Blastocyst Rate In Group Research Articles

Elucidating the Impact of Secretome Derived from Mesenchymal Stem Cell and Uterine Epithelial Cells During <i>In Vitro</i> Blastocyst Production in Buffalo

One challenge that needs to be addressed in animal embryo production is to create the appropriate in vitro culture to improve the blastocyst rate and produce high-quality embryos. Buffalo Mesenchymal Stem Cells (MSCs) were derived from Wharton’s jelly and expanded in vitro. Conditioned media (secretome) was collected from well-characterized WJMSCs at 3rd passage. Similarly, buffalo Uterine Epithelial Cells (UECs) were derived from nongravid uteri and expanded in vitro. The secretome was collected from a well-characterized first passage UECs monolayer primed with steroid hormones (progesterone 3.14ng/ml and estradiol-17β 5 31pg/ml). Culture media was replaced with non-serum media, and the media was collected after 72h. Day 4 IVF-derived embryos were cultured in three groups: in regular mSOF media (Group I), mSOF replaced with 50% CM derived from MSCs (Group II), and mSOF replaced with 50% CM from steroid-treated UECs (Group III). Blastocyst rates were evaluated on day 09 post IVF. The blastocyst rate in group II was significantly higher (p < 0.05) than the control group, which was further enhanced in group III. In vitro co-culture of embryos with the secretome derived from mesenchymal stem cells or steroid-treated UECs improved the blastocyst rate. UECs and their secretions are essential to establish uterine receptivity and to mimic the internal in vivo environment.

The influence of balanced complex chromosomal rearrangements on preimplantation embryonic development potential and molecular karyotype

BackgroundBalanced complex chromosome rearrangements (BCCR) are balanced chromosomal structural aberrations that involve two or more chromosomes and at least three breakpoints. It is very rare in the population. The objective is to explore the difference of influence of three types of BCCR on early embryonic development and molecular karyotype.ResultsTwelve couples were recruited including four couples of three-way rearrangements carriers (group A), three couples of double two-way translocations carriers (group B) and five couples of exceptional CCR carriers (group C). A total of 243 oocytes were retrievedin the seventeen preimplantation genetic testing (PGT) cycles, and 207 of these were available for fertilization. After intracytoplasmic sperm injection, 181oocytes normally fertilized. The rates of embryos forming on day3 in three groups were 87.88, 97.78 and77.14%, which was significantly different (P = 0.01). Compared with group B, the rate of embryo formation was statistically significantly lower in group C (P = 0.01). Furthermore, the rates of high-quality blastocysts in three group were 14.71, 48.15 and 62.96%, respectively, which was significantly different (P = 0.00). Compared with group B andC, the rate of high-quality blastocysts in group A was statistically significantly lower (P = 0.00;P = 0.00).Comprehensive chromosome analysis was performed on 83 embryos, including 75 trophectodermcellsand 8 blastomeres. Except 7 embryos failed to amplify, 9.01%embryos were diagnosed as euploidy, and 90.91% were diagnosed as abnormal. As for group A, the euploid embryo rate was 10.71%and the abnormal embryo rate was 89.29%. In group B,the euploid embryo rate was 3.85%, the abnormal embryo rate was 96.15%. The euploid embryo rate was 13.04%, the abnormal embryo rate was 86.96% in group C. There were no significant differences among the three groups (P = 0.55).ConclusionsThe lowest rate of high quality blastocysts has been for three-way rearrangements and the lowest rate of euploidy has been for double two-way translocations, although no significant difference. Different types of BCCR maybe have little effect on the embryonic molecular karyotype. The difference of influence of BCCR on early embryonic developmentandmolecular karyotypeshould be further studied.

Embryo developmental potential of in vitro matured mi from stimulation cycles depends on the timing of nuclear maturation rather than the length of mii arrest

