Lower Blastocyst Formation Research Articles

P-151 Embrionary self-correction by excluding multinucleated cells improve the euploidy rates

Abstract Study question Could the exclusion of multinucleated cells from an embryo be a mechanism to dispose of cells with aberrant genetic content? Summary answer Exclusion of multinucleated cells could be a self-correction mechanism that would allow some embryos to exclude aneuploid cells. What is known already Multinucleation represents a poor prognosis morphologic trait related to low blastocyst formation rates and implantation rates. Moreover, it has been correlated with an increased rate of aneuploidies and chromosomal abnormalities, thus increasing misscarriage rates. Traditional morphokinetics ensure that excluding blastomeres during the compaction, the embryo could reduce its potential to achieve an euploid blastocyst. According to our previous studies multinucleated embryos excluding multinucleated cells during the blastocyst formation increase their reproductive potential. These studies assessed the clinical outcomes based on the morphokinetics of multinucleated blastomeres, without taking into account the chromosomal status of these embryos. Study design, size, duration Retrospective cohort study involving data from 157 PGT-cycles, performed between 2017 and 2019, with at least one multinucleated embryo. This trial included 678 embryos cultured until blastocyst stage using one-step culture media in time-lapse incubators (Embryoscope, Vitrolife) up to D + 5/6 when PGT-A was performed by trophectoderm biopsy using the NGS analysis technique in good quality embryos (≥3BB) according to the Gardner Score. Chi-square test for a contingency table was performed to compare all groups. Participants/materials, setting, methods Two main groups were considered: Control Group (CG; n = 474), embryos without multinucleation and Multinucleation Group (MNC; n = 204), embryos with at least one blastomere multinucleated on D + 2/3. Multinucleation Group was subdivided in three groups according to the multinucleation cell location using time-lapse technology to track them. MNC-1 (N = 87), no cells excluded; MNC-2 (N = 31), mononucleated cells excluded; MNC-3 (N = 41), multinucleated cells excluded. We had to exclude from the study 45 embryos that could not be follow up. Main results and the role of chance We observed multinucleation in the 20.33% of the embryos. MNC-3 (43.9%) achieved the higher euploidy rate, equivalent to the CG (43.9%); p = 0.998. MNC-1 (26.4%) and MNC-2 (22.6%) had lower euploidy rates than Groups MNC-3 and GC; p < 0.05. Regarding to the aneuploidy rates, MNC-2 (77.4%) showed a higher rate than of the other groups (MNC-1=52.9%; MNC-3=41.5%; CG = 42.0%), being significant compared to the CG and MNC-3; p < 0.05. The mosaicism rate of the MNC-1 (20.7%) is significantly higher than that of the CG (14.0%); p < 0.05. Limitations, reasons for caution Limitations include the retrospective analysis of data, the wide difference on sample size between MNC and CG groups and the amount of embryos excluded due to the impossibility to be monitored. Wider implications of the findings These results prove that embryos excluding multinucleated cells reach equivalent euploidy values than embryos without multinucleation. This outcome, together with previous studies, suggest a self-correction capacity that would allow some embryos to detect and expel cells with aneuploid genetic content, thus improving the global chromosomal status of the embryo. Trial registration number not applicable

P-031 The extent of late paternal effect: does the blastocyst matter? A retrospective analysis of 703 single, frozen-thawed embryo-transfers

