Sequential Embryo Culture Research Articles

Oviduct and endometrial epithelium improve in vitro produced bovine embryo developmental kinetics.

Standard in vitro produced (IVP) bovine embryo culture media limit embryonic development. Culturing IVP bovine embryos in standard IVP bovine embryo culture media conditioned with oviduct and/or endometrial cells improves blastocyst formation and reduces the time to formation. In vitro embryo production in cattle greatly impacts blastomere biochemistry, embryo rate of development and pre- and post-transfer survival. In vivo, the bovine embryo migrates through the oviduct isthmus before entering the uterus on approximately day 4 of development where it remains unattached within the uterine lumen until day 20 of gestation. During this time, the embryo is sequentially exposed to oviduct followed by endometrial secretions that support embryonic development. Considering this, we tested the effect of culturing in vitro produced (IVP) bovine embryos sequentially in oviduct epithelial- (OEp; days 1-3) followed by endometrial epithelial- (EEp) or EEp and fibroblast cell (EEp/F; days 4-8)-conditioned media on embryonic development using a time-lapse monitoring system. Compared to control, culturing IVP embryos in EEp- or EEp/F-conditioned media without prior culture in OEp-conditioned media increased blastocyst formation (P < 0.05) and reduced the time to blastocyst formation (P < 0.05). Culturing IVP bovine embryos in OEp-conditioned media followed by EEp- or EEp/F-conditioned media, however, had the greatest impact on embryo developmental kinetics and increased morula and blastocyst formation (P < 0.05) and reduced time to formation (P < 0.05). Day 8 blastocyst cell numbers, diameter and quality were not significantly different, although, blastocyst quality scores were less (indicative of better quality) for all cell-conditioned media compared to control. In conclusion, IVP bovine embryo development may be improved using a sequential embryo culture system involving bovine oviduct followed by endometrial cell-conditioned media.

Comparison of Single-step and Sequential Embryo Culture Systems: Replacement of Serum with Platelet Lysate

Background: More understanding of the physiological needs of the embryo during its growth from the zygote to the blastocyst stage, as well as the composition of the uterine and fallopian tubal secretions, has led to the development of single-step or sequential culture media. Objectives: The present study aimed to investigate the utilization of mCR2aa culture medium as a sequential culture medium for embryo development, supplemented with platelet lysate (PL) to decrease the concentration of fetal bovine serum (FBS) in the embryo culture medium. Methods: After maturation and fertilization of sheep oocytes obtained from a slaughterhouse, potential zygotes were cultured in mCR2aa medium without FBS for two days (C1, 1 - 3 days of culture). In the second two days of culture (C2, 3 - 5 days of culture), 2.5% or 5% of FBS and 5% or 10% of PL were used. In the remaining days of culture (C3, 5 - 9 days of culture), 2.5%, 5%, or 10% of FBS, and 0%, 2.5%, 5%, or 10% of PL were used. These percentages were compared to the commercial BO-IVC™ medium and mCR2aa medium, both of which contained 10% of FBS. Results: No significant difference was observed in the number of produced blastocysts between different treatments and the control. However, the number of hatched blastocysts in BO-IVC™, as a single-step culture medium, significantly differed from that of the other treatments. The number of hatched blastocysts in the sequential culture medium was higher in media supplemented with 2.5% FBS and 10% PL in the C2 mCR2aa medium, and 2.5% FBS without PL in the C3 mCR2aa medium, but no significant difference was observed in other treatments. However, increasing the concentration of FBS and PL in the C3 mCR2aa medium significantly decreased the number of hatched blastocysts. Conclusions: An increase in the percentage of PL to 10% necessitated a decrease in FBS to 2.5% in the C2 mCR2aa medium, while PL in the C3 mCR2aa medium was not required in the presence of 2.5% FBS.

The impact of single-step and sequential embryo culture systems on obstetric and perinatal outcomes in singleton pregnancies: the Massachusetts Outcomes Study of Assisted Reproductive Technology