The reduced potential of IVM oocytes has been attributed to a lack of synchrony between nuclear maturation and a loss or excess of cytoplasmic maturation. However, there is no direct evidence to describe the duration of MII arrest in order for the meiotic spindle to function normally without negatively effecting of the oocyte’s potential. The objective is to know what is more important between the timing of the first polar body (1PB) extrusion (nuclear maturation; NM) and duration of MII arrest to rescue the developmental potential of in vitro matured (IVM) human oocytes of stimulated cycles? This is a retrospective study of patients who had metaphase I (MI) oocytes at retrieval at the MUHC Reproductive Center between January and December 2013 (n = 30 cycles). MI oocytes were divided into two IVM MI groups according to the different timing of NM (1PB extrusion) and ICSI was performed after different durations of MII arrest. The embryo developments were compared between the two groups. The spindle of the oocytes was examined using the Polscope TM. Group I in which 45 out of 109 MI oocytes became MII after 1∼ 4 h of in vitro culture. Culture was extended for ICSI until next morning because spindle in early telophase I was observed by the Polscope. Group II where 53 IVM MI oocytes became MII during overnight culture and were inseminated by ICS next morning. The rates of fertilization and blastocyst formation in Group I were 84.4 and 36.8% which are not significantly different with those in Group II which were 75.5 and 22.5% respectively. However, the freezable good quality blastocyst rate in Group I was significantly higher than that in Group II (18.4 vs. 2.5 %). Freezable good quality blastocyst is significantly higher in early NM groups with longer MII arrest than that of late NM with shorter MII arrest. This suggests that the success of IVM depends on the NM timing rather than the duration MII. Therefore if it is necessary to increase the number of available embryos for transfer using IVM MI, the optimal timing of ICSI for them may be the next morning.

138 COMPARISON OF KINETICS AND PATTERNS OF THE FIRST CLEAVAGE OF IN VIVO AND IN VITRO-MATURED HOLSTEIN OOCYTES AFTER IN VITRO FERTILIZATION WITH X-SORTED SPERM

Previously, it was reported that a high rate of good quality blastocysts were produced by IVF of in vivo-matured oocytes, obtained by ovum pick up (OPU) after superstimulation in Holstein cows, using X-sorted sperm (Matoba et al. 2012 Reprod. Domest. Anim. 47, 515). In this system, an early first cleavage within 28 h after IVF was found to be a potent marker for the selection of embryos with high developmental competence (Matoba et al. 2013 Reprod. Fertil. Dev. 25, 266). However, we have limited knowledge on the timing and normality of embryonic cleavages in in vitro-matured oocytes after IVF. The purpose of the present study was to compare the kinetics and patterns of the first cleavage of in vivo- and in vitro-matured bovine oocytes after IVF with X-sorted sperm. In vivo-matured oocytes (Group A) were collected by OPU from non-lactating Holstein cows just before ovulation after superstimulation. Immature oocytes were either collected by OPU without hormonal treatment or by aspiration of ovaries at the local abattoir and matured in vitro (Group B or C). All the oocytes were inseminated with 5 × 106 sperm mL–1 of X-sorted sperm, except half of oocytes in Group C inseminated by non-sorted sperm (Group D) and cultured in CR1aa supplemented with 5% calf serum and 0.25 mg mL–1 of linoleic acid albumin at 38.5°C in 5% CO2, 5% O2, and 90% N2 for 216 h. Embryo kinetics were observed individually using a microwell culture dish and time-lapse cinematography (Sugimura et al. 2010 Biol. Reprod. 83, 970–978). Photographs of each embryo were taken in every 15 min during the IVC period and analysed by time-lapse cinematography software. Cleavage pattern was categorized as normal (2 even blastomeres without fragment or protrusion) or abnormal (2 uneven blastomeres, with fragment or protrusion and those dividing into 3–5 blastomeres) at the first cleavage. Data were analysed by ANOVA, chi-squared, or discriminant function. A total of 268 cleaved embryos were used. The blastocyst rate in Group A was higher than in Groups B and C (61.3 v. 40.0 and 25.0%, respectively; P < 0.05). The timing of first cleavage was longer in Group A compared with Groups C and D (28.3 ± 3.8 v. 27.6 ± 3.8 and 26.7 ± 1.9 h, respectively) and in Group B (28.1 ± 4.0 h) compared with in Group D (P < 0.05). Higher rates of normal cleavage were observed in Groups A, B, and D than in Group C (53.5, 44.4, and 54.8 v. 16.7%, respectively; P < 0.01). The frequency of blastocysts derived from the early (28.3 h) and normal pattern cleaving oocytes were greater in Groups A and B than in Group C (29.0 and 20.0 v. 8.3%, respectively; P < 0.05) and similar in Group D (22.6%). Our results reveal that IVF embryos produced from in vivo-matured oocytes with sex-sorted sperm had superior normality than those produced from in vitro-matured oocytes and similar normality to embryos inseminated with non-sorted sperm. Supported by the Research and Development projects for application in promoting new policy of agriculture, forestry and fisheries (22016) and by JSPS and HAS under the Japan-Hungary Research Cooperative Program.

