P–262 Comparison of embryo aneuploidy rate and reproductive outcomes of ART cycles using fresh and vitrified donor oocytes

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
A Garci. Sifre ◽  
L Orteg. Lopez ◽  
L Va. Os ◽  
A Parrella ◽  
M Enciso ◽  
...  
Keyword(s):  
Clinical Outcomes ◽  
Previous History ◽  
Time Lapse ◽  
Developmental Competence ◽  
Embryo Morphology ◽  
Oocyte Vitrification ◽  
Aneuploidy Rate ◽  
Significant Difference ◽  
Blastocyst Rate ◽  
Donor Oocytes

Abstract Study question Is there any difference in blastocyst morphology, embryo aneuploidy rate and ART clinical outcomes when using fresh or vitrified donor oocytes? Summary answer Frequency of good quality blastocyst obtained from fresh oocytes is significantly higher compared to vitrified.No difference in embryo aneuploidy rates nor clinical outcomes were found. What is known already Oocytes vitrification is an efficient method that allows non only fertility preservation but also the creation of donor oocytes banks, optimizing clinical resources for patients undergoing Assisted Reproductive Technology. Although the benefits of donor oocytes vitrification are well known, some studies have shown that this cryopreservation process can induce spindle abnormalities and chromosomal changes, leading to aneuploidy. Comparative studies between fresh and vitrified oocytes to evaluate embryo developmental competence, aneuploidy and clinical pregnancy rate (CPR) are needed. Study design, size, duration This retrospective study includes ICSI cycles with fresh donor oocytes(N = 2795) and vitrified donor oocytes (N = 1225) between January 2019 and September 2020. Pre-implantation Genetic Testing for Aneuploidy (PGT-A) was performed on Day 5 and Day 6 blastocysts. Fertilization rate, blastocyst morphology, aneuploidy status and CPR were analysed and compared between the groups. Recipients were equally distributed in terms of maternal age (40.86 years) and previous history, sperm samples were also similar in profile and origin (fresh-frozen). Participants/materials, setting, methods A total of 266 subfertile couples participated in the study, ICSI was carried out in all cycles. Vitrification and warming protocols were performed with a commercial kit. All embryos were cultured to blastocyst stage in a Time-Lapse incubator and assessed by Gardner’s blastocyst grading scale. PGT-A testing was performed on trophectoderm biopsies by Next Generation Sequencing (NGS). Single/double embryo transfers were performed in all cases. Odd-ratios were calculated,and Chi-square was performed for the statistical analysis. Main results and the role of chance A total of 266 patients underwent 289 donor oocyte cycles yielding an overall of 4557 oocytes. ICSI was performed on 2795 fresh and 1225 vitrified mature oocytes. Similar fertilization rates were achieved with fresh and vitrified oocytes (75.9% (2122/2795) and 75.2% (921/1225), respectively (P = 0.6)) yielding a significant difference in blastocyst rate of 71.7% (1522/2122) and 62.5% (576/921) (OR 1,519; 95% CI 1,290–1,789; p < 0.001). In addition, when blastocysts morphology was analysed, a significant difference was shown in the frequency of good quality embryos that decreases from 56.6% (861/1522) with fresh oocytes to 51% (294/576) with vitrified oocytes (OR 1,249; 95% CI 1,031–1,514; P < 0.02). PGT-A testing of blastocysts revealed not significant differences in euploidy rates (73.6% in fresh oocytes vs 76.8% vitrified oocytes, P = 0.2). With regards to clinical outcomes, similar results were found between the groups. A total of 322 embryo transfers were performed (237 from fresh and 85 vitrified) achieving a CPR of 48.9% (116/237) with fresh oocytes and 54% (46/85) with vitrified (P = 0.7) and a pregnancy loss of 6.7%(16/237) in fresh oocytes and 11.7%(10/85) vitrified oocytes (P = 0.1). Limitations, reasons for caution The study was conducted on a small number of cases. Further studies are needed to confirm our findings. Moreover, although the same stimulation protocol was used, donors from different background were included. Wider implications of the findings: This study supports the use of vitrified oocytes in the laboratory routine without compromising clinical outcomes. Although oocyte vitrification may have an influence on embryo morphology, blastocyst rate, no impact of this cryopreservation process is seen on embryo aneuploidy, developmental competence and CPR. Trial registration number Not applicable