To compare the obstetric and perinatal outcomes of deliveries conceived with embryos from single-step vs. sequential culture media systems. Historical cohort of Massachusetts vital records linked to assisted reproductive technology clinic data from the Society for Assisted Reproductive Technology Clinic Outcome Reporting System and laboratory embryology data from two large academic hospital fertility centers. Not applicable. Patients with singleton live birth deliveries between 2004 and 2017 conceived with autologous assisted reproductive technology cycles with fresh blastocyst transfer using either single-step (n = 1,058) or sequential (n = 474) culture media systems. None. Associations of single-step vs. sequential culture with obstetric outcomes (mode of delivery, placental abnormalities, pregnancy-induced hypertension, and gestational diabetes) and perinatal outcomes (preterm birth, low birthweight, small-for-gestational-age, and large-for-gestational-age [LGA]) were assessed with multivariate logistic modeling, adjusted for maternal age, race/ethnicity, education, parity, insurance type, protein supplementation, oxygen concentration, fertilization method, and number of transferred embryos. Compared with sequential culture, single-step culture was associated with increased odds of LGA (adjusted odds ratio 2.1, 95% confidence interval 1.04-4.22). There were no statistically significant differences between single-step and sequential culture media systems in the odds of placental abnormalities, pregnancy-induced hypertension, gestational diabetes, prematurity, small-for-gestational-age, or low birthweight. Single-step culture is associated with increased odds of LGA, indicating that embryo culture media systems may affect perinatal outcomes.

P–168 RCT comparing the effect of Continuous ( Single Step ) embryo culture system versus a Sequential embryo culture system on the outcome of IVF/ICSI cycles

Abstract Study question Is the outcome of IVF/ICSI cycles done with continuous (single step ) embryo culture system different from that with sequential embryo culture system ? Summary answer Yes the outcome of IVF / ICSI cycles done with continuous (single step ) embryo-culture system is better than that with sequential embryo-culture system . What is known already Embryo culture media are important factors in IVF, which can significantly influence the clinical outcome of IVF/ICSI cycles. However it is not clear which formulation is most optimal and whether sequential or continuous media (single step) should be favored. Sequential media complies with embryo demands based on developmental stage , taking into account metabolic changes embryos undergo in-vivo, while moving from the oviduct to the uterus. The embryos in the early cleavage stage prefer to use pyruvate to produce energy, whereas once development nears the blastocyst stage , the embryos start using glucose in the process of glycolysis . Study design, size, duration A prospective RCT was carried out at our centre between 2018–2019 and IVF-ICSI patients meeting inclusion criteria (at least six normal MII - Oocytes) were included in this study. The aim of study was to compare blastocyst formation rates after embryo-culture in two different culture media systems. 436 metaphase II Oocytes from 62 women were randomly and equally divided to be fertilized and cultured to the blastocyst stage in either sequential media or single-step media. Participants/materials, setting, methods In this prospective trial with sibling oocytes, 436 metaphase II oocytes from 62 women were randomly and equally divided to be fertilized and cultured to the blastocyst stage in either sequential media ( n = 218 MII oocytes) or a single medium ( n = 218 MII oocytes). In both groups, embryos were cultured in an interrupted fashion with media changes on day 3. Embryo transfer was performed on day 5. Main results and the role of chance Blastocyst formation rates on day 5 were significantly higher following culture in single step media 60.55% (132 / 218 ) as compared to sequential media 34.86% ( 76 / 218) . The percentage of good quality blastocysts was also significantly higher in single step media. In conclusion, culture in single step media was associated with higher blastocyst formation rates compared to sequential media , suggesting that the single medium may provide better support to the developing embryo. The proportion of poor quality embryos was significantly higher in the sequential media group. Results indicate that embryo culture in continuous media could be as efficient as embryo culture in sequential media. A significant difference observed was the proportion of poor quality embryos on day 5 , which was significantly higher when the embryos were cultured in sequential media. Our results suggest that the type of embryo culture media can influence the quality of embryos both at the cleavage stage and blastocyst stage. The use of continuous embryo culture media does not seem to cause an adverse effect; in fact, their use can lower the workload in busy IVF labs and lower the stress that embryos are exposed to during handling. Limitations, reasons for caution Although single-step-medium for extended culture has practical advantages and blastocyst formation rates appear to be higher, there is insufficient evidence to recommend either sequential or single-step media as being superior for the embryo-culture to days 5/6. Further studies comparing these two media systems in well-designed trials should be performed. Wider implications of the findings: When employing sequential media for embryo culture , it is necessary to transfer the embryos from one medium to another ( cleavage stage medium to blastocyst stage medium) which increases stress related embryo damage . Therefore, single-step media is beneficial as the embryos can develop undisturbed till blastocyst stage. Trial registration number Not applicable

P–221 Prospective randomized sibling study on gamete preparation, insemination and subsequent culture of human oocytes in a time-lapse system using media systems with and without antioxidants