Establishment and characterization of fetal fibroblast cell lines for generating human lysozyme transgenic goats by somatic cell nuclear transfer

This study was performed to qualify goat fetal fibroblast (GFF) cell lines for genetic modification and somatic cell nuclear transfer (SCNT) to produce human lysozyme (hLYZ) transgenic goats. Nine GFF cell lines were established from different fetuses, and the proliferative lifespan and chromosomal stability were analyzed. The results suggested that cell lines with a longer lifespan had stable chromosomes compared with those of cells lines with a shorter lifespan. According to the proliferative lifespan, we divided GFF cell lines into two groups: cell lines with a long lifespan (GFF1/2/7/8/9; group L) and cell lines with a short lifespan (GFF3/4/5/6; group S). Next, a hLYZ expression vector was introduced into these cell lines by electroporation. The efficiencies of colony formation, expansion in culture, and the quality of transgenic clonal cell lines were significant higher in group L than those in group S. The mean fusion rate and blastocyst rate in group L were higher than those in group S (80.3±1.7 vs. 65.1±4.2% and 19.5±0.6 vs. 15.1±1.1%, respectively, P<0.05). After transferring cloned embryos into the oviducts of recipient goats, three live kids were born. PCR and Southern blot analyses confirmed integration of the transgene in cloned goats. In conclusion, the lifespan of GFF cell lines has a major effect on the efficiency to produce transgenic cloned goats. Therefore, the proliferative lifespan of primary cells may be used as a criterion to characterize the quality of cell lines for genetic modification and SCNT.

128 CULTURE CONDITIONS AFFECT THE SEX RATIO OF IN VITRO PRODUCED BOVINE EMBRYOS

Most systems for producing bovine embryos in vitro use glucose as an energy source despite putative toxic effects. Glucose has a selective embryotoxicity towards female embryos, due to the higher expression of the X-linked glucose-6-phosphate dehydrogenase gene (Kimura et al. 2005 Mol. Reprod. Dev. 72, 201–207). Recently, the replacement of glucose with citrate and myo-inositol in SOF medium supplemented with 5% bovine serum (BS) increased the percentage of female embryos (Rubessa et al. 2011 Theriogenology 76, 1347–1355). Serum also affects the sex ratio of in vitro-produced (IVP) bovine embryos, favoring the male gender (Gutierrez-Adan et al. 2001 Theriogenology 55, 1117–1126). The aim of this work was to evaluate the effect of glucose replacement with myo-inositol during in vitro culture, in the presence of either BS or BSA, on bovine embryo sex ratio. Abattoir-derived oocytes (n = 1164, over 4 replicates) were matured and fertilized in vitro as previously described (Rubessa et al. 2011). After 20 to 22 h of gametes co-incubation, zygotes were denuded and cultured for 7 days in SOF with: group A) 0.34 mM trisodium citrate + 2.77 mM myo-inositol + 5% BS (n = 287); group B) 0.34 mM tri-sodium citrate + 2.77 mM myo-inositol + 8 mg mL–1 BSA(n = 290); group C) 1.5 mM glucose + 5% BS (n = 302) and group D) 1.5 mM glucose + 8 mg mL–1 BSA (n = 285). Representative samples of blastocysts produced in each group (n = 96, 58, 99, and 70, respectively in groups A, B, C, and D) were sexed by PCR as previously described (Rubessa et al. 2011). Differences among groups in blastocyst yields were analyzed by ANOVA. The percentages of female embryos were analyzed by chi-square test. Blastocyst rates in group C were lower (28.1%) than those recorded in groups A, B, and D (35.9, 41.0 and 36.1%, respectively; P &lt; 0.01). A higher (P &lt; 0.05) percentage of female embryos was observed in group A (61.5%) compared to group C (45.5%), with intermediate values in groups B (51.7%) and D (60.0%). Therefore, the replacement of glucose with citrate and myo-inositol favored the development of female embryos in the presence of BS but was ineffective in the presence of BSA. Furthermore, when glucose was the energy source, a tendency to greater incidence of female embryos was observed when the medium was supplemented with BSA rather than BS (P = 0.06). As a small amount of glucose is present in the BS, we hypothesize an additional glucose-dependent toxic effect on female embryos in group C. However, we cannot rule out that other factors present in the BS may interact with the energy source, playing a role in determining the sex ratio. Furthermore, the shift in sex ratio in favor of males or females embryo can be due to a better development of embryo of one sex, or to the delayed development or degeneration of embryos of the other sex. In conclusion, these results suggest that manipulating the metabolic profile of the embryos during culture may have an impact on both blastocyst production and sex ratio.

