Blastocyst Research Articles

Reanalysis of blastocysts previously diagnosed as aneuploid demonstrates that one in four are rediagnosed as euploid

Reanalysis of blastocysts previously diagnosed as aneuploid demonstrates that one in four are rediagnosed as euploid

Preliminary observations on embryo morphokinetics and sperm DNA fragmentation assessed using the halo sperm kit

Sperm DNA fragmentation has been suggested to be a contributor to male infertility and lower pregnancy outcomes. The degree to which DNA fragmentation influences outcomes in IVF/ICSI cycles is however still controversial. Integration of time-lapse microscopy (TLM) in to IVF labs allows more in depth analysis of embryo developmental kinetics than was previously possible with conventional microscopy. This study examines sperm DNA fragmentation and its relationship to embryo morphokinetics, multinucleation and cleavage anomalies. Pilot study looking at DNA fragmentation (DF) and embryo morphokinetics. This study encompassed 77 couples undergoing IVF. Sperm specimens were prepared for ICSI using density gradient centrifugation. Mature oocytes were fertilized by ICSI and immature oocytes were incubated overnight with sperm. Normally fertilized oocytes were cultured in the EmbryoScope time lapse chamber. Timing of specific events from the point of insemination was determined using time lapse (TL) imaging and expressed in hours. The following kinetic markers were assessed: time to syngamy (t-syn), time to 2c (t2), 3c (t3), 4c (t4), 5c (t5), 8c (t8), morula (Mor-t), start of blastulation (tSB), blastocyst (BL-t), and expanded blastocyst (EBL-t). Durations of the second (cc2) and third (cc3) cell cycles as well as time to complete synchronous divisions s2 and s3 were calculated. Embryos were also observed for presence or multinucleation and cleavage anomalies. Sperm DNA fragmentation testing was performed on an aliquot of the unused ICSI sperm preparation using the Halosperm kit (HaloTech/Spectrum Technologies). The kit enabled microscopic detection of sperm chromatins dispersion. Sperm were embedded in agarose coated slides and exposed to acid denaturation and a lysis buffer. Following alcohol dehydration and staining with DiffQuik, slides were examined to detect presence of halos surrounding sperm. Sperm cells with large/medium sized halos characteristic of dispersed DNA loops were considered to have intact DNA. Sperm with small, degraded or absent halos were considered to have fragmented DNA. Cases were divided according to % DF in ICSI specimens. Morphokinetic parameters were compared. P value of <0.05 were considered to be statistically significant. Only parameters that were significantly different are shown in the table.Tabled 1Morphokinetics and DNA fragmentationDNA Fragmentation <15%DNA Fragmentation>=15%P ValueTotal embryos63354Multinucleation47%69%<0.01Irregular chaotic division25%46%<0.001Morula formation79%66%<0.05Start of blastulation74%60%<0.05Early blastocyst formation66%48%<0.01Expanded blastocyst formation52%32%<0.01t439.6+ 0.342.2 + 1.2<0.01t4-t32.7 + 0.24.8 + 0.7<0.01t859.5 + 0.564.7 + 2.2<0.01 Open table in a new tab The Halosperm kit provided an easy inexpensive means to assay DNA fragmentation. Further study is needed on the association between sperm DNA fragmentation and embryo quality markers made possible thru time lapse imaging systems.

The ability of warmed blastocysts to re-expand and the pre-vitrification trophoectoderm grade significantly correlated with implantation

The ability of warmed blastocysts to re-expand and the pre-vitrification trophoectoderm grade significantly correlated with implantation

Why looks matter: morphology predicts success in embryo banking (EB) with preimplantation genetic testing for aneuploidy (PGT-A)