Abstract Study question Does the addition of antioxidants for gamete preparation, insemination and embryo culture lead to differences in embryo development and clinical outcome Summary answer Using an antioxidant-containing media system for sperm preparation, insemination and embryo culture imparts significantly higher good-quality blastocyst rates and improved clinical outcome in elderly patients. What is known already A previous study showed that adding combined antioxidants for sequential embryo culture in conventional incubators (interrupted culture) improves embryo viability and clinical outcome, especially for elderly patients. Here we investigated the combined effect of three antioxidants Acetyl-L-Carnitine (10 µM), N-Acetyl-L-Cysteine (10 µM), and α-Lipoic Acid (5 µM) during sperm preparation, insemination, and time-lapse culture in a single step medium on human embryo development and clinical outcome. Study design, size, duration Prospective randomized single center study including 143 couples for IVF/ICSI between August 2018 and December 2019. Inclusion required at least eight cumulus-oocyte-complexes (COCs) after retrieval. Cycles involving PGT, split IVF/ICSI, and surgically retrieved sperm were excluded. Immediately after retrieval oocytes were randomly distributed to a study or control media system with or without antioxidants (Vitrolife). Similarly, ejaculates were split and prepared with and without antioxidants. Participants/materials, setting, methods Sibling oocytes were inseminated in the respective group with accordingly prepared sperm. Single step embryo culture was conducted in medium with (Gx-TL) and without (G-TL) antioxidants in the EmbryoScope+. Embryo quality and clinical outcome were assessed in relation to maternal age (&amp;lt;35/&amp;gt;35 years). Good-quality embryos on day 3 were defined as 8- to 10-cells with even cells and low fragmentation; good-quality blastocysts as &amp;gt; 3BB. Clinical outcome was assessed after single vitrified blastocyst transfer (SVBT). Main results and the role of chance From 143 participants (female age, 34.7±3.2 years), a total of 2424 COCs were collected; 1180 COCs/916 metaphase-II (MII) oocytes were allocated to Gx-TL media and 1244 COCs/981 MII oocytes to G-TL media. Age-related analysis in Gx-TL compared with G-TL in relation to allocated MII oocytes revealed a trend for higher fertilization rates in Gx-TL for both age groups (&amp;lt;35: 72.1% vs. 66.9%; &amp;gt;35: 70.7% vs. 64.9%, P &amp;lt; 0.1). Good-quality day 3 embryo development/MII oocytes was higher, albeit not significant, in the elderly patients in Gx-TL (&amp;lt;35: 35.9% vs. 34.4%; &amp;gt;35: 31.1% vs. 27.9%). Overall day 5/6 blastocyst rate was similar for both media (&amp;lt;35: 48.2% vs. 49.9%; &amp;gt;35: 42.3% vs. 39.5%). Day 5/6 GQB rate was comparable for younger patients (&amp;lt;35: 23.8% for Gx-TL vs. 26.0% for G-TL) but significantly higher in Gx-TL in elderly patients (&amp;gt;35: 20.7% vs. 14.4%; P &amp;lt; 0.05). A total of 200 SVBT were performed; 99 in the Gx-TL- and 101 in the G-TL-arm. We noted almost similar implantation and ongoing pregnancy rates between Gx-TL vs G-TL in the younger (&amp;lt;35) age group (50.0% vs. 55.4%; 50.0% vs. 55.6%) but higher albeit not significant rates for Gx-TL in older (&amp;gt;35) patients (44.1% vs. 33.3%; 44.1% vs. 33.3%). Limitations, reasons for caution In almost 95% of the cycles, oocytes were inseminated by ICSI; thus results may not equally apply for cycles with IVF. The use of a closed time-lapse system may have prevented from some environmental oxidative stress. Therefore results may come out different with a similar study using standard incubation. Wider implications of the findings: Supplementation of antioxidants to media for gamete isolation and preparation, as well as subsequent single step time-lapse culture may improve GQE/B rates and clinical outcomes in certain age groups, plausibly through the reduction of oxidative stress. Further studies in selected sub-groups (severe OAT syndrome / testicular cases) may be indicated. Trial registration number UMIN000034482

Elimination of stress factors by continuous embryo culture and its influence on in vitro fertilization outcomes