291 INFLUENCE OF THE BREED OF BULL (BOS INDICUS × BOS TAURUS) IN TOLERANCE TO HEAT SHOCK IN BOVINE EMBRYOS PRODUCED IN VITRO

There is evidence that deleterious effects of heat shock (HS) on fertility are less pronounced in breeds tolerant to high temperatures, due mainly to differences in their thermoregulatory capacity. In vitro experiments have shown that Bos indicus embryos are more resistant to HS than Bos taurus. In order to better understand the differences related to HS resistance between Bos indicus and Bos taurus, the main objective of this study was to determine if tolerance to HS is caused by genetic contribution from the oocyte, spermatozoa, or both. Additionally, the influence of the time between collection of ovaries in the abattoir and oocyte aspiration in the laboratory on early embryo development was ascertained. In experiment 1, oocytes from Nellore and crossbreed Holstein cows (cHOL) were collected in a local abattoir, matured and fertilized using semen (n = 6 for each breed) from Nellore (NEL), Angus (ANG), Brahman (BRA,) and Gir (GIR) bulls. In experiment 2, oocytes from Nellore and Holstein (HOL) cows were collected in an abattoir and the oocytes were aspirated in the laboratory 4 (group 4 h) or 6.5 h (group 6.5 h) later, matured and fertilized using semen (n = 6 for each breed) from NEL, GIR, and HOL. In both experiments, 96 h post-insemination (hpi), embryos with &gt; 16 cells were separated in 2 groups: control and HS. In the control group the embryos were cultured at 39°C, whereas in the HS group the embryos were submitted to 41°C for 12 h, and then returned to 39°C. In experiments 1 and 2 the results were analyzed by ANOVA (Proc MIXED, SAS Institute, Cary, NC, USA). In experiment 1, there was no effect of HS on blastocyst and hatched blastocyst rates in all breeds studied. The percentage of oocytes that cleaved and reached the morula stage was significantly lower (P &lt; 0.05) in cHOL × GIR compared with the other breeds. Additionally, blastocyst rate was higher in cHOL × NEL than in cHOL × ANG and cHOL × GIR (P &lt; 0.05). In experiment 2, cleavage, morula, and blastocyst rates in group 4 h were higher (P &lt; 0.05) compared with group 6.5 h. The HS decreased blastocyst rates in all breeds (NEL × NEL, HOL × HOL, and HOL × GIR), and in both time intervals (4 and 6.5 h). The breed NEL × NEL had higher cleavage rate (P &lt; 0.05) for both time intervals compared with HOL × HOL and HOL × GIR. In addition, Nellore oocytes fertilized with Nellore semen (NEL × NEL) originated higher blastocyst rates (P &lt; 0.05) in control and HS group than the other breeds. We conclude that (a) embryos from Holstein are more susceptible to HS than embryos from crossbred Holstein; (b) the oocyte is more important than the spermatozoa for the development of thermotolerance, because the breed of the bull did not influence embryo development after HS; (c) in vitro early embryonic development was impaired by increasing (from 4 to 6.5 h) the time interval between ovary collection and oocyte aspiration. Fellowships to T. Nabhan from CAPES and to R. A. Satrapa, R. A. L. Simoes, and E. M. Razza from FAPESP. Funding from FAPESP (Sao Paulo, Brazil).