EB is increasingly common as more couples delay childbearing. Moreover, performing PGT-A before EB provides confidence that stored embryos are genetically competent and have a high chance of live birth (LB). We aimed to assess the specifics of blastocyst (BL) morphology in determining LB after euploid embryo (EE) transfer and develop counseling tools for patients electing EB+PGT-A. Retrospective cohort at a large university-based program. We reviewed all IVF cycles from 1/2014 - 2/2018 where fresh oocytes served as the female gamete and a single cryopreserved EE was transferred. We assessed age at retrieval, BL expansion (BE), inner cell mass (ICM) and trophectoderm (TE). BE, ICM and TE grades were based on Gardner’s criteria. Outcomes were ongoing pregnancy (>12 wks gestation) / live birth rate (LBR), implantation rate (IR) and spontaneous abortion rate (SABR). Fischer’s exact test was used. See Table. We reviewed 1641 transfers (142 donor+1499 autologous; median age at retrieval 37y, IQR 33-40y, range 21-46y). Overall IR was 66%, SABR was 12%, and LBR was 57%. There were 12 pregnancy terminations (TOP) and 7 ectopics. When stratified by BE, no differences in IR, SABR, or LBR were noted. When stratified by ICM grade, EE with ICM-C had a lower IR than with ICM-A and B (35% vs. 71% and 68%, p<0.01) and EE with ICM-A had a lower SABR than with ICM-B (6% vs. 13%, p=0.02); notably, EE with ICM-A had a higher LBR than with ICM-B and C (66% vs. 58% and 34%, p<0.03) and EE with ICM-B had a higher LBR than with ICM-C (58% vs. 34%, p<0.01). When stratified by TE grade, EE with TE-C had a lower IR than with TE-A and B (51% vs. 80% and 69%, p<0.01) and EE with TE-A had a lower SABR than with TE-C (3% vs. 18%, p=0.03); notably, EE with TE-C had a lower LBR than with TE-A and B (41% vs. 73% and 60%, p<0.01). Outcome differences re: ICM and TE were maternal-age independent. When comparing LBRs re: ICM+TE grade combinations, ICM-A+TE-A= 19/26 (73%), ICM-A+TE-B= 125/195 (64%), ICM-B+TE-A= 13/19 (68%), ICM-B+TE-B= 658/1101 (60%), ICM-B+TE-C= 91/205 (44%), ICM-C+TE-B= 23/57 (40%), and ICM-C+TE-C= 8/36 (22%). Only one EE had ICM-A+TE-C and one ICM-C+TE-A (both LBs). Euploidy is not the sole determinant when selecting embryos. Our data suggest that ICM and TE each play individual and critical roles related to IR, SABR and LBR - even with EE. In our study, EE with the best morphology had a LBR of 73% while EE with the worst had a LBR of 22%. These data add to realistic counseling for patients using EB+PGT-A for future pregnancy. Further, they help elucidate the biology of implantation and pregnancy, suggesting TE (placenta precursor) is as important as ICM (future fetus).Tabled 1Embryo morphologic parameters and outcome dataEmbryo ParameterIRSABRLBRTOP + Ectopic PregnanciesBE: <2 (n=110)73/110 (66%)14/73 (19%)58/110 (53%)1+0/110 (1%)BE: 3 (n=1389)928/1389 (67%)104/928 (11%)808/1389 (58%)9+7/1389 (1%)BE:4 (n=63)40/63 (63%)6/40 (15%)32/63 (51%)2+0/63 (3%)BE: >5 (n=79)47/79 (59%)6/47 (13%)41/79 (52%)0+0/79 (0%)ICM: A (n=221)157/221(71%)10/157 (6%)145/221 (66%)1+1/221(1%)ICM: B (n=1325)898/1325 (68%)119/898 (13%)762/1325 (58%)11+6/1325 (1%)ICM: C (n=95)33/95 (35%)1/33 (3%)32/95 (34%)0+0/95 (0%)TE: A (n=45)36/45 (80%)1/36 (3%)33/45 (73%)0+2/45 (4%)TE: B (n=1353)928/1353 (69%)107/928 (12%)806/1353 (60%)10+5/1353 (1%)TE: C (n=243)124/243 (51%)22/124(18%)100/243 (41%)2+0/243 (1%) Open table in a new tab

Rate of development of euploid blastocyst affects clinical outcomes of frozen embryo transfer

