Differences In Embryo Quality Research Articles

Reproductive outcomes of oocyte donation in patients with uterine Müllerian anomalies

To evaluate live-birth rates per embryo transfer in patients with uterine Müllerian anomalies (UMAs). Secondary objectives were to compare reproductive outcomes between the normal uterus group, the different UMA types, and UMA subgroups with and without required surgery. This retrospective study compared two cohorts, one with UMAs and other with normal uteri of our oocyte donation program at 12 Instituto Valenciano De Infertilidad/Reproductive Medicine Associates University affiliated clinics from January 2000 to 2020. The oocyte donation reduces confounding because of differences in embryo quality. The primary outcome was the live-birth rate per embryo transfer. Secondary outcomes included the rates of implantation, clinical pregnancy, miscarriage, and ongoing pregnancy. We calculated odds ratios with 95% confidence intervals. Infertile women undergoing oocyte donation with UMAs. None. The rates of implantation, clinical pregnancy, miscarriage, ongoing pregnancy, and live birth. We analyzed 58,337 cycles of oocyte donation: 57,869 patients had no uterine malformation, and 468 women had UMAs. Compared with patients with normal uteri, patients with UMAs had lower rates of live births (36.67% [32.84-40.65] vs. 38.1% [95% confidence intervals {CI}: 37.82-38.42]) and ongoing pregnancy (39.74% [35.93-43.66] vs. 41.5% [41.24-41.83]). The miscarriage rate was higher in patients with UMAs (19.5% [16.55-22.85] vs. 16.6% [16.47-16.92]). Specifically, patients with a unicornuate uterus (n=29) had lower rates of implantation (24.07% [13.49-37.64] vs. 42.85% [95% CI: 42.6-43.09]), pregnancy (41.86% [27.01-57.87] vs. 59.51% [59.22-59.81]), ongoing pregnancy (16.67% [6.97-31.36] vs. 41.54% [41.24-41.83]), and live births (16.67% [6.97-31.36] vs. 38.12% [37.83-38.42]). In addition, patients with a partial septate uterus (n=91) had a higher miscarriage rate (26.50% [18.44-34.89] vs. 16.7% [16.47-16.92]). Compared with the normal uterus group, the live-birth rates were lower in the UMA without surgery group (33.09% [27.59-38.96] vs. 38.12% [37.83-38.42]). Among patients who received embryos derived from donated oocytes, live birth and ongoing pregnancy rates were lower in patients with UMAs compared with patients with normal uteri. A higher miscarriage rate was found in patients with UMAs. Patients with a unicornuate uterus had worse reproductive outcomes. Our results show that the uterus is less competent in patients with UMAs. This study was registered at clinicaltrial.gov (NCT04571671).

P-126 Investigation of the deterministic value of presence of visible spindle on fertilization and blastocytes utilization potential in frozen/thawed oocyte cycle

Abstract Study question Does presence or absence of birefringent spindle after warming of the oocytes has deterministic value on fertilization and early embryonic development potentiaL? Summary answer The warmed oocytes with birefringent spindle seem to have a higher potential to fertilize and become good quality blastocytes. What is known already To date, characteristics of the meiotic spindles in human oocytes were studied by several authors by using a non-invasive method (PolScope) and shown that they are associated with fertilization, embryo quality, pregnancy, and live birth rates. Also, it has been known that meiotic spindle structures are very susceptible to cryodamage and can be disassembled and re-assembled depending on the change in temperature. Thus, studying spindle characteristics of the vitrified-warmed oocytes is important and can provide information about the full recovery of the oocytes, fertilization, embryo development, and pregnancy, which the data is limited in the literature. Study design, size, duration A retrospective study consisting 40 egg donation cycles by using vitrified-warmed oocytes between December 2017 and December 2019 at British Cyprus IVF Hospital. Participants/materials, setting, methods Spindle morphologies of vitrified-warmed oocytes were analyzed by PolScope (RI) combined with an image analysis system after approximately 1.5 hours post-warming. The data is divided into two according to the presence or absence of visible nucleus and correlation with embryonic development analyzed accordingly. To make a comparison in groups a new numerical scoring system for embryo quality on days 5 and 6 was created where embryos scored depending on the stage of expansion, Main results and the role of chance In total, 587 out of 648 vitrified/warmed oocytes survived (90.6%) and 447 of them were fertilized (75.0%). Among the survived oocytes, 503 (85.5%) oocytes were containing detectable spindle (Group I) while spindles were missing in 84 oocytes (Group II). The results showed that the fertilization rate (78.1% vs. 64.3%, p < 0.01), the proportion of good PN morphology (Z Score: Z1 and Z2 over other parameters: 50.4% vs 35.2%, p = 0.036), and viable embryo count (40.6% vs 15.1%, p < 0.01) which is defined as embryos that good enough to transfer or freezing, were higher in group I. Similarly, the degree of expansion on day 5/6 (p = 0.013, p = 0.015 respectively) and the quality of the cells in ICM and TE (p < 0.01) were found higher in group I. We further investigated the differences in embryo quality between groups by converting the embryo grades to numerical value starting from arrested (Score=0) to 6AA (Score=22) and found that embryo scores on day 5 (p = 0.01) and day 6 (p = 0.013) were higher in group I. Limitations, reasons for caution The main limitation of this study is the lack of data regarding the spindle status of the frozen oocytes before vitrification where some of the oocytes might not have visible spindle due to incomplete maturation rather than cryodamage. Wider implications of the findings The present data showed that the presence of birefringent meiotic spindles in warmed oocytes is a very important sign for a full recovery and early embryonic development potential, which can be useful for egg banks and IVF clinics. Trial registration number not applicable