Abstract Study question Do the blastocysts derived from severe male infertility show different ongoing pregnancy rates(OPR) compared to blastocysts of couples requiring assisted reproduction techniques(ART) for other indications? Summary answer First, single, frozen-thawed embryo transfers of blastocysts derived from severe male infertility did not affect OPR in-vitro fertilization (IVF)/ intracytoplasmic sperm injection (ICSI) cycles. What is known already Several studies reported a negative impact of severe male infertility on fertilization rate, embryo morphology and embryo cleavage speed, due to the so-called early paternal effect, caused by sperm cytoplasm deficiencies. Nuclear sperm defects are responsible for the late paternal effect, causing lower blastocyst formation rates and, theoretically, reducing implantation potential of the obtained blastocysts. However, the effects of male infertility on OPR and live birth rates (LBR) are still debated. Additionally, the heterogeneity in the definition and estimation of male infertility makes the existing data difficult to interpret. Study design, size, duration Retrospective analysis of first IVF/ICSI cycles performed between January 2019 and December 2021 in the Reproductive Sciences Unit of Gynaecology/Obstetrics Department of San Raffaele Hospital in Milan, Italy. Participants/materials, setting, methods First, single, frozen-thawed embryo transfers of infertile couples first IVF/ICSI cycles were included. Embryo transfers obtained by frozen semen were excluded. Couples characteristics, semen parameters and controlled ovarian stimulation (COS) data were collected. Semen quality was assessed using total motile count (TMC) and grouped into quartiles of TMC for the analyses. The effect of severe male factor infertility evaluated through TMC over OPR was the primary outcome of the study. Main results and the role of chance N = 703 transfers were analysed. Performing a logistic regression analysis adjusted for confounding factors (maternal age, COS protocols, number of oocytes retrieved and quality of transferred blastocysts), OPR was not influenced by TMC values [odds ratio (OR) = 0.999; confidence interval (CI) = 0.986-1.013; p = 0.932]. After grouping male infertility population into quartiles of TMC, no significant differences in OPR were found between extreme quartiles (TMC ≤2.55 million and TMC ≥20 million respectively), even when adjusted for confounders [OR = 1.007; CI 0.609-1.663; p = 0.98]. Therefore, the implantation potential of the obtained blastocysts, once formed, seems to be independent from sperm quality. Limitations, reasons for caution The major limitations of the present study are the retrospective design and sample size. Additionally, the estimation of male infertility as well as the routinary use of TMC in clinical practice are not yet standardized. However, having analysed only the first transfers of the included couples makes our results reliable. Wider implications of the findings Further prospective studies with larger sample size are needed to confirm our results and to validate the use TMC as a standard tool to classify male factor and to guide clinical choices. Pregnancy and obstetrical outcomes are also necessary to better understand the role of male infertility in human reproduction. Trial registration number not applicable

P-200 An addition of nicotinamide adenine dinucleotide (NAD+) precursor to oocyte maturation medium improves the developmental competence of bovine oocytes after IVF

Abstract Study question Does an increase of nicotinamide adenine dinucleotide (NAD+) level in oocytes lead to an improvement of blastulation after IVF? Summary answer The addition of NAD+ precursor to the oocyte maturation medium increased NAD+ levels in the oocytes, improving the developmental competence to the blastocyst after IVF. What is known already With maternal ageing, the enzyme that degrades NAD+ (CD38) increases in cells and the amount of NAD+ in cells, including oocytes, decreases. The degradation of NAD+ is also increased when cells are isolated from tissues. NAD+, which is an electron carrier used by all eukaryotes, is reduced to NADH via beta-oxidation of fatty acids and the TCA cycle, enhancing ATP synthesis in the mitochondrial electron transfer system. In addition, an enzyme in the inner mitochondrial membrane donates protons from NADH to NADP+ to synthesize NADPH, which maintains the intracellular redox state. Study design, size, duration This study is a prospective study using bovine oocytes retrieved from ovaries obtained from a local slaughterhouse in Osaka city. A total of 2,501 bovine oocytes were used in this study. Participants/materials, setting, methods Bovine cumulus-oocyte-complexes (COCs) were matured in vitro in the culture (IVM) medium supplemented with 0-100 μM nicotinamide mononucleotide (NMN). Matured oocytes were inseminated with the washed spermatozoa and cultured to assess the blastulation. Matured oocytes were also used to examine their NAD+, reactive oxygen species (ROS) and ATP levels. Main results and the role of chance The addition of NMN to the IVM medium increased intracellular NAD+ levels of bovine mature oocytes at 1 µM and above. As a result, it increased intracellular ATP levels and decreased ROS levels at 10 µM and above. It also improved blastocyst formation rates after IVF at 100 µM (P < 0.05; 35%) compared with control (22.2%). Moreover, the supplementation of NMN to the IVM medium decreased the percentage of apoptotic cells and intracellular ROS levels in cumulus cells surrounding oocytes. Limitations, reasons for caution This study was conducted in vitro using bovine oocytes. Thus, studies using human samples should be performed before clinical application. Wider implications of the findings By replenishing NAD+, we have found a clue to improving the IVM and IVF outcome, which has been shown to have a lower blastocyst formation rate than conventional IVF methods using in vivo matured oocytes. It may also improve the developmental potential of oocytes retrieved from older women. Trial registration number not applicable

P–291 Searching for a suitable serum progesterone range at triggering day to achieve optimal cumulative live birth rate in high responders – Which range is better?