Preimplantation genetic testing for aneuploidy (PGT-A) has been increasingly used to identify euploid embryos. The present study evaluates clinical outcomes of euploid frozen embryo transfers (FET) in 2016-2017, following trophectoderm (TE) biopsy (BX) on day 5 (D5), day 6 (D6), or day 7 (D7). Retrospective study. Trophectoderm biopsy was performed on embryos once they reached blastocyst (BL) stage on D5, D6, or D7, where the day of biopsy indicates the rate of development. We analyzed a total of 1889 FETs following D5, D6, or D7 TE BX. Comparative genomic hybridization array, SNP microarray, or Next-generation sequencing was used for PGT-A. Transfer was performed following vitrification and warming. Clinical outcomes of implantation rate (IR), ongoing pregnancy/delivered (OGD), and spontaneous abortion (SAb) were compared among different BX days per patient age groups, using Kruskal-Wallis testing for IR and Chi-squared analysis for OGD and SAb. Statistical significance was defined as P <0.05. For patients of all ages, all clinical outcomes (IR, OGD, and SAb) showed statistical significance correlated to the rate of embryo development. In patients under 35 years of age, OGD was significantly higher in D5 euploid embryos compared to D6 or D7. For patients 38 years of age and older, IR and OGD were statistically higher in D5 euploid embryos.Tabled 1Clinical Outcomes Post TE BX/FETD5 BX/FET#FETAll Ages: 684<35: 21535-37: 17538-40: 167>40: 127IR72.5%∗∗68.2%74.4%77.0%∗∗71.2%∗OGD+429 (63%)∗128 (60%)∗113 (65%)113 (68%)∗75 (59%)∗SAb (losses/CP;%)65 (10%)∗20 (9%)13 (7%)17 (10%)15 (12%)D6 BX/FET#FETAll Ages: 1113<35: 31635-37: 24838-40: 318>40: 231IR60.9%∗∗60.8%64.1%61.1%∗∗57.5%∗OGD+540 (49%)∗155 (49%)∗131 (53%)149 (47%)∗105 (45%)∗SAb (losses/CP;%)129 (12%)∗38 (12%)25 (10%)38 (12%)28 (12%)D7 BX/FET#FETAll Ages: 92<35: 2035-37: 1538-40: 41>40: 16IR43.2%∗∗45.5%68.8%31.7%∗∗43.8%∗OGD+32 (35%)∗8 (40%)∗8 (53%)11 (27%)∗5 (31%)∗SAb (losses/CP;%)8 (9%)∗2 (10%)2 (13%)2 (5%)2 (13%)*: P < 0.05 ; **: P < 0.01 ; + :Ongoing pregnancies were defined as those with fetal cardiac activity at or beyond 8 weeks of gestational age. Open table in a new tab *: P < 0.05 ; **: P < 0.01 ; + :Ongoing pregnancies were defined as those with fetal cardiac activity at or beyond 8 weeks of gestational age. Overall, D5 euploid BL showed improved clinical outcomes compared to D6 or D7, suggesting that delayed embryo development may have an adverse effect on clinical outcomes, despite the proven euploid status. Interestingly, for younger patients (<35), D5 euploid BL improved continuation of pregnancy (OGD), while other clinical outcomes were not significantly different among D5, D6, or D7. However, for older patients (≥38), implantation rates as well as ongoing pregnancy rates were improved for D5 embryos compared to D6 or D7. Even though D7 euploid BL result in much lower IR and OGD compared to D5 or D6, they still result in 35% OGD, suggesting that slow developing embryos can still be beneficial for the patients, and therefore should not be overlooked.

Embryotropic effects of vascular endothelial growth factor on porcine embryos produced by in vitro fertilization

Current research suggests that supplementing in vitro culture (IVC) media with vascular endothelial growth factor (VEGF) may have beneficial effects on the development of porcine embryos in vitro. However, the molecular signaling mechanisms underlying this effect are unclear. Therefore, we aimed to investigate the effects of VEGF on molecular signaling events during in vitro embryonic development of porcine embryos. Porcine oocytes matured in vitro were fertilized, and the resultant zygotes were cultured with 5 ng/mL of VEGF supplemented with or without fetal bovine serum from day 4 till day 7. Without VEGF and/or FBS served as the control group. Real-time quantitative PCR was used to detect expression patterns of apoptosis- and oxidative stress-related genes in day 7 blastocysts (BLs). Early-stage apoptosis was detected by annexin-V assays in day 2 and day 7 embryos. We found that the addition of VEGF throughout the culture period with or without FBS supplementation significantly improved embryo survival and development. Supplementation with VEGF in the IVC medium significantly increased early BL formation (p < 0.05), although addition of FBS on day 4 significantly increased hatched BL formation (p < 0.05) regardless of VEGF supplementation. However, supplementation of media with both VEGF and FBS increased the formation of expanded BLs synergistically. The average total cell numbers per BL were significantly (p < 0.05) higher in embryos supplemented with VEGF and FBS than in those supplemented with either VEGF or FBS alone. We also found that accumulation of reactive oxygen species in VEGF-treated embryos was significantly lower (p < 0.05) than that in untreated embryos. The mRNA levels of caspase-3 were significantly lower (p < 0.05), and those of Bcl-2 and Nrf-2 were significantly higher (p < 0.05) in embryos grown in VEGF-supplemented media than in embryos grown in non-supplemented media. Furthermore, on day 2, the numbers of viable embryos (44.06 ± 3.94%) and blastomeres (67.18 ± 3.60%) were significantly higher (p < 0.05), and the numbers of early apoptotic embryos (55.94 ± 3.94) and blastomeres (23.23 ± 4.22) were significantly lower (p < 0.05) in VEGF-treated BLs than in controls. Furthermore, the numbers of early apoptotic cells in BLs on day 7 were also significantly lower (p < 0.05) in VEGF-treated BLs than in controls. Overall, our results indicate that supplementing IVC media with VEGF during in vitro culture of porcine embryos increases their developmental potential.

