP + BSP1 group: Fert-TALP medium with heparin (5 UI/ml) and BSP1 (5 µg/ml for ejaculated sperm; 20 µg/ml for epididymal sperm) and determined in vitro capacitation rates in different interval times (0, 15, 30 and 60 min) using the chlortetracycline fluorescence (CTC) method. Also, we evaluated the development rates of oocytes fertilized with ejaculated or epididymal sperm into the same treatments. Capacitation was greater and faster when ejaculated sperm were treated for 60 min with heparin compared with other treatments. However, developmental rates were similar in all treatments. For epididymal sperm, the treatments with BSP1 presented higher capacitation and fertilization rates compared with heparin (P < 0.05). The effects of heparin + BSP1 on capacitation and developmental rates did not cause any increase in capacitation or blastocyst rates compared with other groups for ejaculated or epididymal sperm. In conclusion, this study confirmed that either BSP1 and heparin can be used as capacitator agents for bovine ejaculated sperm during IVF. However, BSP1 seems to be more efficient compared with heparin for epididymal sperm. Furthermore, BSP1 and heparin have no synergic effects on sperm capacitation.

154 CRYO-ACROSOME REACTION/CAPACITATION OF EJACULATED AND EPIDIDYMAL BOVINE SPERM AND THEIR IN VITRO FERTILIZATION RATES

2012 ◽  
Vol 24 (1) ◽  
pp. 189
Author(s):  
M. A. Stout ◽  
J. R. Saenz ◽  
J. F. Chenevert ◽  
G. T. Gentry ◽  
K. B. Bondioli ◽  
...  
Keyword(s):  
Acrosome Reaction ◽  
Fertilization Rate ◽  
Fluorescent Staining ◽  
Epididymal Sperm ◽  
Vitro Fertilization ◽  
Mean Values ◽  
Fertilization Rates ◽  
Bovine Sperm ◽  
The Mean

Seminal plasma has been shown to affect the composition and function of sperm. This exposure may alter the ability of sperm to endure cryopreservation, undergo capacitation and fertilize oocytes. Earlier studies demonstrated that the freezing response of epididymal and ejaculated sperm from the bulls utilised in these studies was similar (Alapati et al. 2009). Subsequent studies are designed to investigate the response of ejaculated and epididymal bovine sperm to cryopreservation and their ability to undergo capacitation. Ejaculated and epididymal sperm from the same bulls (n = 4) were collected by an artificial vagina and retrograde caudal epididymal flush, respectively and were cryopreserved in standard egg-yolk glycerol extender. Sperm was compared by level of cryo-acrosome reaction/capacitation and their fertilization rates in vitro with and without the capacitating agent heparin. Upon evaluation, a sample was thawed and washed and the viable sperm population was isolated through a discontinuous Percoll® gradient. Sperm viability and acrosome status were assessed by fluorescent staining with propidium iodine and fluorescein isothiocyanate-PNA followed by evaluation with flow cytometry. Capacitated sperm were then induced to undergo the acrosome reaction by exposure to lysophosphatidylcholine (10 μg mL–1). Cryo-capacitation was determined by the difference between cryo- and lysophosphatidylcholine-induced acrosome-reacted sperm levels. Differences in the mean values between groups were analysed by ANOVA. Ejaculated and epididymal sperm differed by level of cryo-acrosome reaction (15 vs 4%, respectively; P < 0.001), but did not differ by level of cryo-capacitation (6.2 vs 5.9%, respectively; P = 0.92). The ability of epididymal and ejaculated sperm to fertilize oocytes in vitro with and without heparin was also investigated. Sperm were thawed and washed and the viable population was isolated through a Percoll® gradient. Oocytes were washed and randomly assigned to a treatment group, either ejaculated ± heparin or epididymal ± heparin. Oocytes and sperm were added to a capacitating medium (TALP) with and without heparin. Following 18 h of incubation, fertilization rate was determined by pronuclear fluorescent staining with Hoechst 33342 followed by confirmation with aceto-orcein staining. Differences in the mean values among treatment groups were analysed by one-way ANOVA followed by Holm-Sidak pairwise multiple comparisons. Fertilization rates of epididymal sperm with and without heparin and ejaculated sperm with heparin were similar (76, 80 and 70%, respectively). Ejaculated sperm without heparin was lower than all other groups, with a fertilization rate of 42% (P < 0.001). In summary, epididymal sperm displayed lower levels of cryo-acrosome reaction but were similar by level of cryo-capacitation. In addition, epididymal and ejaculated sperm differed in the need for a capacitating agent such as heparin when used in vitro. This may be useful to increase efficiency when using epididymal sperm in assisted reproductive techniques.