Recently, infertility has become one of the most important endemic conditions, affecting approximately 15–20 % of couples worldwide. Among others, the careerist lifestyle, the increasing maternal age and the parallel increment in the aneuploidy rate of embryos play a crucial role in this phenomenon. In this study, embryological parameters and pregnancy outcomes were investigated in IVF cycles using either sequential embryo culture or a single step culture system. By sequential media, oocytes/embryos are needlessly exposed to the potentially negative effects of light exposure, temperature decrement and altered oxygen tension. In comparison with sequential media, single step media induced 1.28, 1.21 and 1.21-fold increments in implantation, biochemical pregnancy and clinical pregnancy rates, respectively. Pregnancy outcomes showed strong maternal age-dependency, so the difference between the two investigated culture systems was equalized by the increasing maternal ages (35–44 years) and the supposed incidence of embryo aneuploidy. Nevertheless, the significant enlargements in the outcomes of the younger ages (25–34) induced by the single step cultures suggest that, beside the resultant maternal aneuploidy, aneuploidy (reduced pregnancy rates) may evolve from exposure to the mentioned environmental stress factors.

Comparative study on effect of different embryo culture media on in vitro blastocyst production in goats

The present study was aimed to assess the efficacy of different culture media for blastocyst development. Oocytes (2,539) from 1,059 ovaries recovered by follicle puncture technique were utilized for present investigation. In experiment 1, effect of different embryo culture media (Gr. 1; mKSOMaa; Gr. 2; TCM-199+OEC; Gr. 3; mSOFaa and Gr. 4; mCR2aa) and in experiment 2, effect of sequential embryo culture medium, Gr. 1: mKSOMaa containing 5% FBS (1–3 days) and 10% FBS (4–10 days); Gr. 2: 0.8% BSA (1–3 days) and continuous embryo culture medium, 10% FBS (4–10 days); Gr. 3: 0.8% BSA (1–10 days) and Gr. 4: 10% FBS (1–10 days) on in vitro blastocyst development potential was evaluated. Significant higher (P&lt;0.05) blastocyst production rate was observed in TCM– 199 co-culture with OEC and mKSOMaa medium compared to mSOFaa embryo culture medium. Similarly, significantly higher morula production rate was observed in mKSOMaa medium compared to mSOFaa and mCR2aa medium. However, numerically higher number of embryos cleaved in mSOFaa medium compared to TCM-199 co- culture with OEC. Results indicated that embryos cultured in mKSOMaa, TCM-199+OEC and mCR2aa embryo development media are equally effective in supporting pre-implantation development.While considering the risk factor associated with OEC co-culture, mKSOMaa is proved to be efficient medium for obtaining higher in vitro embryo development rate. In experiment 2, significantly higher morula and blastocyst formation was observed in sequential embryo culture medium compared to BSA supplemented continuous embryo culture medium. Further, the present findings indicated that the use of mKSOMaa+0.8% BSA or 5% FBS for first 3 days of embryo culture resulted in increased rate of blastocyst stage embryos, if initial 3 days of culture in mKSOMaa+0.8% BSA was followed by culture in mKSOMaa containing 10% FBS. However, no significant difference was observed in sequential embryo culture medium and continuous embryo culture medium supplemented with 10% FBS.

Effect of growth factors on oocyte maturation and allocations of inner cell mass and trophectoderm cells of cloned bovine embryos.

This study was conducted to determine the additive effects of exogenous growth factors during in vitro oocyte maturation (IVM) and the sequential culture of nuclear transfer (NT) embryos. Oocyte maturation and culture of reconstructed embryos derived from bovine granulosa cells were performed in culture medium supplemented with either epidermal growth factor (EGF) alone or a combination of EGF with insulin-like growth factor-I (IGF-I). The maturation rates of oocytes matured in the presence of EGF or the EGF + IGF-I combination were significantly higher than those of oocytes matured in the presence of only fetal calf serum (FCS) (P 0.05). IGF-I alone or in combination with EGF in sequential embryo culture medium significantly increased the ratio of inner cell mass (ICM) to total blastocyst cells (P < 0.05). Our results showed that the addition of growth factors to IVM and sequential culture media of cloned bovine embryos increased the ICM without changing the total cell number. These unknown and uncontrolled effects of growth factors can alter the allocation of ICM and trophectoderm cells (TE) in NT embryos. A decrease in TE cell numbers could be a reason for developmental abnormalities in embryos in the cloning system.