Enhanced development of mouse single blastomeres into blastocysts via the simultaneous inhibition of TGF-β and ERK pathways in microdroplet culture.

Optimization of an in vitro culture that supports blastocyst (BL) development from single blastomeres (SBs) is essential to generate additional embryos for farm animals and humans and unravel the mechanisms that underlie totipotency. In this study, we have examined BL development from SBs that were derived from 2-cell and 4-cell mouse embryos in different media. Moreover, BLs were assessed for inner cell mass (ICM) by staining with Oct4. We found that BL development was improved in a lower volume of medium (1 µL) compared with a higher volume (5 µL). Furthermore, the supplementation of medium with the inhibitors of ERK1/2 and TGFβ (R2i) signaling pathways in 1 µL droplets of T6 medium improved BL development. The co-culture of SBs with intact embryos in the presence of R2i showed more BL development and ICM to trophectoderm cell number ratio in comparison with SB culture and SB group culture. We also observed reduced total cell number, ICM, and trophectoderm cell numbers in all of the SB culture conditions versus intact embryo development. These findings might facilitate the successful generation of additional embryos for biomedical applications and elucidate the mechanisms that underlie totipotency.

Author response: G9a regulates temporal preimplantation developmental program and lineage segregation in blastocyst

Author response: G9a regulates temporal preimplantation developmental program and lineage segregation in blastocyst

Lysophosphatidic acid increases in vitro maturation efficiency via uPA-uPAR signaling pathway in cumulus cells

Lysophosphatidic acid (LPA) is a phospholipid-derived signaling molecule with biological activities, such as stimulating cell proliferation, differentiation and migration. In the present study, we examined the effect of LPA on porcine oocytes during in vitro maturation (IVM) and subsequent embryonic development following parthenogenetic activation (PA) and in vitro fertilization (IVF). During IVM, the maturation medium was supplemented with various concentrations of LPA (0, 10, 30, and 60 μM). After 42 h of IVM, the 30 μM LPA-treated group showed a significant (P <0.05) increase in nuclear maturation and intracellular glutathione (GSH) levels compared with the other groups. The 30 μM LPA-treated group exhibited a significant decrease in intracellular reactive oxygen species (ROS) levels compared with the other groups. In PA, the 30 μM LPA-treated group had significantly higher cleavage (CL) and blastocyst (BL) rates compared with those of the other LPA-treated groups. In IVF, the 30 μM LPA-treated group had significantly higher CL and BL rates than the other LPA-treated groups. The expression of the developmental competence gene (proliferating cell nuclear antigen, PCNA) in the oocytes and cumulus cells of the individuals in the 30 μM LPA-treated group was significantly increased compared with the control group. In addition, the specific expression of urokinase Plasminogen Activator (uPA) and uPA Receptor (uPAR) in cumulus cells was significantly increased in the 30 μM LPA-treated group. The western blotting results revealed that LPA improves the activities of p38 mitogen-activated protein kinase (MAPK) and epidermal growth factor (EGF) by enhanced phosphorylation. In conclusion, treatment with 30 μM LPA during IVM promotes enhances the EGF-EGFR signaling pathway, resulting in cumulus cell expansion. And then, this treatment improves the developmental potential of PA and IVF porcine embryos by enhancing nuclear and cytoplasmic maturation and reducing ROS.

Blastocyst only culture-are we throwing the baby out with the bathwater?

Blastocyst only culture-are we throwing the baby out with the bathwater?

Effects of different activation protocols on cleavage rate and blastocyst production of caprine oocytes.