scholarly journals Purification of binder of sperm protein 1 (BSP1) and its effects on bovine in vitro embryo development after fertilization with ejaculated and epididymal sperm

2016 ◽  
Vol 85 (3) ◽  
pp. 540-554 ◽  
Author(s):  
P. Rodríguez-Villamil ◽  
V. Hoyos-Marulanda ◽  
J.A.M. Martins ◽  
A.N. Oliveira ◽  
L.H. Aguiar ◽  
...  
Keyword(s):  
Embryo Development ◽  
Epididymal Sperm ◽  
Sperm Protein

296 EFFECT OF GROWTH HORMONE ON EMBRYO DEVELOPMENT AND ULTRASTRUCTURE OF BOS TAURUS IVP BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 304
Author(s):  
L. M. C. Pegoraro ◽  
M. N. Dode ◽  
C. F. Weissheimer ◽  
F. G. Leivas ◽  
A. Vieira ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Culture Medium ◽  
Production Systems ◽  
Bos Taurus ◽  
In Vitro Production ◽  
Assisted Reproductive Technique ◽  
Development Rates ◽  
Developmental Rates ◽  
Chi Square Analysis

Bovine in vitro production systems are one of the most used assisted reproductive technique. However, this technique has some limitations especially in Bos taurus breeds, because of the low percentage of viable blastocysts produced (around 40% of oocytes inseminated) and higher cryosensitivity due to higher lipid content. Growth hormone (GH) can be a promising additive to increase in vitro embryo production. The aim of this study was to evaluate the embryo developmental rates (blastocyst/oocytes cleaved and blastocyst/oocytes inseminated) and ultrastructural features in Bos taurus embryos produced in SOFaa medium with or without GH. Cumulus oocyte complexes (COC) were recovered from slaughterhouse-derived ovaries (Angus Red crosses) and by ovum pick-up (OPU) from Jersey donors. After IVM and IVF, the presumptive zygotes were allocated in the SOFaa medium without (control) or with addition of GH (100 ng mL-1), for culture at 39°C in an atmosphere of 5% CO2. The cleavage and viable blastocyst rates were recorded 2 and 8 days after initiation of IVF, respectively. The results were compared by chi-square analysis. Similar (P > 0.05) cleavage rates were found in different culture medium and bovine breeds (61 v. 63% for Jersey control and Jersey GH; 71 v. 72% for cross-breed control and cross-breed GH). The development rates (blastocyst/oocytes cleaved and blastocyst/oocytes inseminated) did not differ in culture medium with or without GH within breeds (35 v. 30% for Jersey control and GH; 52 v. 56% for cross-breed control and GH; 21 v. 20% for Jersey control and GH; 36 v. 41% for cross-breed control and GH, respectively; P > 0.05). However, when breeds were compared, higher development rates were observed in cross-breed obtained from slaughterhouses than Jersey donors obtained by OPU (35 v. 52% for Jersey v. cross breed control; 30 v. 56% for Jersey v. cross-breed GH; 21 v. 36% for Jersey v. cross-breed control; 20 v. 41% for Jersey v. cross-breed GH. P < 0.001). The analyses of ultrastructure demonstrated no difference in the lipid proportion and organelle distribution of embryos produced with or without GH. We concluded that GH addition to SOFaa medium did not increase developmental rates for cross-breed or Jersey IVP embryos.