196 A NEW APPROACH TO PREDICT MOUSE EMBRYONIC DEVELOPMENTAL COMPETENCE USING A TIME-LAPSE SYSTEM AND THE 'SHORTEST HALF' analysis procedure

2007 ◽  
Vol 19 (1) ◽  
pp. 214 ◽  
Author(s):  
S. Yavin ◽  
A. Aroyo ◽  
Z. Roth ◽  
A. Arav
Keyword(s):  
Embryonic Development ◽  
Time Lapse ◽  
Time Frame ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Embryo Morphology ◽  
Cell Stage ◽  
Developmental Potential ◽  
Additional Tool ◽  
Embryonic Cleavage

Embryonic development is a dynamic process in which embryo morphology may change immensely within several hours. Therefore, identifying and selecting embryos with the highest probability of developing and achieving a pregnancy is a major challenge. The timing of embryonic cleavage may serve as an additional indicator for the identification of quality embryos. The aim of this study was to characterize the cleavage timing of mouse embryos and to identify the stage that is most indicative of blastocyst formation. Mated mice (CB6F1) were sacrificed 20 h after hCG administration; putative zygotes were recovered and cultured (50 embryos in each 20-µL drop of M16) in a time-lapse system (EmbryoGuard; IMT, Ltd., Ness-Ziona, Israel) inside the incubator. The time-lapse system was programmed to take photos at half-hour intervals such that culture dishes were not removed from the incubator. The ‘shortest half’ statistical procedure of JMPIN (SAS Institute, Inc., Cary, NC, USA) was utilized to evaluate the period during which at least 50% of the embryonic population cleaves within the shortest time frame. Captured images made it possible to search along the time axis for the densest 50% of cleavage observations. Developing embryos were categorized into 3 groups according to the time of cleavage after hCG administration: before, during, and after the ‘shortest half’ for each developmental stage. Two hundred thirty putative zygotes cleaved and created 2-cell-stage embryos, of which 55 arrested at various stages and 175 progressed to the blastocyst stage. During embryonic development, cleavage timing appeared to become less uniform and the ‘shortest half’ became longer for each successive cell division: Whereas the shortest period in which 50% of the 2-cell-stage embryos cleaved was a 2-h interval, cleavage into the 4-cell, 8-cell, and blastocyst stages took 2.5, 3.5, and 5 h, respectively. The ‘short half’ for the first cleavage appears to be a predictive time frame for subsequent embryonic development, because cleavage was closely synchronized with 80% of the embryos developing to the blastocyst stage. Note that only a small number of embryos were actually cleaving early, while the ‘shortest half’ consisted of 50% of the embryonic population. Moreover, late-cleaving embryos in the 2-cell stage expressed inferior developmental potential relative to those that cleaved within the ‘shortest half’ (see Table 1). In summary, 2-cell-stage embryos that cleaved within the ‘shortest half’ seemed to be better synchronized and consequently more competent than the rest of the embryonic population. Embryonic cleavage timing using the ‘shortest half’ parameter can be considered a biological indicator of embryo potential. It may be useful as an additional tool for selecting embryos for transfer and cryopreservation. Table 1. Cleavage timing distribution into the 2-cell stage according to the shortest half

Early embryo morphokinetics is a better predictor of post-ICSI live birth than embryo morphology: speed is more important than beauty at the cleavage stage

Zygote ◽  
2021 ◽  
pp. 1-8
Author(s):  
Alessandro Bartolacci ◽  
Mariabeatrice Dal Canto ◽  
Maria Cristina Guglielmo ◽  
Laura Mura ◽  
Claudio Brigante ◽  
...  
Keyword(s):  
Multivariate Analysis ◽  
Live Birth ◽  
Live Birth Rate ◽  
Roc Curves ◽  
Time Lapse ◽  
Early Embryo ◽  
Developmental Competence ◽  
Embryo Morphology ◽  
Roc Curve Analysis ◽  
Morphokinetic Parameters