Parallel comparison of parthenogenetic development following oocyte cryopreservation vitrification vs. slow freezing

Parallel comparison of parthenogenetic development following oocyte cryopreservation vitrification vs. slow freezing

286 INFLUENCE OF REVERSIBLE MEIOSIS INHIBITION ON SWINE EMBRYOS PRODUCED BY IN VITRO FERTILIZATION AND PATHENOGENETIC ACTIVATION

The present work evaluated the reversible meiosis inhibition effect on the development of swine embryos produced by in vitro fertilization (IVF) or parthenogenetic activation (PA). The efficiency of PZM3 and NCSU23 embryo culture media was also evaluated. Oocytes from ovaries collected at a slaughterhouse were subjected to IVM in two different groups: CHX (cycloheximide 5 µM for 10 h) and control, both with TCM-199 + 3.05 mM glucose + 0.91 mM sodium pyruvate + 10% porcine follicular fluid (pFF) + 0.57 mM cystein + 10 ng epidermal growth factor (EGF)/mL + 10 IU eCG/mL + 10 IU hCG/mL for the initial 22 h. In the remaining period (20 h for CHX and 22 h for control), medium without hormones was utilized. After IVM, oocytes were denuded and fertilized for 6 h (IFV) or the matured oocytes were submitted to activation by electric pulses (PA) (2 DC of 1.5 kV/cm for 30 µs), incubated for 1 h in culture medium with 10 μM of CHX, and again submitted to the same electric pulses for 60 µs. Embryo development was evaluated by cleavage rate on Day 3 and blastocyst rate and blastocyst cell number on Day 7 of culture. Cleavage and blastocyst rates were analyzed by the equality-of-two-ratios test and cell number by the Kruskal-Wallis and Mann-Whitney tests (P &lt; 0.05). In relation to IVF, the PZM3 medium was more efficient than NCSU23 for cleavage rate in the CHX group (PZM3: 68.4%, NCSU23: 44.4%) and had a better blastocyst rate in the control group (PZM3: 13.4%, NCSU23: 5.6%). With reference to PA, NCSU23 presented better cleavage and blastocyst rates than PZM3 in the CHX group (NCSU23: 89.5%, PZM3: 78.5% and NCSU23: 20.4%, PZM3: 13.0%, respectively). In the control group, only the NCSU23 blastocyst rate was higher than that for PZM3 (NCSU23: 22.5%, PZM3: 10.8%). No culture medium effect on cell number mean of IVF and PA blastocysts was observed. Maturation block improved cleavage rates in IVF groups cultured with PZM3 (68.4% and 50.6%, respectively, for CHX and control) and in PA groups cultured with NCSU23 (89.5% and 80.3%, respectively, for CHX and control), but no improvement of blastocyst rates in both groups (IVF and PA) was verified. Table 1 below shows that maturation block decreased the IVF and increased the PA blastocyst cell numbers. As older oocytes are more effectively activated, oocytes blocked with CHX achieved the maturation stage faster than the control group, therefore resulting in high-quality PA blastocysts. In conclusion, PZM3 was more efficient for IVF embryo production in contrast to NCSU23, whereas NCSU23 can be indicated for PA embryo production. Moreover, maturation blockage with CHX influenced blastocyst cell number, decreasing in IVF embryos and increasing in PA embryos. Table 1. Mean (±SD) of blastocyst cell numbers for IVF or PA groups after in vitro maturation without (control) or with cycloheximide (CHX) and cultured in NCSU23 or PZM3 medium This work was supported by FAPESP 02/10747–1.

Correlation between in vitro maturation and expression of LH receptor in cumulus cells of the oocytes collected from PCOS patients in HCG-primed IVM cycles

The aim of this study was to investigate whether in vitro maturation (IVM) and blastocyst development of oocytes collected following HCG-primed IVM cycles of PCOS patients are correlated with their cumulus cell (CC) patterns and further to investigate mRNA expression of the receptors for FSH, LH and epidermal growth factor (EGF) in the CCs with each pattern. Patients who underwent IVM were primed with 10,000 IU of HCG 36 h before oocyte aspiration. The isolated cumulus-oocyte complexes were divided into three groups according to the CC patterns: oocytes with dispersed CCs (group A), oocytes with compacted CCs (group B) and oocytes with sparse CCs (group C). Oocyte maturation and blastocyst development were compared among three groups. The expression of the mRNA for FSH, LH and EGF receptors in group A and B was analysed by semi-quantitative RT-PCR. The maturation rate of group A was significantly higher than those of group B and C. The rate of blastocysts in group A was significantly higher than those of group B and C. mRNA expression of the LH receptor in group A was more abundant than that of group B. These results suggest that the presence of dispersed CCs at oocyte collection may be positively correlated with the rates of oocyte maturation and blastocysts in HCG-primed IVM cycles. In addition, the expression of LH receptor in CCs may be correlated with the CC pattern of oocytes at collection.