The present study was undertaken to assess the effect of different chemical activators along with 6-DMAP on in vitro matured caprine oocytes. From 4332 ovaries, 14235 cumulus oocyte complexes (COCs) were collected which were matured in TCM-199 medium containing follicle stimulating hormone (FSH) (5 µg/ml), Leutinizing hormone (LH) (10 µg/ml), oestradiol-17β (1 µg/ml) supplemented with 10% fetal bovine serum, 10% follicular fluid and 3 mg/ml bovine serum albumin (BSA) at 38.5°C and 5% CO2 in an incubator under humidified air for 27 h. In group 1 (control), 3117 in vitro matured oocytes were co incubated with sperms for 18 h in ferttalp medium. In group 2, 3563 in vitro matured oocytes were activated with 7% ethanol for 5-7 min followed by treatment with 2.0 mM DMAP for 4 h in mCR2aa medium. In group 3, 3109 in vitro matured oocytes were activated with 5 μM ionomycin for 5-7 min followed by treatment with 2.0 mM DMAP for 4 h in mCR2aa medium. In group 4, 3455 in vitro matured oocytes were activated with 5 μM calcium ionophore for 5-7 min followed by treatment with 2.0 mM DMAP for 4 h in mCR2aa medium. Oocytes were cultured in 50 µL drops of research vitro cleave (RVCL) medium for embryo development. The cleavage rate, morula and blastocyst production in group 1, 2, 3 and 4 were 26.07 ± 2.37%, 14.91 ± 2.91 & 1.45 ± 0.71%, 49.57 ± 3.79%, 20.07 ± 2.38% & 5.29 ± 1.42%, 50.18 ± 3.59%, 15.26 ± 2.87% & 1.85 ± 0.72% and 80.26 ± 2.30%, 35.33 ± 2.67 & 7.10 ± 0.89%, respectively. These results indicated that the activation of in vitro matured oocytes by 5 μM calcium ionophore for 5-7 min followed by treatment with 2.0 mM DMAP for 4 h is most favorable for parthenogenetic caprine embryos production.

128 Evidence that Pregnancy-Associated Serum Protein A (PAPP-A) Plays Role on Bovine In Vitro Embryo Production

The aim of present work was to assess the effects of pregnancy-associated serum protein A (PAPP-A) during oocyte in vitro maturation (IVM) on meiosis progression, DNA fragmentation, IGF-1 free bioavailability, as well as effects on embryo yield and transcriptional profile of matured cumulus–oocyte complexes (COC). First, the COC from a local abattoir were submitted to IVM for 24 h with TCM-199 serum-free medium supplemented with PAPP-A (100 ng mL−1: P100 group) or not (control group). The matured oocytes were submitted to evaluation of DNA fragmentation (TUNEL assay) and meiosis progression (Hoechst 33342; n = 5 replicates; 20 COC/replicate per group), and maturation medium was collected to measure levels of free IGF-1. Then, the oocytes were separated from their respective cumulus cells and followed for the transcriptional profile of 96 genes (3 reference genes; ACTH, GAPDH, PPIA) by RT-qPCR using Taqman® assays in the HD-Biomark System® (Fluidigm Corp., South San Francisco, CA, USA). Further, the matured oocytes were submitted to in vitro fertilization followed by in vitro culture for 7 days. On Days 3 and 7, the cleavage and blastocyst (BL) rates were verified. On Day 7, BL (3 BL/pool; control: n = 4 pools; P100: n = 5 pools) were collected to analyse the transcriptional pattern of 96 genes (4 reference genes; ACTH, GAPDH, PPIA, and SDHA) as described above for COC. The DNA fragmentation, meiosis progression, cleavage, and BL rates were calculated as percentages and transformed to arcsine. The mRNA abundance of target genes was normalized by geometric mean of reference genes and data were transformed to fold change. The free IGF-I concentration also was transformed to fold change. All data were tested by ANOVA and means were compared with t-test or Wilcoxon tests using JMP software (SAS Institute Inc., Cary, NC, USA). Differences were considered significant when P ≤ 0.05. The addition of PAPP-A increased free IGF-I concentration 1.27-fold in IVM medium. There were no alterations in the percentage of oocytes in metaphase II or oocyte DNA fragmentation. In cumulus cells, the genes BCL2, GPX1, RPLP0, and RPS25 (anti-apoptotic and anti-oxidative stress) was higher in the P100 group, whereas DICER, GREM1, GUCY1B3, and FOXO3 (cell proliferation, cumulus expansion, cGMP regulator, and apoptotic initiator, respectively) were higher in the control group. In oocytes, the mRNA relative abundance of ACACA, BCL2, H1FOO, TXNRD1, and VCAN (related with fatty acid synthesis, anti-apoptotic effect, chromatin regulation, oxidative stress processes, and cell proliferation, respectively) was higher in the P100 group. There was no difference in cleavage rate or embryo yield. The mRNA abundance of genes related to cellular stress (ATF4, GPX4, and HIF1A) and lipid metabolism (FASN and SREBF1) was lower in embryos of the P100 group. On the other hand, genes involved in cellular proliferation/differentiation (MAPK1) and pluripotency (POU5F1) were up-regulated in embryos of the P100 group. In conclusion, the addition of PAPP-A during the IVM increased free IGF-I and modulates the gene expression in COC and blastocysts, which could modify oocyte competence and embryo development.