7 THE EFFECT OF A NOVEL RECOMBINANT PROTEASE TREATMENT ON BOVINE SPERM FERTILIZING CAPACITY AND EMBRYO DEVELOPMENT IN VITRO

2009 ◽  
Vol 21 (1) ◽  
pp. 104
Author(s):  
J. T. Aaltonen ◽  
K. J. Mattson ◽  
N. M. Loskutoff
Keyword(s):  
Embryo Development ◽  
Disease Transmission ◽  
Bos Taurus ◽  
Gradient Centrifugation ◽  
Control Group ◽  
Bovine Embryo ◽  
Human Sequence ◽  
Bovine Sperm

As described in the IETS Manual (Stringfellow and Seidel, 1995), and endorsed by the OIE, trypsin can be used (for specific pathogens and livestock) to effectively remove certain infectious agents from in vivo-derived embryos for international transport. Because of the multimillion-dollar AI industry for livestock, the OIE has encouraged more research in developing similar decontamination techniques for semen as an added safeguard to animal quarantine for the prevention of disease transmission. Most or all of the earlier studies on embryos used a porcine pancreatic-derived trypsin. Because of more stringent guidelines from international regulatory agencies on the use of animal products, several serine protease recombinants are now available. Previous experiments comparing the porcine pancreatic extract with a recombinant bovine sequence trypsin developed in corn resulted in no statistical difference in cleavage or morula/blastocyst rates. (Mattson et al. 2008 Theriogenology 69, 724–727). An additional in vivo study treating bovine sperm with a yeast-derived human-sequence trypsin resulted in significantly more transferable-quality embryos after the AI of superovulated cows as compared with sperm not treated with trypsin (Blevins et al. 2008 Reprod. Fertil. Dev. 20, 84). The goal of this experiment was to examine the in vitro development of bovine embryos produced from sperm treated with a recombinant trypsin found in a commercially available density gradient centrifugation (DGC) product (Bovipure, Nidacon, Sweden) compared with DGC without trypsin. Oocyte aspiration, maturation, fertilization, and embryo culture were performed using standard methods in 5 replications (n = 2220 oocytes). Semen was collected and pooled from 2 Bos taurus bulls and frozen in an egg-yolk cryodiluent (Biladyl, Minitube). The semen was processed using Bovipure DGC composed of 2 mL of 40% colloid of silane-coated silica particles containing either a yeast-derived human sequence recombinant trypsin containing no animal by-products (n = 1126 oocytes) or the same colloid without trypsin as the control group (n = 1094 oocytes). Both 40% concentrations were layered over 2 mL of an 80% concentration of the same colloid without any additives. The density gradients were centrifuged at 300g for 20 min, after which time the pellets were washed in 5 mL of prewarmed TL Hepes solution (Cambrex) and centrifuged at 500g for 10 min. The resulting sperm pellets were then resuspended in a volume calculated to provide 1 × 106 sperm mL–1, to be used for in vitro inseminations. Results were compared using a 2-tailed unpaired t-test. Cleavage rates for the trypsin-treated sperm (n = 969, 35.8%) and the control (n = 950, 44.3%) groups were not statistically different (P = 0.20). Although more embryos reached the morula to blastocyst stages in the control group (n = 421, 61.0%) than in the trypsinized group (n = 347, 54.7%), these differences also were not statistically significant (P = 0.85). In conclusion, trypsinized Bovipure DGC of sperm before insemination showed no detrimental effects on IVF-derived bovine embryo development.

322 EFFECT OF SODIUM NITROPRUSSIDE ON BUFFALO SPERM CAPACITATION IN VITRO

2007 ◽  
Vol 19 (1) ◽  
pp. 276 ◽  
Author(s):  
L. Boccia ◽  
L. Attanasio ◽  
A. De Rosa ◽  
G. Pellerano ◽  
R. Di Palo ◽  
...  
Keyword(s):  
Sodium Nitroprusside ◽  
Acrosome Reaction ◽  
Production Efficiency ◽  
Control Group ◽  
Sperm Capacitation ◽  
Promoting Effect ◽  
Vitro Fertilization ◽  
Domestic Species ◽  
Frozen Semen