Recipient outcomes in an oocyte donation programme: should very young donors be excluded?

Research questionDoes the oocyte donor's age affect live birth rate (LBR) in recipients? DesignRetrospective study of 3766 oocyte recipient cycles carried out between January 2009 and December 2018. Cycles were categorized into groups according to donor's age: <20 years (4.7%); 20–25 years (41.1%); ≥26 years (54.2%). Chi-squared test was used to evaluate differences in LBR and analysis of variance was used to test differences in embryo quality, and fertilization and embryo development rates. A generalized linear mixed model was applied to estimate the odds for each end point. ResultsLBR was 40.7%. When analysed according to donors’ age, significant differences were found: 33.9% for the youngest group, 39.1% for the group aged 20–25 years, and 42.5% for donors aged ≥26 years (P = 0.022). When adjusting for confounding factors (recipient age, number of transferred embryos and day of embryo transfer), LBR was lower in the group aged <20 years (OR 0.70; CI 95% 0.50 to 0.99) and in the group aged 20–25 years (OR 0.85; CI 95% 0.74 to 0.98) compared with the group aged ≥26 years. No significant differences were observed in fertilization rates (74.2%, 76.1% and 77.5%) or embryo development rates (57.0%, 61.4% and 62.0%). The number of good-quality embryos transferred was significantly lower in the group aged <20 years (1.03 ± 0.71; 1.18 ± 0.69; 1.19 ± 0.67; P = 0.015). ConclusionsLBR is significantly lower when donors are younger than 25 years and, especially, when they are younger than 20 years.

Sire contribution to fertilization failure and early embryo survival in cattle

Despite passing routine laboratory tests of semen quality, bulls used in artificial insemination (AI) exhibit a significant range in field fertility. The objective of this study was to determine whether subfertility in AI bulls is due to issues of sperm transport to the site of fertilization, fertilization failure, or failure of early embryo or conceptus development. In experiment 1, Holstein-Friesian bulls (3 high fertility, HF, and 3 low fertility, LF) were selected from the national population of AI bulls based on adjusted fertility scores from a minimum of 500 inseminations (HF: +4.37% and LF: -12.7%; mean = 0%). Superovulated beef heifers were blocked based on estimated number of follicles at the time of AI and inseminated with semen from HF or LF bulls (n = 3-4 heifers per bull; total 19 heifers). Following slaughter 7 d later, the number of corpora lutea was counted and the uteri were flushed. Recovered structures (oocytes/embryos) were classified according to developmental stage and stained with 4',6-diamidino-2-phenylindole to assess number of cells and accessory sperm. Overall recovery rate (total structures recovered/total corpora lutea) was 52.6% and was not different between groups. Mean (± standard error of the mean) number of embryos recovered per recipient was 8.7 ± 5.2 and 9.4 ± 5.5 for HF and LF, respectively. Overall fertilization rate of recovered structures was not different between groups. However, more embryos were at advanced stages of development (all blastocyst stages combined), reflected in a greater mean embryo cell number on d 7 for HF versus LF bulls. Number of accessory sperm was greater for embryos derived from HF than for LF bulls. The aim of experiment 2 was to evaluate the effect of sire fertility on survival of bovine embryos to d 15. Day 7 blastocysts were produced in vitro using semen from the same HF (n = 3) and LF (n = 3) bulls and transferred in groups of 5-10 to synchronized heifers (n = 7 heifers per bull; total 42 heifers). Conceptus recovery rate on d 15 was higher in HF (59.4%,) versus LF (45.0%). Mean length of recovered conceptuses for HF bulls was not affected by fertility status. In conclusion, while differences in field fertility among AI sires used in this study were not reflected in fertilization rate, differences in embryo quality were apparent as early as d 7. These differences likely contributed to the higher proportion of conceptuses surviving to d 15 in HF bulls.