32 ALLOCATION OF INNER CELL MASS AND TROPHECTODERM CELLS OF NUCLEAR TRANSFER EMBRYOS CULTURED IN MEDIUM SUPPLEMENTED WITH EPIDERMAL GROWTH FACTOR AND INSULIN-LIKE GROWTH FACTOR-I

This study was conducted to determine the additive effects of exogenous growth factors and different macromolecules during in vitro oocyte maturation (IVM) and sequential embryo culture of nuclear transfer (NT) embryos. Oocytes were matured in TCM-199 supplemented with 10% fetal calf serum (FCS), 50 mg mL–1 sodium pyruvate, 1% penicillin/streptomycin (10 000 U mL–1 penicillin G, 10 000 mg mL–1 streptomycin), 5 mg mL–1 LH, and 0.5 mg mL–1 FSH without growth factors (Treatment 1) or with 50 ng mL–1 epidermal growth factor (EGF; Treatment 2) or with 50 ng mL–1 EGF and 100 ng mL–1 insulin-like growth factor-I (IGF-1; Treatment 3). Cloned bovine embryos were produced by transferring granulosa cells into enucleated meiosis II oocytes. Following activation, reconstructed embryos were cultured in Quinn's Advantage Cleavage Medium (QACM) supplemented with 8 mg mL–1 essentially fatty-acid free (FAF) BSA for 72 h. Then, developing embryos from granulosa cells were cultured in Sequential Quinn's Advantage Blastocyst Medium (QABM) supplemented with 4 mg mL–1 essentially FAF-BSA (Sigma-Aldrich, St. Louis, MO, USA) + 5% FCS (Group 1), 4 mg mL–1 BSA + 5% FCS+100 ng mL–1 IGF 1 (Group 2), and 4 mg mL–1 BSA + 5% FCS+100 ng mL–1 IGF-1+50 ng mL–1 EGF (Group 3) for an additional 5 days under low oxygen tension (5% CO2, 5% O2, 90% N2) at 38.5°C in high humidity conditions. Maturation rates of oocytes matured in the presence of EGF (75.5%) and EGF+IGF-I combination (75.0%) were significantly higher than those of oocytes matured (63.8%) in the presence of only FCS (P &lt; 0.05). The developing NT embryos derived from granulosa cells of the Anatolian Grey Cattle showed no significant differences in fusion (53.62%, 53.25%, 57.36%), cleavage (67.98%, 74.20%, 66.80%), or blastocyst rates (32.65%, 29.47%, 41.77%) among culture groups (P &gt; 0.05). When 13 to 23 embryos per group were examined by using differential staining, the results showed that the IGF-I alone and combination with EGF in the sequential embryo culture medium (Group 2: 46.61%. and Group 3: 41.37%) significantly increased the number of inner cell mass (ICM)/total blastocyst cell ratio in comparison with Group 1 (29.32%, no IGF-I and EGF; P &lt; 0.05). Our results showed that the addition of growth factors to IVM medium and sequential culture medium changed the cell ration of cloned bovine embryos to the advance of ICM without changing total cell number. Supplementation of media with growth factors can alter the allocation of ICM and trophectoderm cells in NT embryos.

Hybrid Embryo Rescue: A Non-Conventional Breeding Strategy in Horticultural Crops

Production of interspecific and intergeneric hybrids is useful for transfer of desirable genes from wild species into cultivated species. In many instances, progeny from wide crosses is difficult to produce owing to several barriers. Post-zygotic barriers such as endosperm abortion and, at later stages, embryo degeneration are of common occurrence, leading to low fertility; but these have been overcome through the use of embryo rescue and several hybrids have been developed. This approach is especially useful in horticultural crops, more so in fruit crops. In our laboratory, we have developed protocols for hybrid embryo rescue in several top-of-the-line fruit crops that suffer from an inability to cross naturally (e.g., distant crosses, use of seedless parent/s) or instances where initial fruit drop is very high. Thus, interspecific, intergeneric and intervarietal hybrids have been generated in mango, banana, seedless grape, papaya and seedless citrus using embryo rescue. Culture of embryos has also been demonstrated in rose, capsicum, hot pepper, onion and tomato. Among the very important strategies under non-GM biotechnologies figure techniques of hybrid embryo rescue, and related applications like ovule/ovary/placental cultures through sequential embryo culture. Embryo culture applied to practical problems is a tissue culture technique that has proven to be of greatest value to breeders.