Abstract Study question Is there a suitable range of serum progestereone level at triggering day to optimize the cumulative live birth rate (LBR) in high responders? Summary answer Fom the point of view of cLBR, the optimal P4 range for triggering is between 1.5 to 2.5 ng/ml generally and in the high responders. What is known already It is well established that premature progesterone rise (PPR) affect adversely the pregnancy outcome in fresh embryo transfer cycle. It is inferred that PPR alters synchrony between endometrium and the embryos. However, detailed study of the effect of PPR on efficiency of oocyte retrieval , embryo quality and the subsequent cumulative pregnancy outcome is still lacking. Hence we sort to analyze the effect of PPR on the final cumulative LBR in our program especially focused on high responders. Study design, size, duration ART Database in our center was retrospectively reviewed. Total 1523 cycles between 20160101 and 20191231 were recruited under the condition of GnRH antagonist cycle with duration of ovulation induction for more than 5 days and available serum P4 level data on triggering day for data analysis for the relationship between serum P4 value and final cumulative LBRs. Participants/materials, setting, methods Cycles with serum P4 level < 1.5 ng/ml were defined as without PPR (Group A: n = 1383). Cycles with serum P4 level >1.5 were defined as with PPR: P4 between 1.5 and 2.5 as Group B (n = 113), P4 > 2.5 as Group C (n = 27). Those high responding cycles (n = 404) were analyzed similarly and separately as Group A’ (n = 304), B’ (n = 81) and C’(n = 19). The statistics were carried out by SPSS-PC ver. 22.0 with p < 0.05 as statistical significance. Main results and the role of chance Group A had significantly lower number of oocytes (9.8 + 8.0) retrieved as compared to Group B (19.3 + 11.2) and Group C (18.2 + 9.9). However there were no differences in fertilization rate, good embryo rates and BC formation rates between groups. The cumulative LBR (cLBR) were significantly higher in Group B (65.1%) as compared to Group A (40.9%, p < 0.001) and Group C (37.0%, p = 0.008). For the high responding cycles, Group B’ also had marginally significant higher cLBR (75.3%) as compared to group A’(63.8% ; p=0.051) and Group C’ (52.6%; p = 0.050). Comparisons between Group A’ and C revealed significantly less oocytes retrieved but significantly higher blastocyst formation rates in Group A’ and the resultant cLBR were comparable between these two groups. Comparisons between Groups B’ and C’ revealed comparable oocytes retrieved but significant lower blastocyst formation rates and cLBRs in Group C’. The baseline of the first part analysis revealed higher age and lower AMH in Group A, but comparable age and AMH between groups B and C.The lower cLBR in group A could be due to selection bias.The second part (high responders) showed comparable baselines between three groups. However, the case numbers are too few in group C’ which might also result in uncertainty. Limitations, reasons for caution Although the data revealed interesting, significantly different results between groups, this is only a retrospective analysis from our ART patient series. Selection bias could not be precluded. Analysis restricted to high responders could have a more balanced population for comparisons. However, more cases are needed to affirm the findings. Wider implications of the findings: We still do not know the tolerable ceiling of serum P4 at the triggering day in high responders if future FET already planned. Pushing P4 value too high not only could not increase mature oocyte yields and possibly may decrease the number of available good blastocysts for optimizing final cLBRs. Trial registration number Not applicable

Transferrin and antioxidants partly prevented mouse oocyte oxidative damage induced by exposure of cumulus-oocyte complexes to endometrioma fluid