Abstract Given the importance of embryo developmental competence assessment in reproductive medicine and biology, the aim of this study was to compare the performance of fertilization and cleavage morphokinetics with embryo morphology to predict post-ICSI live birth. Data from embryos cultured in a time-lapse microscopy (TLM) incubator and with known live birth outcomes (LB: embryos achieving live birth, n = 168; NLB: embryos not achieving live birth, n = 1633) were used to generate receiver operating characteristic (ROC) curves based on morphokinetic or morphological scores, and the respective areas under the curve (AUC) were compared. The association between live birth and 12 combinations of four morphokinetic quality degrees (A–D) with three morphological quality degrees (A–C) was assessed using multivariate analysis. Morphokinetic parameters from tPNa to t8 were reached earlier in LB compared with NLB embryos. The ROC curve analysis indicated that morphokinetic information is more accurate than conventional morphology to predict live birth [AUC = 0.64 (95% CI 0.58–0.70) versus AUC = 0.58 (95% CI 0.51–0.65)]. The multivariate analysis was in line with AUCs, revealing that embryos with poor morphokinetics, independently of their morphology, provide lower live birth rates (P < 0.001). A considerable percentage of embryos with top morphology presented poor morphokinetics (20.10%), accompanied by a severely reduced live birth rate in comparison with embryos with top morphology and morphokinetics (P < 0.001). In conclusion, TLM-derived early morphokinetic parameters were more predictive of live-birth achievement following ICSI than conventional morphology.

scholarly journals No association of mitochondrial DNA levels in trophectodermal cells with the developmental competence of the blastocyst and pregnancy outcomes

2019 ◽  
Author(s):  
G Ritu ◽  
Geetha Veerasigamani ◽  
Mohammed C. Ashraf ◽  
Sankalp Singh ◽  
Saniya Laheri ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Clinical Outcomes ◽  
Live Birth ◽  
Maternal Age ◽  
Small Sample ◽  
Developmental Competence ◽  
Significant Difference ◽  
Blastocyst Quality ◽  
Implantation Rates ◽  
Ploidy Status

AbstractStudy questionCan mitochondrial DNA (mtDNA) levels in trophectodermal cells of the blastocyst predict the blastocyst quality, ploidy status, implantation rate and clinical outcomes?Summary answermtDNA levels in trophectodermal cells of the blastocyst do not associate with the blastocyst quality, ploidy status, implantation potential and clinical outcomes, but can differentiate between aneuploid and euploid blastocysts.What we already knowmtDNA levels in the trophectodermal cells have been suggested to be associated with blastocyst morphology, ploidy and implantation rates, and has been proposed as biomarker to access blastocyst quality and predict clinical outcomes. However, discrepancies exist if mtDNA levels could serve as a marker for the same.Study design and durationRetrospective analysis of mtDNA levels in trophectodermal cells obtained from blastocysts undergoing preimplantation genetic testing for aneuploidy (PGT-A) at Craft Hospital & Research Center, Kerala from January 2016-July 2017.Participants/materials and methodsStudy included data from 287 blastocyst from (61) couples who underwent PGT-A using next generation sequencing (NGS). Levels of mtDNA in trophectodermal cells of the blastocyst were estimated by the NGS. Comparison of mtDNA levels with maternal age, blastocyst morphology, ploidy status, implantation rates, miscarriage rates and live birth rate was done.Main resultsThe levels of mtDNA in the trophectoderm of the blastocyst did not correlate with maternal age. There was no significant difference in the mtDNA levels between grade 1 and grade 2 blastocyst. Euploid blastocyst had significantly lower amounts of mtDNA levels in trophectodermal cells of the blastocyst were compared to aneuploid blastocyst. No significant differences were seen between mtDNA levels and implanting and non-implanting blastocysts or those resulted into miscarriage or live birth.LimitationsThe study is limited by a small sample size and hence type II error cannot be ruled out.Wider ImplicationsThe study does not support the potential use of mtDNA levels in the trophectodermal cells as biomarker for blastocyst quality and predicting clinical outcomes needs.Study funding/competing interest(s)There is no external funding for the study. There is no conflict of interest.