Maintained MPF Level after Oocyte Vitrification Improves Embryonic Development after IVF, but not after Somatic Cell Nuclear Transfer.

Levels of maturation-promoting factor (MPF) in oocytes decline after vitrification, and this decline has been suggested as one of the main causes of low developmental competence resulting from cryoinjury. Here, we evaluated MPF activity in vitrified mouse eggs following treatment with caffeine, a known stimulator of MPF activity, and/or the proteasome inhibitor MG132. Collected MII oocytes were vitrified and divided into four groups: untreated, 10 mM caffeine (CA), 10 μM MG132 (MG), and 10 mM caffeine +10 μM MG132 (CA+MG). After warming, the MPF activity of oocytes and their blastocyst formation and implantation rates in the CA, MG, and CA+MG groups were much higher than those in the untreated group. However, the cell numbers in blastocysts did not differ among groups. Analysis of the effectiveness of caffeine and MG132 for improving somatic cell nuclear transfer (SCNT) technology using cryopreserved eggs showed that supplementation did not improve the blastocyst formation rate of cloned mouse eggs. These results suggest that maintaining MPF activity after cryopreservation may have a positive effect on further embryonic development, but is unable to fully overcome cryoinjury. Thus, intrinsic factors governing the developmental potential that diminish during oocyte cryopreservation should be explored.

How many does it take? achievement of euploid blastocyst (BL) as the primary predictor of live birth (LB) in oocyte cryopreservation (OC)

Current data suggests that LB following transfer of a euploid embryo (EE) is independent of age or type of ART treatment. At our center, OC is routinely performed and trophectoderm biopsy-preimplantation genetic screening (TEBX-PGS) is now being used in conjunction with autologous OC thaw (AOCT) cycles. From our novel data, we estimated the no. of MII oocytes required to achieve LB at different ages. Retrospective study and theoretical model. Data from 152 patients (202 retrievals, 167 thaws) who underwent AOCT with intended TEBX-PGS aged 25-40y were reviewed. To calculate the no. of MII oocytes needed to achieve one EE the following data were assessed: total no. of MII oocytes that survived, 2-pronuclear (2PN) fertilization, development to BL, BL achieving quality for TEBX, and BL ploidy status after TEBX-PGS. The no. of MII oocytes needed to achieve one EE was calculated as no. MII thawed/EE. Because combining these technologies is relatively new, many pregnancies are currently ongoing (ONG). Thus “LB” included >1st trimester ONG pregnancies and babies born (BB), with the LB rate calculated as total no. ONG+BB/EE transferred. The theoretical no. of MII oocytes needed for one LB was then calculated as no. thawed MII needed to achieve one EE/LB rate per EE. See Table. Of the 1,839 MII oocytes that survived, 2PN fertilization rate was 75% and of those, 50% formed BL. 83% of BL were suitable for TEBX. The no. of MII oocytes needed to achieve one EE was 8, 12, and 13 for age groups of 25-34y, 35-37y, and 38-40y; LB rate per EE was 59.8% across all ages. Thus, the no. of MII calculated to achieve one LB was 13, 19, and 22 in the age groups, respectively. EE have a relatively uniform efficiency and therefore, achievement of an EE after OC, thaw, and fertilization can help to estimate the theoretical no. of cryopreserved MII oocytes required to achieve a LB. One criticism of OC is that it provides women with a false sense of security; because of varying cycle outcomes, OC cannot guarantee future birth. Our findings serve as a counseling tool by providing women with realistic expectations of OC outcomes at our center.Tabled 1Oocyte development and ploidy status following AOCT+TEBX-PGS by ageAge (y)25-34 (mean 30.7; n=23)35-37 (mean 36.5; n=61)38-40 (mean 38.9; n=118)Total (mean 37.2; n=202)Oocytes retrieved574104516013220MII thawed38569810882171MII survived32955895218392PN fertilized2554246931372BL124215353692BL biopsied110186279575Euploid486084192Aneuploid59122193374No. MII to achieve euploid8.02 (385/48)11.6 (698/60)13.0 (1088/84)No. MII to achieve LB13.4 (8.02/0.598)19.4 (11.6/0.598)21.7 (13.0/0.598)EE LB rate: 59.8% (52/87) Open table in a new tab