BackgroundExposure of oocytes to the endometrioma fluid has an adverse effect on embryonic quality. To determine whether adding transferrin and antioxidants to culture medium could counteract detrimental effects on mouse cumulus-oocyte complexes (COCs) induced by exposure to endometrioma fluid or not, we conducted an in vitro cross-sectional study using human and mouse COCs.MethodsEighteen women who had their oocytes exposed to endometrioma fluid during oocyte retrieval were enrolled. COCs from superovulated ICR female mice were collected. They were first exposed to human endometrioma fluid and then treated by transferrin and/or antioxidants (cysteamine + cystine). Subsequently, COCs function was assessed by molecular methods.ResultsThis study observed that human COCs inadvertently exposed to endometrioma fluid in the in vitro fertilization (IVF) group led to a lower good quality embryo rate compared to intracytoplasmic sperm injection (ICSI) group. Exposure of mouse COCs to endometrioma fluid accelerated oocyte oxidative damage, evidenced by significantly reduced CCs viability, defective mitochondrial function, decreased GSH content and increased ROS level, associated with the significantly higher pro-portion of abnormal spindles and lower blastocyst formation (p < 0.05, respectively). This damage could be recovered partly by treating COCs with transferrin and antioxidants (cysteamine + cystine).ConclusionsTransferrin and antioxidants could reduce the oxidative damage caused by COCs exposure to endometrioma fluid. This finding provides a promising new possibility for intervention in the human oocyte oxidative damage process induced by endometrioma fluid during oocyte pick-up.

Importance of oil overlay for production of porcine embryos in vitro.

Technologies to edit the zygote genome have revolutionized biomedical research not only for the creation of animal models for the study of human disease but also for the generation of functional human cells and tissues through interspecies blastocyst complementation technology. The pig is the ideal species for these purposes due to its great similarity in anatomy and physiology to humans. Emerging biotechnologies require the use of oocytes and/or embryos of good quality, which might be obtained using in vitro production (IVP) techniques. However, the current porcine embryo IVP systems are still suboptimal and result in low monospermic fertilization and blastocyst formation rates and poor embryo quality. During recent years, intensive investigations have been performed to evaluate the influence of specific compounds on gametes and embryos and to avoid the use of undefined supplements (serum and serum derivate) in the incubation media. However, little consideration has been given to the use of the mineral oil (MO) to overlay incubation droplets, which, albeit being a routine component of the IVP systems, is a totally undefined and thus problematic product for the safety of gametes and embryos. In this review, we provide an overview on the advantages and disadvantages of using MO to cover the incubation media. We also review one important concern in IVP laboratories: the use of oils containing undetected contamination. Finally, we discuss the effects of different types of oils on the in vitro embryo production outcomes and the transfer of compounds from oil into the culture media.

Calcium-sensing receptor (CASR) is involved in porcine in vitro fertilisation and early embryo development.

It has been demonstrated that extracellular calcium is necessary in fertilisation and embryo development but the mechanism is still not well understood. The present study mainly focussed on the extracellular calcium effector called the calcium-sensing receptor (CASR) and examined its expression in porcine gametes and embryos and its function during fertilisation and early embryo development. By using reverse transcription polymerase chain reaction, CASR was found to be expressed in porcine oocytes, spermatozoa and embryos at different developmental stages. Functionally, medium supplementation with a CASR agonist or an antagonist during in vitro fertilisation (IVF) and in vitro culture (IVC) was tested. During fertilisation, the presence of a CASR agonist increased sperm penetration rate and decreased polyspermy rate leading to an increased normal fertilisation rate. During embryo development, for the IVF embryos, agonist treatment during IVC significantly increased cleavage rate and blastocyst formation rate compared with the control group. Furthermore, parthenogenetically activated embryos showed similar results with lower cleavage and blastocyst formation rates in the antagonist group than in the other groups. It was concluded that CASR, as the effector of extracellular calcium, modulates porcine fertilisation and early embryo development.

The fertilization ability and developmental competence of bovine oocytes grown in vitro.

In vitro growth culture systems for oocytes are being developed in several mammalian species. In these growth culture systems, in vitro grown oocytes usually have lower blastocyst formation than in vivo grown oocytes after in vitro fertilization. Furthermore, there have been a few reports that investigated the fertilization ability of in vitro grown oocytes in large animals. The purpose of this study was to investigate the fertilization process and developmental competence of bovine oocytes grown in vitro. Oocyte-granulosa cell complexes collected from bovine early antral follicles (0.4−0.7 mm in diameter) were cultured for growth with 17β-estradiol and androstenedione for 14 days and matured in vitro. These oocytes were then inseminated for 6 or 12 h, and further cultured for development up to 8 days in vitro. After growth culture, oocytes grew from 95 µm to around 120 µm and acquired maturation competence (79%). Although fertilization rates of in vitro grown oocytes were low after 6 h of insemination, 34% of in vitro grown oocytes fertilized normally after 12 h of insemination, having two polar bodies and two pronuclei with a sperm tail, and 22% of these oocytes developed into blastocysts after 8 days of culture. The fertilization and blastocyst formation rates were similar to those of in vivo grown oocytes. In addition, blastocyst cell numbers were also similar between in vitro and in vivo grown oocytes. In conclusion, in vitro grown bovine oocytes are similar to in vivo grown oocytes in fertilization ability and can develop into blastocysts.