16 EFFECT OF DONOR CELL AND TRICHOSTATIN A ON DEVELOPMENT OF CLONED DAIRY CATTLE EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 119
Author(s):  
Z. Mei-Ling ◽  
Z. Yun-Hai ◽  
T. Yong ◽  
L. Ya ◽  
C. Hong-Guo ◽  
...  
Keyword(s):  
Trichostatin A ◽  
Donor Cell ◽  
Developmental Competence ◽  
Donor Cells ◽  
Significant Difference ◽  
Blastocyst Rate ◽  
Different Origins ◽  
Vitro Development

The objective of present study was to investigate the effects of treatments to donor cells with fresh digestion (FD), cryopreservation/thawing (CT), trichostatin A (TSA) and durations of culture using TSA-CR1aa medium on in vitro development of dairy cow cloned embryos. In addition, some somatic cell cloned embryos were transferred to surrogates in heat to evaluate the in vivo developmental competence. The results (Table 1) showed that pretreatment of donor cells using TSA could significantly increase both cleavage and blastocyst rates of embryos (P < 0.05) compared with FD and CT group, whereas no significant difference was found between FD and CT group. When cloned embryos were subjected to TSA treatment in CR1aa for different times (0, 24, 48 and 60 h), the results showed that the blastocyst rate in the 60-h group was the highest (36.11 ± 1.78%) compared with the other groups (P < 0.05). Whereas the reconstructed embryos derived from donor cells treated with TSA for 24 h were continually cultured in TSA for different times (24, 48 and 60 h), the results showed that the blastocyst rate (37.39 ± 1.78%) in the 60-h group was significantly higher than that of the 24-h (25.48 ± 1.34%) group (P < 0.05). Finally, when the cloned embryos from different groups were respectively transferred to 40 natural oestrus recipients, no significant difference in terms of pregnancy rate among groups was found; however, a viable cloned calf was successfully obtained from TSA-treated donor cells and cloned embryo. Therefore, cloned embryos treated with optimized methods can develop to term. Table 1.Pregnancy results established from embryos of different origins

159 RELATIONSHIP BETWEEN CLEAVAGE PATTERNS OF FIRST CELL CYCLE AND POST-TRANSFER VIABILITY IN BOVINE EMBRYOS OBTAINED BY OVUM PICKUP AND IN VITRO FERTILIZATION

2012 ◽  
Vol 24 (1) ◽  
pp. 191
Author(s):  
K. Imai ◽  
S. Sugimura ◽  
T. Somfai ◽  
Y. Inaba ◽  
Y. Aikawa ◽  
...  
Keyword(s):  
Cell Division ◽  
Calf Serum ◽  
Development Program ◽  
Time Lapse ◽  
The Other ◽  
Developmental Competence ◽  
Vitro Fertilization ◽  
Significant Difference ◽  
Conception Rates

More than 300 000 embryos have been transferred all over the world (Stroud 2010 IETS Newsl. 27(4), 11–21). We have reported that embryos that showed the abnormal cleavage pattern at the first cell division can develop to the blastocyst stage (Somfai et al. 2010 J. Reprod. Dev. 56, 200–207). However, we have limited knowledge about the consequences of the pattern of first embryonic cleavage on their post-transfer developmental competence. The present study was conducted to determine the developmental competence of bovine blastocysts showing different cleavage patterns at their first cell division. Cumulus–oocyte complexes were collected by ovum pickup from Japanese Black cows and were subjected to in vitro maturation and IVF as reported previously (Imai et al. 2006 J. Reprod. Dev. 52, S19–S29 suppl). Inseminated oocytes were cultured in CR1aa medium supplemented with 5% calf serum covered by mineral oil at 38.5°C in 5% CO2 in air with micro-droplets or 5% CO2, 5% O2 and 90% N2. The kinetics of embryo development were analysed by time-lapse cinematography for 168 h after IVF by using a Cultured Cell Monitoring System (CCM-M1.4ZS, Astec, Fukuoka, Japan). A total of 673 photographs of each embryo were taken (1 photograph in every 15 min) during in vitro culture. Image stacks were analysed by the CCM-M1.4 software. Embryos were classified in 5 groups according to the pattern of first cleavage as normal cleavage (NC), direct cleavage from 1 cell to 3 to 4 blastomeres (3–4BL), unequal blastomeres (UB), multiple fragments (MF) and protrusion formation (PT). Blastocysts developing from each group were transferred into the ipsilateral uterine horn of each synchronized recipient on Day 7 or 8 after oestrus. Data on conception at Day 60, abortion and delivery were then recorded. Data were analysed by chi-square test and Student's t-test. In total, 43 embryos were transferred, 17 conceptions (39.5%) were established and 16 recipients (94.1%) were delivered. Only 1 abortion was detected at Day 223 in the NC group. The highest conception rate was observed in the NC group (55%, n = 20) and the 3–4BL (n = 12), UB (n = 6) and PT (n = 3) groups showed similar conception rates of 33.3% (1 implanted embryo belonged to 2 classes in UB and PT) and none of the embryos derived from the MF group (n = 3) could cause conception. There was a significant difference (P < 0.05) in conception rates between the NC group and totals of each of the other cleavage groups. No significant difference was found in gestation lengths and birth weights between the NC group (282.2 ± 4.4 days, 30.6 ± 3.8 kg, respectively) and totals of each of the other cleavage groups (282.8 ± 5.3 days, 30.3 ± 1.9 kg, respectively). These results indicate that embryos showing abnormal cleavage patterns at first cell division can develop to normal calves with normal gestation lengths and birth weights; however, their post-transfer viability is lower than for NC embryos. This work was supported by the Research and Development Program for New Bio-industry Initiatives.