Blighted ovum in subfertile patients undergoing assisted reproductive technology

To explore the incidence and risk factors of blighted ovum in subfertile patients undergoing assisted reproductive technology (ART). This retrospective analysis was conducted among 2378 patients who were pregnant following embryo transfer at our center from January, 2012 to December, 2015, including cases of early pregnancy losses and simultaneous live births. The cases with early pregnancy losses were divided into embryonic pregnancy and blighted ovum groups based on the presence or absence of an embryonic pole before dilation and curettage. The clinical data of the 3 groups were analyzed for comparisons of the maternal age, paternal age, BMI, AFC, basal FSH, bFSH/bLH, duration of infertility, Gn dosage, Gn days, serum estradiol on the day of HCG administration, endometrium thickness, number of oocyte retrieved, proportion of high-quality embryos transferred, serum β-HCG value on the 10th to 14th days of embryo transfer, infertility type and miscarriage times. The incidences of blighted ovum were compared between cases with different cycles, embryo stages, infertile factors and methods of fertilization. Maternal age and paternal age, BMI, duration of infertility, infertility type and miscarriage times differed significantly between cases with blighted ovum and those with live births. Serum β-HCG level was the lowest in blighted ovum group followed by embryonic pregnancy group and then by live birth group. Blastocyst transfer was associated with a significantly higher incidence of blighted ovum as compared with cleavage embryo transfer (11.6% vs 5.6%, P=0.000). No significant difference was found in the other parameters among the 3 groups (P>0.05). Adjusted logistic regression analysis showed that maternal age, β-HCG level and blastocyst transfer were risk factors of blighted ovum. Advanced maternal age, low β-HCG level and blastocyst transfer may increase the risk of blighted ovum possibly in association with gene imprinting errors during the early stage of embryo development.

Study of TALP and TRIS citrate medium on caprine sperm capacitation and subsequent in vitro embryo production.

The aim of the present investigation was to compare Tyrode's albumin lactate pyruvate (TALP) medium and TRIS citrate medium for capacitation of caprine sperm. In experiment 1 capacitation was assessed by chlortetracycline assay and in experiment 2 with in vitro fertilization and embryo development. In experiment 2, cumulus oocyte complexes (COCs) recovered by slicing the caprine ovaries were matured in maturation medium for 27 h in humidified atmosphere at 38.5°C with 5% CO2. After 27 h of culture a total of 2480 in vitro matured oocytes were selected and randomly divided into two groups. Group 1 (n=1124) matured oocytes were fertilized by the spermatozoa capacitated in TALP medium and in group 2 (n=1356) matured oocytes were fertilized by the spermatozoa capacitated in TRIS citrate medium. The results of experiment 1 indicated a comparatively more number of sperms with Chlortetracycline (CTC) Pattern B in TRIS citrate than TALP medium (55.32 ± 0.91% vs 47.96 ± 0.20%). In experiment 2, the cleavage rate and blastocyst production were higher following capacitation of spermatozoa in TRIS citrate than TALP medium. In conclusion, TRIS citrate can be used as an alternative and effective media for sperm capacitation to get higher cleavage rate and blastocyst production in goat.

Cryo-injury of human blastocysts in a closed system, re-vitrification model: comparison of unstable and metastable solutions

Commercial vitrification solutions (VS) possessing 30-32% cryoprotective agents (EG-DMSO or EG-PPG; 4.4 to 4.7M) are relatively unstable solutions, susceptible to ice-induced cryo-injury under insufficient cooling/warming conditions. Alternatively, a more concentrated EG-Glycerol VS (I.C.E.; >7.9M, Schiewe et al., 2015) is metastable under lower cooling/warming rates. We have recently shown that the latter VS mixture is equally non-toxic to blastocysts (BL) for up to a 10 min exposure. The aim of this study was to evaluate the physical stability of an unstable versus metastable VS when challenged to repeated re-vitrification (rVTF).