The overall in vitro embryo production efficiency in buffalo is hampered by the poor fertilization rate. It is known that the quality of the frozen semen may affect fertilization efficiency. However, it is not possible to rule out that the process of capacitation, required by spermatozoa to acquire the fertilizing ability, is impaired in the in vitro fertilization (IVF) system. Although several agents have been proven to induce sperm capacitation in vitro, heparin treatment is still the most efficient method in most of the domestic species. There is evidence that capacitation is part of an oxidative process and that nitric oxide (NO) acts as a capacitation inducer in human (Herrero et al. 1999 Biol. Reprod. 61, 575–581) and bovine (Rodriguez et al. 2005 Anim. Reprod. Sci. 85, 231–242) spermatozoa. The aim of the present study was to evaluate whether sodium nitroprusside (SNP), a well-known generator of NO in vitro, improves buffalo sperm capacitation in vitro. Frozen–thawed sperm from a bull previously tested for IVF were treated by swim-up in order to select only the motile population. Spermatozoa were incubated in the presence of 0.01 mM heparin (control group) for 1 h (n = 266), 2 h (n = 270), and 3 h (n = 306), and in the presence of 10 �M SNP for 1 h (n = 302), 2 h (n = 286), and 3 h (n = 260). The concentration of SNP was chosen on the basis of a preliminary dose-response trial (0.1 �M, 1 �M, and 10 �M). Following incubation with these agents, sperm were exposed for 15 min to 60 �g mL-1 of lysophosphatidylcholine, an agent known to induce acrosome reaction only on capacitated spermatozoa. Trypan blue was used first to differentiate live from dead spermatozoa and the dried smears were then fixed in 37% formaldehyde and stained with Giemsa for acrosome evaluation by microscopic examination. The proportion of acrosome-reacted spermatozoa in each group was used to assess the efficiency of capacitation under different incubation conditions. Differences between groups were analyzed by chi-squared test. No dead spermatozoa were found in all groups. Following 1-h sperm treatment with either heparin or SNP, the proportion of acrosome-reacted spermatozoa was similar (35.3% vs. 28.5%, respectively). However, extending the incubation time to 2 h, SNP significantly (P &lt; 0.01) increased the incidence of acrosome reaction compared to heparin (60.1% vs. 44.1%, respectively). Analogously, when the sperm treatment was prolonged to 3 h, SNP gave a significantly (P &lt; 0.01) higher percentage of acrosome reaction compared to the control (68.8% vs. 36.6%, respectively). In conclusion, sperm treatment with SNP for either 2 or 3 h significant improved the efficiency of buffalo sperm capacitation in vitro compared with heparin, that is, the capacitating agent currently used in the IVF system. The promoting effect of SNP indirectly indicates that NO acts as a capacitation inducer in buffalo spermatozoa. Finally, these results suggest the need to evaluate the effect of SNP on the fertilizing capability of buffalo spermatozoa in vitro.

26 Drugs that Modify Epigenetics...What do they do to Porcine Clones?

2018 ◽  
Vol 30 (1) ◽  
pp. 152
Author(s):  
C. P. Buemo ◽  
A. Gambini ◽  
L. N. Moro ◽  
N. Canel ◽  
D. F. Salamone
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Electric Pulse ◽  
Donor Cell ◽  
T Test ◽  
Control Group ◽  
Developmental Rates ◽  
Student’S T ◽  
Student’S T Test

Although somatic cell nuclear transfer (SCNT) technology was developed more than 20 years ago, cloning efficiency remains low. Failures in the reprogramming of the donor cell result in embryos with aberrant epigenetic patterns and low developmental rates. In this study, we assessed whether the use the inhibitor of DNA (cytosine 5) methyltransferase 5-azacitidine (5Aza) combined with the MEK inhibitor in the MAPK pathway PD0325901 (PD) could improve SCNT efficiency in pigs. In vitro maturation of cumulus–oocyte complexes was performed in TCM for 44 h at 39°C under 5% CO2. Cumulus cells and zona pellucida was removed from matured oocytes, followed by enucleation of the metaphase plate previously stained with Hoëchst 33342. Each enucleated oocyte was attached to a donor cell by phytohemagglutinin treatment followed by an electric pulse of 80V for 30 μs. After fusion, reconstituted embryos were activated by an electric pulse followed by an incubation in 2 mM 6-DMAP for 3 h. Cloned embryos were cultured in vitro in a modified well-of-well system in SOF medium, where 3 cloned embryos were placed per microwell (3X). The experimental group 3X + drugs was exposed for the first 3 days to 1 μM PD and 1 μM 5Aza in SOF medium. After washing, embryos were cultured until Day 7 in regular SOF medium. The control group (3X) was cultured in regular SOF medium for 7 days. In vitro embryo developmental rates, gene expression, histone acetylation, and DNA methylation status were studied. The use of epigenetic modifying drugs significantly increased blastocyst rates (40.9% v. 29%; Fisher’s test, P < 0.05) and embryo size (41.46% v. 28.56%; Student’s t-test, P < 0.05) compared with the control group. Regarding gene expression, an increase of the relative expression of genes related to cell differentiation (Igf2 and Cdx2), antiapoptotic pathways (Bcl-xl) and DNA methylation modulation (Mapk1) was observed (P < 0.05). Pluripotency genes Oct4 and Nanog did not show differences between groups. The Bax proapoptotic gene significantly decreased its expression after drug treatment, as did the Klf4 gene (P < 0.05). Results were analysed by Student’s t-test. According to Histone H3K27ac, which is associated with enhancers or gene promoters, its marker was located mainly in the nuclear periphery respect to the control group with a uniform dispersion, indicating that the treatment could be activating certain genes by locating them near the periphery. Histone H3K4me1 was more uniformly localised throughout the nucleus in both groups. The intensity of the fluorescence was measured by quantitative confocal microscopy using a histogram produced by the ImageJ program (National Institutes of Health, Bethesda, MD, USA). Regarding DNA methylation by bisulphite sequencing, the 2 genes studied (Oct4 and DNMT1) showed a higher demethylation status for the treated group. Our results indicate that the combination of 5Aza+PD during early pre-implantation development dramatically increase blastocyst rates and embryo quality. This novel combination could be used as a strategy to improve the efficiency of SCNT in pigs and potentially other animals.