Effects of Sperm Pretreatments and In vitro Culture Systems on Development of In vitro Fertilized Embryos Derived from Prepubertal Boer Goat Oocytes in China

Use of oocytes from prepubertal animals for in vitro embryo production holds potential application for reducing generation intervals and increasing genetic progress through embryo transfer. The objective of these studies was to compare the effect of three sperm pretreatments (prior to in vitro fertilization) and seven embryo culture protocols on fertilization rate and (or) subsequent development of in vitro fertilized embryos derived from oocytes harvested from ovaries of 1-6 month old prepubertal Boer goats in China. Cleavage rates were highest for embryos fertilized with heparin-treated versus calcium ionophore- or caffeine-treated sperm. Similar rates of blastocyst development were observed using heparin- and ionophore-treated sperm, which were higher than obtained with caffeine-treated sperm. No differences in cleavage or blastocyst rates were observed following embryo culture in basal medias (synthetic oviductal fluid (SOF), Charles Rosenkrans 1 (CR1) or tissue culture medium-199 (TCM-199)) containing 10% fetal bovine serum (FBS). Cumulus or oviductal cell co-culture did not enhance cleavage or blastocyst rates relative to culture in SOF+10% FBS. Replacement of FBS in SOF medium with 0.3% BSA increased cleavage rates, but did not increase rates of blastocyst development. Sequential culture in SOF+0.3% BSA followed by SOF+10% FBS increased blastocyst yield versus continuous culture in SOF+10% FBS and tended to increase blastocyst yield versus continuous culture in SOF+0.3% BSA. These results demonstrate a pronounced effects of sperm pretreatments and in vitro embryo culture systems on rates of blastocyst development and provide a potential protocol (sperm pretreatment with heparin and sequential embryo culture in SOF+0.3% BSA followed by SOF+10% FBS) for generation of the significant numbers of in vitro produced blastocysts from oocytes of prepubertal Boer goats necessary for application of embryo transfer in rural regions of China for distribution of Boer goat genetics.

121 DEVELOPMENT OF IN VITRO-PRODUCED BOVINE EMBRYOS CULTURED IN SEQUENTIAL OR CONTINUOUS MEDIA

Slaughtered bovine females have different characteristics including age, nutritional status, breed, and management system, all of which may affect the results obtained in in vitro embryo production. Another key consideration is that early embryos move from the oviduct to a slightly different environment in the uterus, which has led to development of sequential embryo culture media (e.g. Lane M et al. 2003 Theriogenology 60, 407–419). However, the benefits and importance of using sequential media are not fully known. Therefore, the aim of the present study was to compare developmental rates of oocytes obtained from slaughterhouse-derived ovaries from cows or heifers after culture in sequential media (CDM-1, CDM-2) or in a continuous medium (C-CDM). The experiment was a 3 × 2 × 2 factorial design [bulls (A, B, or C), source (cows or heifers), and medium (sequential or continuous)]. Cumulus–oocyte complexes were aspirated, within 5 h of slaughter, from 3- to 8-mm ovarian follicles of cows (1482 oocytes) and fattened heifers usually fed melengesterol acetate (2818 oocytes). Embryos were produced in vitro as described by De La Torre-Sanchez et al. 2006 Reprod. Fertil. Devel. 18, 585–596, with slight modifications. Presumptive zygotes were vortexed to remove cumulus cells and cultured for 2.5 d in C-CDM (CDM supplemented with 5.0 mm L-lactate, essential and nonessential amino acids, and 0.5% FAF-BSA, or in CDM-1 (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Devel. 18, 585–596) at 39°C in a humidified incubator under 5% CO2, 5% O2, and 90% N2. Cleavage was assessed after 2.5 d; 2- to 6-cell embryos were considered as cleaved, but were not cultured further. Embryos at the 7- to 8-cell stage were cultured for an additional 4.5 d in fresh C-CDM or CDM-2. The percentage blastocysts per oocyte was assessed after 7 and 8 days of culture. Data were arcsin-transformed and evaluated by ANOVA. There was a significant interaction between bull and ovary source for both 8-cell embryos and cleavage rate (P &lt; 0.05); however, this interaction was no longer significant for blastocysts. No other interactions were significant nor a source of ovaries. Culturing embryos in CDM-C refreshed after cleavage evaluation (continuous) or culturing embryos in CDM-1 early and CDM-2 after cleavage evaluation (sequential) resulted in similar cleavage and blastocyst rates (Table 1). We conclude that bovine embryos can be produced using a single chemically defined medium (+BSA) with similar efficacy as a system using 2 sequential media. Table 1. Effects of media on embryonic development (mean ± SE)

Blastocyst development in an extended embryo culture system after intracytoplasmic sperm injection