Unusually Large, Unruptured Tubal Ectopic Pregnancy Mass in a Woman With Bilateral Tubal Obstruction Treated by Laparoscopy: A Case Report

A tubal ectopic pregnancy mass usually measures 1.5 - 3.5 cm, and ruptures if it grows beyond this size. The author encountered a case of an unruptured, tubal ectopic pregnancy mass measuring up to 7.3 cm, containing an embryo with a crown-rump length (CRL) of 2.02 cm, corresponding to 8 weeks, 4 days. To the author’s knowledge, this is the largest reported singleton tubal ectopic pregnancy mass. A 36-year-old gravida 0, para 0 woman presented with a history of mild vaginal bleeding, lower abdominal pain, and 8 weeks of amenorrhea. Vaginal bleeding started 3 weeks before, but she mistook this for menstruation. Her menstrual cycle ranged between 28 and 30 days and lasted 6 days. Her last menstrual period was about 8 weeks prior. She had an appendectomy 22 years prior. She was diagnosed with infertility due to tubal obstruction and underwent laparoscopic fimbrioplasty at another general hospital 13 years prior. She was told that her Fallopian tubes were still obstructed on hysterosalpingography after fimbrioplasty. She had not used contraception. On arrival in the emergency room, her serum beta-human chorionic gonadotropin (hCG) level was 15,944 mIU/mL. The transvaginal ultrasound scan (TVUS) demonstrated an ectopic pregnancy mass measuring 7.3 × 4.0 cm in the left adnexa. An embryo with CRL of 2.02 cm corresponding to 8 weeks, 4 days was noted in the ectopic pregnancy mass. Upon entering the pelvic cavity, a dark reddish-colored unruptured tubal ectopic pregnancy mass was noted. Laparoscopic salpingectomy was carried out. The right Fallopian tube was adherent to the right pelvic wall. Even though the Fallopian tubes are obstructed, pregnancy is still possible. Laboratory studies should always include a pregnancy test. A large tubal ectopic pregnancy mass could have developed when a blastocyst was implanted in a fused Fallopian tube that was adherent to the pelvic peritoneum and uterus. J Med Cases. 2016;7(9):396-398 doi: http://dx.doi.org/10.14740/jmc2613w

Easy, quick and cheap technique to cryopreserve Welsh B pony blastocyst

Easy, quick and cheap technique to cryopreserve Welsh B pony blastocyst

Effect of the cryopreservation method used, the embryonic stage and the use of conjugated linoleic acid isomers on the cryotolerance of in vitro-produced bovine embryos

Conjugated linoleic acid (CLA) might be able to improve the cryotolerance of in vitro-produced (IVP) embryos. The effect of two CLA isomers on the cryotolerance of bovine IVP embryos, as well as that of the stage of embryonic development and the method used for cryopreservation was evaluated by three experiments. In Experiment 1, oocytes (n = 3,917) were fertilized in vitro and cultured with 0, 50, 100, or 200 ?M trans-10, cis-12 (t10, c12 CLA). In Experiment 2, fertilized oocytes (n = 2,131) were cultured with 100 ?M t10, c12 or cis-9, trans-11 (c9, t11 CLA), or a combination of both isomers. The embryos were vitrified at the blastocyst (BL) or the expanded blastocyst (EB) stage. In Experiment 3, oocytes (n = 1,720) were fertilized and cultured with or without 100 ?M t10, c12 CLA, and the blastocysts were vitrified or frozen. Blastocyst development rate as well as the rates of re-expansion and hatching after thawing was recorded. Moreover, the mean cell number and mRNA expression of acetyl-CoA carboxylase (ACC1) and stearoyl-CoA desaturase (SCD1) as well as fatty acid synthase (FASN) multienzyme complex were determined. In Experiment 1, the highest concentration of t10, c12 CLA that did not reduce blastocyst development rate was 100 ?M. In Experiment 2, the rates of re-expansion and hatching among the EBs obtained through IVP after supplementation with t10, c12 CLA (73.1% and 57.7%), with c9, t11 CLA (80.0% and 68.6%), with the combination (78.3% and 52.2%), and with the control group (85.4% and 58.3%) were similar. At the BL stage, the rates of re-expansion and hatching were lower than those at the EB stage, and CLA combination allowed a hatching rate (8.0%) lower than that observed in the control group (40.0%). In Experiment 3, the hatching rates for vitrified EBs (vitrified control; 67.4%) and vitrified CLA EBs (65.8%) were higher than those obtained for frozen EBs, exposed (13.3%) or not exposed (28.6%) to CLA. In addition, in Experiment 3, the hatching rate was higher at the EB stage in vitrified groups, while the rates of BL and EB were similar in frozen groups, thus proving that vitrification was more efficient than freezing for IVP bovine embryos. In Experiment 3, CLA isomer t10, C12 did not influence the embryonic cell number or mRNA expression of ACC1 and SCD1 enzymes, but decreased the mRNA expression of FASN. In conclusion, 100 ?M CLA did not affect subsequent embryonic development. However, neither CLA isomer improved the cryotolerance of IVP bovine embryos.