77 Development and quality of in vitro bovine hemi embryos produced by blastomere separation and embryo bisection

2019 ◽  
Vol 31 (1) ◽  
pp. 164
Author(s):  
A. E. Ynsaurralde Rivolta ◽  
M. Suvá ◽  
V. Alberio ◽  
C. Vazquez Echegaray ◽  
A. Guberman ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Monozygotic Twin ◽  
Cell Number ◽  
Control Group ◽  
Cell Stage ◽  
Exact Test ◽  
Development Rates

Bovine monozygotic production of twins became popular in the 1980s as a technique to multiply high value genetics. Moreover, it also became a powerful model for research. Different techniques have been used on bovine embryos obtained by superovulation. In this work, we compared the development rates and quality of monozygotic twin embryos produced by blastomere separation (BS) and embryo bisection (EB) of IVF embryos. To this aim, cumulus-oocytes complexes collected from slaughterhouse ovaries were in vitro matured in TCM 199 containing 10% fetal bovine serum, 10µg mL−1 FSH, 0.3mM sodium pyruvate, 100mM cysteamine, and 2% antibiotic-antimycotic for 24h, at 6.5% CO2 in humidified air and 38.5°C. The IVF was performed with 16×106 spermatozoa per mL for 5h. Afterward, presumptive zygotes were cultured in SOF medium for 7 days at 38.5°C and 5% O2. After 24h of culture, blastomeres of 2-cell stage embryos (N=114) were separated and each one was cultured individually in a microwell for 7 days. Embryo bisection (N=179) was performed manually on Day-7 blastocysts previously depleted of their zonae pellucidae, under stereoscopic microscope. Hemi embryos were cultured for 24h and then twins and single blastocyst rates were calculated. For quality assessment, diameter, total and inner cell mass (ICM) cell number of hemi embryos (BS: 6 couples; ES: 10 couples) and the control group (C: 11) were evaluated. The ICM cell number was measured by immunofluorescence staining using SOX2 antibody and the percentage of ICM and trophectoderm (TE) cells was calculated. The results were analysed using Fisher’s exact test and ANOVA with mean comparison using Tukey’s test (P=0.05). No statistical differences were found in blastocyst rates of twins and single hemi embryos produced by BS (28 and 25%) or EB (23 and 32%). Blastocyst diameter was similar between groups and control. Hemi embryos exhibited lower total and ICM cell number than control (BS: 43±18, EB: 57±14v. C: 93±35 and BS: 16±7, EB: 12±8v. C: 34±19). However, BS hemi embryos had higher ICM and lower TE percentage (40/60%) compared with the EB group (20/80%). The control group did not differ with hemi embryo treatments for ICM and TE (30/70%). Our preliminary results have indicated that although the development rates of hemi embryos produced in vitro were similar between both techniques, blastomere separation generates better quality embryos than blastocyst bisection.

scholarly journals In vivo exposure to 17β-estradiol triggers premature sperm capacitation in cauda epididymis