Introduction: The aim of this study was to determine the factors affecting blastocyst formation in an extended (5–6 days) embryo culture system after intracytoplasmic sperm injection (ICSI). Methods: The study included 77 consecutive couples (mean maternal age 34.5 years), undergoing IVF with the ICSI procedure and blastocyst culture for infertility treatment in a private clinic setting. These patients were divided into the following groups: group A, patients with total motile sperm count of 0–5×10 6 ( n=16); group B, patients with total motile sperm count of >5×10 6 ( n=61); group C, patients with normal sperm morphology (Krugers's strict criteria) of 0–5% ( n=55); and group D, patients with normal sperm morphology of >5% ( n=22). A control group of 160 standard IVF patients not undergoing ICSI was also compared. Embryos were cultured for up to 6 days in a sequential embryo culture system (Irvine Scientific, CA). Development of zygotes to blastocysts in vitro was used as the main outcome measure for this study. Results: Overall, 853 oocytes were injected with normal fertilization and embryo cleavage rates of 74.5% and 99.2%, respectively. Overall, 631 supernumerary ICSI embryos were cultured for possible blastocyst development, resulting in 189 blastocysts (30% overall blastocyst development rate). Blastocyst development results in different groups were group A, 26%; group B, 32%; group C, 31%; and group D, 30%. There was no statistically significant difference in blastocyst development rates among these groups of patients. A blastocyst development rate of 38% was observed in the standard IVF group vs. 30% in IVF-ICSI group ( p<0.05). Conclusions: Although the blastocyst development rate of ICSI embryos is statistically lower than the blastocyst development rate of non-ICSI embryos, this decrease is not statistically correlated with any of measured semen parameters. These results suggest that the ICSI procedure may have an adverse effect on the ability of the embryo to form a blastocyst in vitro.

Biochemical pregnancy and miscarriage rates for fresh and frozen cycles following extended embryo culture in non-selected patients

Objective: Recent reports regarding the incidence of biochemical pregnancy and miscarriage rates associated with in-vitro fertilization (IVF) and frozen embryo transfer (FET) procedures have prompted clinics to evaluate the effect of sequential embryo culture on fresh and frozen embryo quality as well as transfer and freezing techniques.

Increased embryo implantation with increased protein quantity and complexity in a sequential embryo culture system

Objective: Since the abandonement of co-cultures, the presence of more complex rather than simple protein molecules in the embryo culture medium is advocated and frequently implicated in improved in vitro embryo performance and subsequent in vivo viability. However, many commercial culture media are recommended for use with human serum albumin (HSA) only. The objective of this study was to evaluate the effect of an increase in the quantity and complexity of the embryo culture medium protein supplement on implantation and clinical pregnancy rates.

Sequential culture medium promotes the in vitro development of Macaca fascicularis embryos to blastocysts.

In vitro production of blastocyst stage embryos from Macaca fascicularis (Mf) has not previously been demonstrated without cell support. Historical data indicates that a large proportion of Mf embryos arrest at the morula stage in nonsequential culture medium (NSM) lacking serum supplementation and/or cell support. Here we report the application of a sequential culture system supporting in vitro production of Mf blastocysts. Mf embryos produced by in vitro fertilization (IVF; n = 69) were subjected to in vitro culture without cell support in either a commercial sequential embryo culture medium (SM) or an NSM. At 24 hr post-insemination (PI) embryos generated from in vivo and in vitro matured oocytes and cultured in the NSM cleaved to two or more cells in significantly greater proportions (15/23; 65%) compared to embryos cultured in SM (14/46; 30%). However, by day 3 PI embryo development beyond eight cells was not different in NSM (9/23; 39%) compared to SM (25/46; 54%). At day 5 PI embryo development to the morula stage was slightly lower in NSM (8/23, 35%) compared to SM (21/46, 45%), and embryo degeneration was slightly higher in NSM (9/23, 39%) compared to SM (9/46, 20%). After 7-9 days of in vitro culture, embryo development to the blastocyst stage and embryo degeneration were significantly lower and higher, respectively, in NSM (0/23, 0%; and 23/23, 100%) compared to SM (9/46, 20%; and 26/46, 56%). In this study the sequential culture system was better able to support in vitro development of Mf embryos compared to nonsequential culture systems.