235 DEVELOPMENT OF IN VIVO-MATURED OOCYTES COLLECTED FROM JAPANESE BLACK CATTLE STIMULATED WITH DIFFERENT DURATIONS OF FOLLICULAR GROWTH

2015 ◽  
Vol 27 (1) ◽  
pp. 207
Author(s):  
T. Yamanouchi ◽  
H. Matsuda ◽  
M. Ohtake ◽  
Y. Aikawa ◽  
S. Kobayashi ◽  
...  
Keyword(s):  
Formation Rate ◽  
Time Lapse ◽  
Early Embryo ◽  
Developmental Competence ◽  
Follicular Growth ◽  
Culture Dish ◽  
Cleavage Rate ◽  
Significant Difference

We demonstrated that in vivo-matured oocytes (mOC) collected by ovum-pick up (OPU) from cows after stimulation of follicular growth (FG) are suitable for producing good quality blastocysts (BL). However, it is not known whether duration of FG affects developmental competence of mOC. The purpose of this study was to examine development of mOC after stimulation with different duration of FG. Japanese black donor cows (n = 4 per each group), were treated with a CIDR at Day 0. Follicle of diameter >8 mm were removed on Day 5. A total 20 AU of FSH was administrated to cows twice daily with decreasing doses from the evening of Day 6 to the morning of Day 10. In the conventional group (48PG), a administration of PGF2α (0.75 mg of cloprostenol), CIDR withdrawal, and administration of GnRH (0.2 mg of fertirelin acetate) were performed on the evening of Day 8, morning of Day 9, and morning of Day 10, respectively. In the experimental group (72PG), administration of PGF2a, CIDR withdrawal, and administration of GnRH were performed on the evening of Day 9, the morning of Day 10, and the morning of Day 11, respectively. The mOC were collected from follicles >5 mm by OPU at 25 to 26 h following GnRH administration. Collected mOC were inseminated with 3 × 106 sperm mL–1 in BO solution on 30 h after GnRH. After 6 h of IVF, presumptive zygotes were cultured for 168 h in 5% CS + CR1aa, using a micro-well culture dish (Dai-Nippon-Print) and time-lapse cinematography (CCM-1.4MZS; Astec) for individual embryo observation. The kinetics of early embryo was analysis by CCM-1.4 software. To assess the quality of BL, prognostic factors were used as follows: (1) less than 27 hpi (hours post-insemination) at the first cleavage (1st CD), (2) 2 blastomeres at the end of 1st CD, and (3) absence of multiple fragments at the end of the 1st CD (Sugimura et al. 2012 PLoS ONE 7, e36627; Imai et al. 2014 Reprod. Fertil. Dev. 26, 182). Data were analysed by Student's t-test or chi-square test. The number of mOC were 12.5 ± 4.7 and 10.3 ± 2.7 (means ± s.e.) oocytes per session in 48PG and 72PG. There was no significant difference in cleavage rate or BL formation rate (97.5 ± 1.5 v. 98.2 ± 1.8%, 66.3 ± 8.2 v. 66.8 ± 3.5%, respectively). The time for 1st CD was shorter in 48PG (26.1 ± 0.3 v. 27.8 ± 0.4; P < 0.01), and the rate of 1st CD less than 27 hpi was superior in 48PG compared with 72PG (74.3 v. 42.9%; P < 0.05). However, the rate of 2 blastomeres and absence of multiple fragments were not different between 48PG and 72PG. The number of BL tended to decrease in 72PG compared with 48PG (28.6 v. 48.6%; P = 0.087). These results indicate that duration of FG did not affect the rate of cleavage and BL formation. However, extension of duration of FG might reduce the quality of BL.