Differences in embryo quality are associated with differences in oocyte composition: a proteomic study in inbred mice.

Current models of early mouse development assign roles to stochastic processes and epigenetic regulation, which are considered to be as influential as the genetic differences that exist between strains of the species Mus musculus. The aim of this study was to test whether mouse oocytes vary from each other in the abundance of gene products that could influence, prime, or even predetermine developmental trajectories and features of derivative embryos. Using the paradigm of inbred mouse strains, we quantified 2010 protein groups (SILAC LC-MS/MS) and 15205 transcripts (RNA deep sequencing) present simultaneously in oocytes of four strains tested (129/Sv, C57Bl/6J, C3H/HeN, DBA/2J). Oocytes differed according to donor strain in the abundance of catalytic and regulatory proteins, as confirmed for a subset (bromodomain adjacent to zinc finger domain, 1B [BAZ1B], heme oxygenase 1 [HMOX1], estrogen related receptor, beta [ESRRB]) via immunofluorescence in situ. Given a Pearson's r correlation coefficient of 0.18-0.20, the abundance of oocytic proteins could not be predicted from that of cognate mRNAs. Our results document that a prerequisite to generate embryo diversity, namely the different abundances of maternal proteins in oocytes, can be studied in the model of inbred mouse strains. Thus, we highlight the importance of proteomic quantifications in modern embryology. All MS data have been deposited in the ProteomeXchange with identifier PXD001059 (http://proteomecentral.proteomexchange.org/dataset/PXD001059).

137 EFFECTS OF X-SORTED SPERM IN QUALITY OF BOVINE BLASTOCYST DERIVED FROM IN VIVO-MATURED OOCYTES

Recently, we reported on a promising system for selecting healthy IVF embryos in cattle using kinetics of early embryo development and oxygen consumption of blastocyst [Sugimura et al. 2012 PLoS ONE 7, e36627]. The present study was conducted to examine the differences in embryo quality of bovine blastocysts obtained after IVF of in vivo-matured oocytes with X-sorted and unsorted sperm. Holstein dry cows (n = 8) were reared under the same feeding and environmental conditions. Two ovum pickup (OPU) sessions were conducted in each cow to fertilize with or without X-sorted sperm. In vivo-matured oocytes were collected by OPU just before ovulation after superstimulation treatment. The oocytes were inseminated with 5 × 106 sperm mL–1 of each sperm, and presumptive zygotes were cultured in CR1aa supplemented with 5% newborn calf serum and 0.25 mg mL–1 of linolenic acid albumin at 38.5 C in 5% CO2, 5% O2, and 90% N2 for 168 h. Embryo kinetics were observed individually using a microwell culture dish (Dai-Nippon Print) and time-lapse cinematography (CCM-1.4MZS; Astec, Fukuoka, Japan; Sugimura et al. 2010 Biol. Reprod. 83, 970–978). Photographs of each embryo were taken every 15 min during the in vitro culture period and images were analysed by CCM-1.4 software (Astec). By assessing the quality of blastocysts, a combination of identified prognostic factors were used: (1) timing of the first cleavage (less than 27 h post-insemination); (2) two blastomeres at the end of the first cleavage; (3) absence of fragments at the end of the first cleavage; and (4) six or more blastomeres at the onset of the lag-phase. Data were analysed by ANOVA. In total, 34.1 ± 18.4 oocytes per session per donor were collected by OPU, and 23.7 ± 13.4 oocytes had an expanded cumulus cell. Oocyte recovery rates were recorded at 77.1 ± 15.1%. After IVF and in vitro culture, 10.6 ± 7.7 blastocysts per session per donor were produced in this study. There was no significantly difference in cleavage rates and blastocyst formation rates between X-sorted sperm and unsorted sperm (87.1 ± 10.8 and 82.6 ± 12.1% and 38.4 ± 23.6 and 57.1 ± 23.4%, respectively). However, blastocysts derived from X-sorted sperm showed significantly (P &lt; 0.05) lower quality in the prognostic factor (1) and combined (1) to (4) than that in unsorted sperm (35.3 v. 54.0 and 14.7 v. 42.9%, respectively). Pregnancy rates were higher for the blastocysts that had a high score in the prognostic factors (1) to (4) compared to those that had a low score (75.0%, n = 8 v. 36.4%, n = 22). These results suggest that quality of blastocysts, based on the prognostic factors studied, derived from X-sorted sperm is lower than that from unsorted sperm. Supported by the Research and Development projects for application in promoting new policy of agriculture, forestry and fisheries (22016).