Effect of Culture Media on In Vitro Development of Cloned Mouse Embryos

The effect of simple and sequential embryo culture media on the preimplantation development of mouse nuclear transfer (NT) embryos reconstructed with cumulus cell nuclei using a mechanical NT technique was studied. Blastocyst formation rate was evaluated using CZB medium and the sequential media G1/G2 and KSOM/G2. Arrested two- and three-cell NT embryos were Hoechst-stained to check for nuclear abnormalities. Nonmanipulated and sham-manipulated parthenogenetic embryos served as controls for, respectively, the medium and the handling technique. Rates of blastocyst formation for medium and handling control embryos were similar in CZB (58% and 61%), in G1/G2 (94% and 85%), and in KSOM/G2 (88% and 84%). Development of NT embryos was significantly impaired from the two-cell stage onwards, reaching the blastocyst stage at a rate of 5% in CZB, 14% in G1/G2, and 28% in KSOM/G2. Arrested two- and three-cell stage NT embryos showed a high rate of binucleation. These data demonstrate not only that NT embryos are more sensitive to in vitro culture conditions than parthenogenetic control embryos but also that selection of culture media can influence the preimplantation development of NT embryos.

The prognostic significance of day 3 embryo cleavage stage on subsequent blastocyst development in a sequential culture system.

To determine prognostic significance of blastomere number on Day 3 of culture upon subsequent blastocyst (BL) development. A retrospective analysis was conducted in 37 IVF subjects undergoing standard protocols and BL transfer after sequential embryo culture in P1 and BL media. Of Day 3 embryos containing 7 or more blastomeres, 68.9% (186/270) developed into BL compared to embryos containing 4-6 blastomeres, 38.1% (56/147), P < 0.0001. The majority of BL, 68.9% (168/244), were observed on Day 5. Extended Day 6 culture represented 31.1% (76/244) of all BLs. The observation of 7 or more blastomeres on Day 3 yielded a significantly greater likelihood of BL development. Embryos containing 4-6 blastomeres are still relatively likely to progress to a BL. Extended culture to Day 6 still yields a significant proportion of BL. Cell cleavage stage on Day 3 appears to be a useful prognostic indicator of subsequent BL development.

Does Blastocyst Transfer Affect Sex Ratio?

Objective: Advances in embryo culture have made blastocyst transfer (BT) a valuable technique in IVF. Embryos with the fastest cleavage rates and the best morphologic appearance are selected for transfer. Evidence exists that male embryos may grow more rapidly than female embryos. Recent published data (Menezo, et. al. Fertil Steril 1999; 72:221) have noted that there is a significant increase in the ratio of male offspring (58%) with fresh blastocysts grown in co-culture. Thus, concern exists regarding whether BT might result in an altered sex ratio favoring males. The objective of this study was to assess the sex ratio of pregnancies resulting from fresh BT in sequential media. Design: Retrospective chart review of pregnancies resulting from fresh BT at two academic institutions. Materials and Methods: Sequential embryo culture was performed using either P1 +10%SSS/Blastocyst medium + 10%SSS (Irvine Scientific) or IVF500/S2 (Scandinavian Science) or G1/G2 (Scandinavian Science.) Based on degree of embryo expansion and the presence of a distinct inner cell mass and trophectoderm the best 2 or 3 blastocysts were chosen for transfer on day 5. Results: 150 pregnancies resulted in 200 offspring with 112 (56%) males and 88 (44%) females. The difference did not reach statistical significance (p>0.05.) Conclusion: Even if a 56/44 male to female ratio such as ours were to be confirmed by a larger series, this is not so drastically different than the ratio of 107/100 (i.e. 51.7%/48.3%) reported in the general population. We believe that this slight trend for male offspring should not deter IVF programs from adopting BT with all of the advantages it offers at optimizing pregnancy rates while minimizing high-order multiple gestation.

Improved blastocyst development by combining media from two different commercial vendors

Low implantation rates and high multiple pregnancy rates have been the impetus for blastocyst embryo transfer. The availability of commercially developed sequential embryo culture media has facilitated the transition from day 3 to day 5 embryo transfers. After several groups reported excellent blastocyst development with these sequential media, we began using G1/G2 (period I, Scandinavian IVF Science). Conversion of embryos from day 3 cleaved to day 5 blastocyst was lower than expected. As a result, we switched to P1/Blastocyst (period II, Irvine Scientific). Blastocyst conversion improved; however, overall embryo cleavage and day 3 pregnancy rates declined. During period III embryos were cultured in G1 for the first 72 hours and then moved to Blastocyst Medium for extended culture. The purpose of this study was to compare conversion of day 3 cleaved embryos and pregnancy rates with extended culture in different growth media combinations.