74 Treatment with Melatonin During In Vitro Culture Enhances Porcine Parthenogenetically Activated Embryo Development

2018 ◽  
Vol 30 (1) ◽  
pp. 175
Author(s):  
G. A. Kim ◽  
J.-X. Jin ◽  
S. Lee ◽  
A. Taweechaipaisankul ◽  
B. C. Lee
Keyword(s):  
Oxidative Stress ◽  
Embryo Development ◽  
Developmental Competence ◽  
Free Radical Scavengers ◽  
Control Group ◽  
Cleavage Rate ◽  
Cell Numbers ◽  
Significant Difference ◽  
Blastocyst Rate

Melatonin and its metabolites are powerful antioxidants and free radical scavengers. Because porcine embryos are vulnerable to oxidative stress in vitro, the addition of various protective chemicals to the culture medium, including melatonin, has been explored. The aim of this study was to investigate the effect of melatonin on in vitro developmental competence of porcine parthenogenetically activated (PA) embryos. Immature cumulus–oocyte complexes (COC) were collected and cultured in medium comprising TCM-199 supplemented with 10 ng mL−1 epidermal growth factor, 0.57 mM cysteine, 0.91 mM sodium pyruvate, 5 μL mL−1 insulin, transferrin selenium solution 100×, 10% porcine follicular fluid, 10 IU mL−1 eCG, and 10 IU mL−1 hCG for 44 h. Then, COC were denuded and PA with electrical stimulation, and PA embryos were cultured in porcine zygote medium 5 (PZM-5) supplemented with melatonin at increased concentrations (10−9, 10−7, 10−5 M) at 39°C in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2 for 7 days. Subsequent embryo development, including cleavage rate, blastocyst rate, and blastocyst cell numbers, was compared between groups (mean no. of embryos; control, 27.14; 10−9 M, 28.86; 10−7 M, 27.71; 10−5 M, 26.43). The experiments were repeated 7 times for each treatment group. Statistical analyses of all data were performed using one-way ANOVA with Dunn’s multiple comparison test. Results are expressed as the mean ± SEM and all differences were considered significant at P < 0.05. No apparent effect on cleavage rate of melatonin treatment of various concentrations was noted. Blastocyst cell number did not show any significant difference between groups. However, the potential of PA oocytes to develop into blastocysts was significantly higher in the group supplemented with 10−9 M melatonin compared with the control group (35.44 ± 3.84 v. 24.71 ± 1.59) and other melatonin treated groups (10−5 M, 21.35 ± 2.82; 10−7 M, 24.01 ± 2.31; P < 0.05). These indicated that treatment with 10−9 M melatonin in embryo culture might reduce the oxidative stress properly compared with other concentrations, which results in improvement of blastocyst rate formation. In conclusion, treatment with 10−9 M melatonin positively promoted the blastocyst formation rate of porcine PA embryos with no beneficial effects on their blastocyst cell numbers or cleavage rate. This study was supported by the National Research Foundation (#2015R1C1A2A01054373; 2016M3A9B6903410), Research Institute for Veterinary Science and the BK21 PLUS Program.