A high incidence of chromosome abnormalities in two-cell stage porcine IVP embryos.

In pigs, in vitro production is difficult with a high occurrence of polyspermy and low blastocyst formation rates. To test the hypothesis that this may, at least in part, be due to chromosomal errors, we employed whole genome amplification and comparative genomic hybridization, performing comprehensive chromosome analysis to assess both cells of the two-cell stage in vitro porcine embryos. We thus described the incidence, nature and origin of chromosome abnormalities, i.e. whether they derived from incorrect meiotic division during gametogenesis or aberrant mitotic division in the zygote. We observed that 19 out of 51 (37%) of two-cell stage early pig IVP embryos had a chromosome abnormality, mostly originating from an abnormal division in the zygote. Moreover, we frequently encountered multiple aneuploidies and segmental chromosome aberrations. These results indicate that the pig may be particularly sensitive to in vitro production, which may, in turn, be due to incorrect chromosome segregations during meiosis and early cleavage divisions. We thus accept our hypothesis that chromosome abnormality could explain poor IVP outcomes in pigs.

Effect of potassium simplex optimization medium (KSOM) and embryo screening on the production of human lactoferrin transgenic cloned dairy goats

In this study, we produced cloned transgenic dairy goat based on dairy goat ear skin fibroblast as donor cells for nuclear transfer (NT), which were modified by human lactoferrin ( hLF ) gene. The developmental competence of NT embryos was compared with either between different embryo culture medium, potassium simplex optimization medium (KSOM) and tissue culture medium (TCM 199), or different classification of NT embryos (48 h after fusion). First we cultured NT embryos to cleavage stage (48 h after fusion) by TCM 199 supplemented with 1 mg/ml bovine serum albumin BSA and KSOM, then used TCM 199 supplemented with 10% FBS to culture them to blastula stage. The results show that the NT embryos in KSOM (19.5%) were superior to TCM 199 (10.6%) in blastulation. In the second experiment, we found that the growth rate of NT embryos (48 h after fusion) was different, then we divided them into four groups: 2-cell, 3- to 4-cell, 5- to 8-cell and >8-cell in stereo microscope and cultured them in vitro respectively. The results show day-2 embryos at 3-4cell and 5-8cell stage (31.9 and 28.2%, P 8-cell (8.3%) stage, and finally three healthy cloned transgenic goat were successfully produced using 3-8 cell embryos at Day-2 (82%). Using Hoechst 33342 staining, we also found that the >8 cells embryos at Day- 2 demonstrated higher frequency of fragmentation, which may be the one cause of the low blastocyst formation rate. This study therefore demonstrates that KSOM medium could be selected as the early embryo culture medium, and 3-8 cell embryos at day-2 (48 h after fusion) may be the suitable embryos for transplantation, which could reduce the nuclei fragmentation and result in good quality blastocysts that may also enhance the efficiency of transgenic cloned dairy goats production, as well as decrease the economic loss due to embryonic mortality when embryos are transferred to synchronized recipients. Key words: Nuclear transfer, KSOM, transgenic, human lactoferrin, dairy goat.

Embryos exhibiting abnormal first cytokinesis phenotypes are associated with poorer embryo development and lower implantation potential

Combined with timing of the 2nd and 3rd cytokinesis, a prolonged 1st cytokinesis duration has been reported to correlate with low blastocyst formation and reduced expression of cytokinesis genes (Wong et al, 2010). This study determined the incidence of a novel abnormal 1st cytokinesis phenotype, evaluated the duration of 1st cytokinesis, and assessed clinical relevance.