Reproduction ◽  
2013 ◽  
Vol 145 (3) ◽  
pp. 255-263 ◽  
Author(s):  
Lukas Ded ◽  
Natasa Sebkova ◽  
Martina Cerna ◽  
Fatima Elzeinova ◽  
Pavla Dostalova ◽  
...  
Keyword(s):  
Reproductive Tract ◽  
Negative Impact ◽  
Reproductive Potential ◽  
Female Reproductive Tract ◽  
Sperm Capacitation ◽  
Epididymal Sperm ◽  
Knock Out ◽  
17Β Estradiol

Estrogens play a crucial role in spermatogenesis and estrogen receptor α knock-out male mice are infertile. It has been demonstrated that estrogens significantly increase the speed of capacitation in vitro; however this may lead to the reduction of reproductive potential due to the decreased ability of these sperm to undergo the acrosome reaction. To date the in vivo effect of estrogens on the ability of sperm to capacitate has not been investigated. Therefore, in this study, we exposed mice (n=24) to 17β-estradiol (E2) at the concentration of 20 ng/ml either during puberty from the fourth to seventh week of age (n=8), or continuously from birth for a period of 12 weeks (n=8) at which age the animals from both groups were killed. The capacitation status of epididymal and testicular sperm was analysed by tyrosine phosphorylation (TyrP) antibody (immunofluorescence and western blot) and chlortetracycline (CTC) assay. According to our results, in vivo exposure to increased E2 concentrations caused premature sperm capacitation in the epididymis. The effect of E2, however, seems reversible because after the termination of the exposure premature epididymal sperm capacitation is decreased in animals treated during puberty. Furthermore the changes in epididymal sperm capacitation status detected by TyrP and CTC positively correlate with plasma levels of E2 and the expression of the estrogen-dependent trefoil factor 1 (Tff1) gene in testicular tissue. Therefore, our data implicate that in vivo exposure to E2 under specific conditions leads to the premature capacitation of mouse sperm in epididymis with a potential negative impact on the sperm reproductive fitness in the female reproductive tract.

scholarly journals In Vitro Fertilization Outcome After Intracytoplasmic Sperm Injection with Fresh and with Frozen-Thawed Epididymal Spermatozoa

KnE Medicine ◽  
2016 ◽  
Vol 1 (1) ◽  
Author(s):  
Hilma Putri Lubis
Keyword(s):  
In Vitro Fertilization ◽  
Intracytoplasmic Sperm Injection ◽  
Oocyte Retrieval ◽  
Epididymal Sperm ◽  
Vitro Fertilization ◽  
Fertilization Rates ◽  
Sperm Aspiration ◽  
Significant Difference ◽  
Epididymal Spermatozoa

<p><strong>Introduction</strong></p><p>Testicular epididymal sperm aspiration (TESA) is one of the method  to retrieve sperm from the testes in men with azoospermia. The aim of the study is to compare the In vitro fertilization (IVF) outcome of intracytoplasmic sperm injection (ICSI)-ET cycles with fresh testicular epididymal spermatozoa obtained on the same day with  oocyte retrieval and with frozen-thawed testicular epididymal spermatozoa.</p><p><strong>Material &amp; Methods</strong></p><p>A retrospective comparative analysis of  patients who underwent fresh TESA and frozen-thawed TESA in ICSI-ET cycles from January 2012 to December 2014 in Halim Fertility Center was done. Fresh testicular epididymal sperm aspiration (fresh TESA) was performed on the same day with oocyte retrieval in 28 cycles and the frozen-thawed testicular epididymal sperm aspiration (frozen-thawed TESA) was used in 30 cycles.  </p><p><strong>Results</strong></p><p>The two groups were comparable in terms of the ages of male and female patients, etiology of infertility and duration of infertility. Fertilization rates in fresh TESA group were 53,5% and in frozen-thawed TESA group, fertilization rates were 50%. There was no statistically significant difference between the groups. Clinical pregnancy rates in fresh TESA group were 35,7%  and in frozen-thawed TESA group, clinical pregnancy rates were 26,7% and statistically there was no significant difference between the groups.</p><p><strong>Conclusion</strong></p>There is no significant difference in the in vitro fertilization outcome of intracytoplasmic sperm injection (ICSI)-ET cycles between fresh TESA and frozen-thawed TESA .

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