292 INHIBITION OF HSP90 AGGRAVATES THE EFFECTS OF HEAT SHOCK ON DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 235
Author(s):  
E. D. Souza ◽  
N. C. Rabelo ◽  
T. D. Araujo ◽  
C. M. Assunção ◽  
C. C. R. Quintão ◽  
...  
Keyword(s):  
Heat Shock ◽  
In Vitro Maturation ◽  
Calf Serum ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Oocyte Developmental Competence ◽  
Significant Difference ◽  
Blastocyst Rate ◽  
Bovine Oocytes

The heat shock protein 90kDa (HSP90) is a chaperone involved in protein homeostasis under normal and stress conditions. Its inhibition by 17-(allylamino)-17-demethoxygeldanamycin (17AAG, Sigma, St. Louis, MO, USA) for 12 or 24 h during in vitro maturation reduces the oocyte's ability to develop after in vitro fertilization (Souza et al. 2014 Reprod. Fert. Dev. 26, 197). This study aimed to evaluate the effect of treatment with 17AAG during the heat shock on oocyte developmental competence. Immature bovine COC were randomly allocated in 4 treatments during IVM: control = no heat shock or 17AAG; HS = heat shock (41.5°C) for the first 12 h of IVM; 17AAG = 2 µM 17AAG for the first 12 h of IVM; and 17AAG + HS = 2 µM 17AAG plus heat shock for the first 12 h of IVM. In vitro maturation was performed in Nunc plate containing 400 µL of TCM199 medium (Invitrogen, Carlsbad, CA, USA) supplemented with porcine FSH (Hertape Calier, Juatuba, Brazil) and 10% oestrus cow serum under 5% CO2 in air, 95% humidity, and 38.5°C for 24 h. Semen was processed by Percoll gradient (Nutricell, Campinas, Brazil) and oocytes were in vitro fertilized for 20 h with 2 × 106 spermatozoa mL–1 under the same IVM atmospheric conditions. Presumptive zygotes were completely denuded in a PBS solution with 0.1% hyaluronidase and then cultured in wells with 500 µL of modified CR2aa medium supplemented with 2.5% fetal calf serum (Nutricell) in an incubator at 38.5°C under 5% CO2, 5% O2, 90% N2, and saturated humidity. Cleavage rate was evaluated 72 h postfertilization and blastocyst rate was evaluated at Day 7 (D7) and 8 (D8). Data from 7 replicates were submitted to analysis of variance and means were compared by Student Newman Keul's test. There was no difference (P > 0.05) on cleavage rate among treatments. Heat shock or treatment with 17AAG, both for 12 h of IVM, decreased (P < 0.05) the blastocyst rate at D7 and D8 when compared to control but no significant difference between HS and 17AAG treatments was found (Table 1). However, the lowest (P < 0.05) blastocyst rate at D7 and D8 was achieved when oocytes were submitted simultaneously to 17AAG and heat shock for 12 h of IVM (17AAG + HS treatment, Table 1). In conclusion, the treatment with 17AAG during IVM worsens the deleterious effect of heat shock on oocyte developmental competence and suggests that HSP90 may also play role on cellular protection during heat shock in bovine oocytes. Table 1.Cleavage and blastocyst (Bl) rates at D7 and D8 for control, 17AAG, Heat Shock (HS), and 17AAG plus HS treatments Financial support comes from CNPq, FAPEMIG, and FAPES.

79 Effect of Polydatin on Development and Cryotolerance of In Vitro-Produced Bovine Embryos

2018 ◽  
Vol 30 (1) ◽  
pp. 178
Author(s):  
C. M. Owen ◽  
M. Barceló-Fimbres ◽  
J. L. Altermatt ◽  
L. F. Campos-Chillon
Keyword(s):  
Lipid Content ◽  
In Vitro Maturation ◽  
Nile Red ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
Treatment Groups ◽  
Significant Difference ◽  
Blastocyst Rate ◽  
Conventional Freezing