Alterations in calcium oscillatory activity in vitrified mouse eggs impact on egg quality and subsequent embryonic development

Cryopreservation of mature eggs is a useful technique that can be applied in assisted reproductive technology. However, the method has some limitations, such as cryodamage induced by biophysical modifications during the cryopreservation process. To assess these biophysical damage, we analyzed the relationship between intracellular calcium ([Ca2+]i) oscillatory activity via type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) distribution after vitrification and efficiency of cryopreservation according to cryoprotectant (CPA) composition. In immunostaining, results of IP(3)R1 with eggs after the vitrification performed using ethylene glycol (EG) alone or EG + dimethylsulfoxide (DMSO) as CPAs, CPA-treated, and fresh eggs displayed a homogeneous IP(3)R1 distribution which is spread uniformly throughout cytoplasm with clusters on the cortex. However, after vitrification and warming process, more than 60% of eggs displayed a heterogeneous distribution which is non-uniform distribution with patches and disconnection of IP(3)R1. In 90-min incubation for recovery from cryodamage, eggs from the EG + DMSO group recovered from with a heterogeneous IP(3)R1 distribution to the homogeneous distribution, but not in EG alone group. In ICSI experiments, vitrified eggs in the EG-alone group presented significantly low blastocyst formation compared to those of the fresh and EG + DMSO groups. These results suggest that the vitrification process influences IP(3)R1 distribution, and subsequently, [Ca2+]i oscillatory activity and embryonic development. Accordingly, we propose that IP(3)R1 distribution and [Ca2+]i oscillatory activity are correlated with egg quality and developmental potential after vitrification, and may thus be applied as an effective indicator to evaluate the efficiency of oocyte cryopreservation methods.

214 EFFECT OF OOCYTE SOURCE ON THE DEVELOPMENTAL CAPACITY OF SOUTH AFRICAN IN VITRO-PRODUCED INDIGENOUS CATTLE EMBRYOS

The present study was undertaken to determine whether the source of oocytes (slaughterhouse ovaries from feedlot cows or naturally grazing indigenous cows) would affect in vitro bovine embryo production. Bovine oocytes (n = 1047), aspirated from slaughterhouse ovaries from feedlot cows and naturally grazing indigenous cows were randomly allocated to Sanyo, Forma, and Thermo 5% CO2 incubators. Oocytes were then in vitro matured in TCM-199 plus 10% fetal bovine serum, 1 μg mL–1 for both FSH and LH at 39°C for 24 h, and fertilized in Brackett and Oliphant (BO) medium per treatment group at 39°C. Presumptive zygotes were cultured in vitro per treatment group. Total cleavage and blastocyst rates were recorded postfertilization. Data were analyzed by ANOVA. Preliminary results demonstrated that there was no effect of incubator or source of oocytes on cleavage and 8-cell embryos. However, the cleavage and embryo developmental rate tended to be lower for the feedlot group, regardless of the incubator used (Table 1). In conclusion, this study suggests that slaughterhouse ovaries obtained from South African indigenous cows from a feedlot resulted in a lower blastocyst formation rate. Further studies are currently underway to count the cell numbers and to conduct embryo transfer. Table 1.Comparison of three different incubators and source of oocytes on embryo development in vitro This work was funded by the South African National Department of Agriculture, DST-PDP, and the National Research Foundation (NRF, Grant. Nos. RT21 and 24000).

59 HIGHER BLASTOCYST FORMATION OF SOMATIC CELL NUCLEAR TRANSFER EMBRYOS DOES NOT GUARANTEE BETTER PREGNANCY IN PIG

Generally, blastocyst (BL) formation and embryo cell number are used as main parameters to evaluate the viability and quality of in vitro-produced somatic cell nuclear transfer (SCNT) embryos. We investigated whether in vitro development of SCNT pig embryos correlates with in vivo viability after transfer to surrogates. For SCNT, cumulus–oocyte complexes (COCs) were matured in TCM-199 supplemented with follicular fluid, hormones, EGF, cysteine, and insulin for the first 22 h and in a hormone-free medium for 18 h. Three sources of pig skin cells were used as nuclear donor: (1) skin fibroblasts of a cloned piglet that were produced by SCNT of fetal fibroblasts from a Landrace × Yorkshire × Duroc F1 hybrid (LYD), (2) skin fibroblasts of a miniature pig having the human decay accelerating factor gene (hDAF-MP), and (3) skin fibroblasts of a miniature pig with a different strain (MP). MII oocytes were enucleated, subjected to nuclear transfer from a donor cell, electrically fused, and activated 1 h after fusion. SCNT embryos were cultured in a modified NCSU-23 (Park Y et al. 2005 Zygote 13, 269–275) for 6 days or surgically transferred (110–150 fused embryos) into the oviduct of a surrogate that showed standing estrus on the same day as SCNT. Embryos were examined for cleavage and BL formation on Days 2 and 6, respectively (Day 0 = the day of SCNT). BLs were examined for their cell number after staining with Hoechst 33342. Pregnancy was diagnosed by ultrasound 30 and 60 days after embryo transfer. Embryo cleavage was not affected by donor cells (82, 81, and 72% for LYD, hDAF-MP, and MP, respectively), but BL formation was higher (P &amp;lt; 0.05) in hDAF-MP (16%) than in LYD (9%) and MP (6%). MP showed higher (P &amp;lt; 0.05) BL cell number (46 cells/BL) than hDAF-MP (34 cells) but did not show a difference from LYD (37 cells). LYD and MP showed higher pregnancy rates (Table 1) on Days 30 and 60, even though they showed lower BL formation in vitro. Due to a relatively small number of embryo transfers through a limited period, we could not exclude any possible effects by seasonal or operational differences. These results indicated that pregnancy did not correlate with in vitro BL formation of SCNT pig embryos but rather were affected by the source of donor cells. Table 1.In vivo development of somatic cell nuclear transfer pig embryos derived from different sources of donor cells This work was supported by the Research Project on the Production of Bio-organs (No. 200506020601), Ministry of Agriculture and Forestry, Republic of Korea.

316 SUPPLEMENTAL CYSTEINE PRESENCE DURING THE DECONDENSATION OF SPERM CHROMATIN IMPROVES FERTILIZATION AND BLASTOCYST FORMATION AFTER INTRACYTOPLASMIC SPERM INJECTION IN PIGS

The effect of a cysteine supplement in culture media for oocytes matured in vitro after intracytoplasmic sperm injection (ICSI) on fertilization and embryo development were examined. In the first experiment, sperm injected oocytes were cultured in NCSU23 (control) or NCSU23 supplemented with 0.57–3.71 mM cysteine (0.57–3.71 Cys) for 12 h after ICSI, and then fixed to observe pronuclear formation. In the second experiment, to examine the appropriate duration time of cysteine supplement to support fertilization, sperm-injected oocytes were transferred into NCSU23 following culture in NCSU23 supplemented with 1.71 mM cysteine for 1, 2, 3, 4, 5, 6, or 9 h after ICSI, and then fixed at 12 h. At the same time, morphological changes of sperm heads in oocytes cultured in NCSU23 (1.71 Cys) were observed. In the third experiment, to examine the developmental ability of ICSI embryos fertilized in NCSU23 (1.71 Cys), sperm injected oocytes were cultured under the following conditions for a total of 168 h; NCSU23 (control), NCSU23 (1.71 Cys) for 3 h followed by transfer into NCSU23 (1.71 Cys-3 h), NCSU23 (1.71 Cys) for 12 h followed by transfer in NCSU23 (1.71 Cys-12 h), or NCSU23 (1.71 Cys) (1.71 Cys). Data were pooled from at least five replicates. Values in each replicate were analyzed using one-way ANOVA. Significance of differences was assessed by Student's t-test. Culture with several concentrations of cysteine for 12 h showed that 1.71–3.71 Cys significantly (P &lt; 0.05) increased fertilization rates above controls or 0.57 Cys (56–60%, 35%, or 48%, respectively). Culture for several duration times with 1.71 Cys showed that fertilization rates increased as the duration time increased to 3 h which was significantly (P &lt; 0.05) higher than controls (68% and 34%, respectively), and culture times of greater than 3 h did not increase fertilization rates (58–68%). At 3 h, 59% of oocytes cultured in NCSU23 (1.71 Cys) had decondensed sperm heads and 16% of those had enlarged sperm heads. At 6 h, 50% of oocytes cultured in NCSU23 (1.71 Cys) had male pronuclei. Blastocyst formation rate in 1.71 Cys-3 h was 29% which was higher than for controls (20%). On the other hand, 1.71 Cys-12 h cultures showed low blastocyst formation rates, and continuous culture in NCSU23 (1.71 Cys) for 168 h (1.71 Cys) significantly (P &lt; 0.05) decreased blastocyst rates (16% and 7%, respectively). We found that the supplement of 1.71 mM cysteine to NCSU23 for culture of oocytes after ICSI improved fertilization rates. However, the presence of 1.71 mM cysteine for 12 h or longer after ICSI had adverse effects on embryo development. Since 1.71 mM cysteine supplement for 3 h after ICSI improved blastocyst formation with the same fertilization rates as when supplemented for 12 h, the presence of cysteine only during the decondensation of sperm chromatin was found to be associated with the improvement of fertilization and also the promotion of blastocyst formation.