In vitro-produced (IVP) cattle embryos have high reactive oxygen species levels resulting in poor development and cryotolerance. Polydatin, a naturally occurring antioxidant, improves embryonic metabolism when added to maturation media; however, it has not been evaluated at other stages of embryo production. We hypothesised that embryos cultured with polydatin during maturation and fertilization would have increased development and cryotolerance. Therefore, IVP embryos were produced in 8 treatment groups supplemented with 1 µM polydatin during in vitro maturation, fertilization, and culture, or a combination of the different production stages, and each assigned a letter (Table 1). Embryos were produced in 7 replicates by oocytes (n = 3320) aspirated from abattoir ovaries, matured for 23 h in TCM-199 plus 10% fetal bovine serum and gonadotropins, fertilized with semen from 1 of 3 bulls, and cultured in SCF1 (SOF for Conventional Freezing 1; Owen et al. 2017 Reprod. Fertil. Dev. 29, 129-130) in 38.5°C in 5% O2, 5% CO2, and 90% N2. Stage 7 blastocysts were stained with 1 µg mL−1 Nile Red for lipid content or 300 nM Mitotracker Red CMX-Rosamine (Thermo Fisher Scientific, Waltham, MA, USA) for mitochondrial activity. Ten images per embryo were acquired using confocal microscopy at 5 µM step size at 40× magnification, and fluorescence was measured by Image Pro software (Media Cybernetics, Rockville, MD, USA). Remaining blastocysts were slow frozen following a 20-min equilibration in conventional freezing medium (1.5 M ethylene glycol and 0.5 M sucrose in holding medium) with 1 mm l-ascorbic acid. Embryos were thawed and assessed for re-expansion at 48 h. Blastocyst rate, Nile Red, Mitotracker, and re-expansion data were analysed by one-way ANOVA and means separated by least significant difference. Results indicate that treatment B had a higher blastocyst rate than treatment H (P < 0.01), lower lipid content than all other treatment groups (P < 0.01 or 0.05), and higher level of mitochondrial polarity than treatments A, D, E, and G (P < 0.01 or 0.05), suggesting enhanced metabolic activity. Additionally, this treatment enhanced cryotolerance compared with treatment H (P < 0.01). These results suggest that adding polydatin to maturation media has the most effect on embryo developmental competence and cryotolerance. Table 1.Effect of polydatin addition during in vitro maturation (IVM), fertilization (IVF), and culture (IVC) on blastocyst rate, lipid content, Mitotracker, and cryotolerance (± SEM)

scholarly journals An Eight Year Experience of Autologous Oocyte Vitrification for Infertile Patients Owing to Unavailability of Sperm on Oocyte Retrieval Day

2021 ◽  
Vol 8 ◽  
Author(s):  
Xiao Fu ◽  
Xiaojie Liu ◽  
Jing Li ◽  
Meng Zhang ◽  
Jingjing Jiang ◽  
...  
Keyword(s):  
Survival Rate ◽  
Clinical Outcomes ◽  
Live Birth ◽  
Descriptive Analysis ◽  
Oocyte Retrieval ◽  
High Quality ◽  
Oocyte Vitrification ◽  
Individual Parameter ◽  
Kaplan Meier ◽  
Significant Difference

Objective: The objective of this study was to provide a descriptive analysis of the clinical outcomes achieved in oocyte vitrification in cases where sperm was unavailable on oocyte retrieval day, and to identify predictors of oocyte survival.Methods: This retrospective cohort study used data from a university-affiliated reproductive medical center. There were 321 cycles in which some of, or all oocytes were vitrified owing to the unavailability of sperm between March 2009 and October 2017. A descriptive analysis of the clinical outcomes including both fresh embryo transfers and cryopreserved embryo transfers was provided. The ability of an individual parameter to forecast oocyte survival per thawing cycle was assessed by binary logistic regression analysis. The cumulative probability of live birth (CPLB) was estimated by using the Kaplan-Meier method according to the total number of oocytes thawed in consecutive procedures.Results: The average survival rate was 83.13%. High-quality embryo rate and blastocyst rate decreased significantly decreased significantly in vitrification oocyte group compared to fresh control oocytes. The comparison of sibling oocytes in part-oocyte-vitrified cycles shows fewer high-quality embryos developed in the vitrified group. The live birth rate per warmed-oocyte was 4.3%. Reasons for lack of sperm availability on oocyte retrieval day and serum cholesterol levels were found to be associated with oocyte survival rate in the present study. Kaplan-Meier analysis showed no significant difference in CPLB between patients ≤35 vs. &gt;35 years.Conclusions: Oocyte vitrification is an indispensable and effective alternative when sperm are not available on oocyte retrieval day. The present study provided evidence that oocytes from infertile couples were more likely to suffer oocyte/embryo vitrification injury. Clinicians need to take this into account when advising patients in similar situations. Further studies will be necessary to clarify the correlation between serum metabolism parameters and human oocyte survival after vitrification.

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