Prolonged culture of human cryopreserved embryos with recombinant human leukemia inhibitory factor.

To evaluate the efficiency of recombinant human leukemia inhibitory factor (LIF) in the prolonged culture of human cryopreserved-thawing embryos. After thawing, all embryos were divided into four groups: (1) Human tubal fluid (HTF), (2) HTF + LIF, (3) M3TH medium, and (4) M3TH medium plus LIF. Following prolonged culture, embryo development in each group was compared. In embryo development from about the 2- to 4-cell to 9- to 16-cell stage, there were nonsignificant differences between each group. There was lower morula formation rate in group 1 (6.9%) than those in other groups (23.2%, 19.7%, 23.1%). The lower blastocyst formation in group 1 and 3 (0%, 0%) than those in group 2 and 4 (11.0%, 12.8%) were noted. LIF is beneficial for preimplantation embryos. LIF does not influence the early embryo development. LIF-supplemented HTF provided a similar culture environment for thawing embryos as LIF-supplemented M3TH medium.

Improved methods for blastocyst formation and culture.

Transfer at the blastocyst stage has been proposed to increase the pregnancy rates after in-vitro fertilization (IVF) and embryo transfer. In the first period of experiments, culture in a single medium, from fertilization to blastocyst led to disappointing results: low blastocyst formation rates and low implantation rates per blastocyst transferred. Then the period of co-culture began (starting with animals in the early 1980s and with humans in the early 1990s). With this technique, using tubal or granulosa cells or layers obtained from established cell lines of transport epithelium origin, blastocyst formation has reached approximately 50% and the overall implantation rate is approximately 25%. The embryos obtained have high numbers of cells (> 200 cells for a day 6 expanded blastocyst). Co-culture with fibroblasts has been found to be useless. This technology has been proven reliable and reproducible: Blastocyst formation is highly dependent on maternal and paternal factors. It has enabled the design of efficient freezing and thawing protocols. Numerous interesting observations have been obtained to reach the third period, i.e. the use of sequential media. A simple medium is used for fertilization, then another one is used from fertilization up to the 4-cell stage (beginning of waves of transcription), then a third medium is used for development up to the blastocyst stage. The results obtained seem very similar to the one obtained with co-culture. Obviously it is now time, in humans, to switch to sequential media.

The protective effects of polymers in the cryopreservation of human and mouse zonae pellucidae and embryos

To evaluate the occurrence of injury due to physical factors in embryo cryopreservation and the effect of the polymers dextran, polyvinylpyrrolidone (PVP), and Ficoll on this mechanical damage. Damage to the zona pellucida (ZP) observed after cryopreservation was taken as indication of cryoinjury caused exclusively by physical factors. Human and mouse ZPs from oocytes remaining unfertilized after previous IVF attempts and mouse two-cell embryos were frozen in the presence of different polymers. After thawing, they were checked carefully for signs of physical damage (cracks). A possible toxicity of the use of the polymers in cryoprotection was evaluated by development to the blastocyst stage of mouse two-cell embryos that survived the freezing and thawing process. Incidences of damaged ZPs in groups of human and mouse ZPs and two-cell embryos frozen without polymers were found to vary between 20% and 29%. The use of any of the tested polymers resulted in significantly lower incidences of damaged ZPs (0% to 15%). Damage to the ZP after freezing and thawing in mouse embryos was accompanied by low survival rates of the embryo itself. Of mouse embryos that survived the cryopreservation process, blastocyst formation was not significantly different in groups frozen without polymer (80%) or in the presence of either dextran (90%) or Ficoll (82%); however, embryos frozen in the presence of PVP showed low blastocyst formation (12%). Polymers can protect embryos against cryoinjury by avoiding mechanical strain occurring during cryopreservation. Polyvinylpyrrolidine is toxic to mouse two-cell embryos when present during freezing and thawing.