Can we improve implantation by cancellation of fresh embryo transfer?

Single embryo transfer is becoming increasingly popular in IVF/ICSI. More IVF/ICSI cycles therefore include freezing of high quality embryos, and the cumulative effect of such cycles becomes more important. To improve the results obtained using frozen–thawed embryos, the predictive value of embryo and patient characteristics such as ovarian reserve, hormone levels and age play an important role in both cases whether the women treated with oestradiol/progesterone or undergo natural cycle transfer. Although, embryo quality indicators revealed sometime morphologically and numerically inferior embryo cohorts after cryopreservation, the clinical pregnancy rate is higher in cycles using thawed embryos compared with fresh. Moreover, subsequent logistic regression analysis controlled for differences in embryo quality and revealed significantly greater probability of clinical pregnancy with thawed embryos when compared with fresh, suggesting a negative effect of ovarian stimulation on endometrial receptivity. The aim of this study is to discuss an idea of cancellation of a fresh embryo transfer and put on an alternative method which is the frozen thawed embryo.

Embryo quality and reproductive outcomes of spermatozoa selected by physiologic-ICSI or conventional ICSI in patients with kruger 4% normo-morphology

Spermatozoa bound to HA show significant reduction of DNA fragmentation and aneuploidy and could offer better results in IVF cycles. The aim of this study was to determine differences in embryo quality and reproductive outcomes in ICSI cycles with the sperm selected using Physiologic -ICSI (PICSI).

Evidence of impaired endometrial receptivity after ovarian stimulation for in vitro fertilization: a prospective randomized trial comparing fresh and frozen-thawed embryo transfers in high responders

Clinical pregnancy rates of 80% and 65% were observed in cycles using thawed and fresh embryos, respectively, although embryo quality indicators revealed morphologically and numerically inferior embryo cohorts after cryopreservation. Subsequent logistic regression analysis controlled for differences in embryo quality and revealed significantly greater probability of clinical pregnancy with thawed embryos when compared with fresh, suggesting a negative effect of ovarian stimulation on endometrial receptivity.

Influence of follicular fluid GDF9 and BMP15 on embryo quality

To evaluate the association between follicular fluid levels of propeptide and mature forms of growth differentiation factor (GDF) 9 and bone morphogenetic protein (BMP) 15 with subsequent oocyte and embryo quality. Prospective clinical study. University hospital. Eighty-one infertile patients who underwent in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI). The expression levels of the propeptide and mature forms of follicular fluid GDF9 and BMP15 were determined by western blot analysis. The levels of follicular fluid hormones (FSH, E2, and P) were measured with automated chemiluminescent enzyme immunoassays. The relationships between the levels of GDF9 and BMP15, hormones, oocyte maturation, and embryo quality. Mature GDF9 levels were significantly correlated with the nuclear maturation of oocytes. The mean mature GDF9 level was 4.87±0.60 in the high-embryo-quality group and 1.45±0.81 in the low-embryo-quality group. There were no statistically significant differences in embryo quality among the patients regarding propeptide GDF9 and BMP15 expression status. There was a negative correlation between follicular fluid levels of P and the mature form of GDF9. Higher mature GDF9 levels in the follicular fluid were significantly correlated with oocyte nuclear maturation and embryo quality.

A giant oocyte in a cohort of retrieved oocytes: does it have any effect on the in vitro fertilization cycle outcome?

To investigate an association between the presence of a giant oocyte and outcome for the cohort in in vitro fertilization (IVF). Retrospective study. Hospital-based academic medical center. IVF and IVF/intracytoplasmic sperm injection (ICSI) cycles having at least one giant oocyte (study group; n = 105), matched with cycles without a giant oocyte (external control group). A further subanalysis compared giant oocyte cycles with the next closest cycles within patients (internal control group; n = 65). Standard IVF protocols. Incidence of giant oocytes, fertilization rate, quality of cohort embryos, and pregnancy outcome. Out of 97,556 oocytes, 117 (0.12%) were giant. Compared with the external control group, cleavage in the study group was abnormal (increased percentage of 1-cell and 10-cell embryos and decreased percentage of 6-cell and 7-cell embryos), although there was no difference for percentage of embryos with perfect symmetry, fragmentation, or implantation, clinical pregnancy, or ongoing/delivered rate. The study group had two cases of pregnancy termination due to chromosomal abnormalities (4.4%) versus none in external controls. No striking differences in embryo quality existed between the study and internal control groups. A giant oocyte is associated with abnormal cleavage in cohort embryos, but not with implantation or pregnancy rates. Giant oocytes occur sporadically and appear not to reflect quality of remaining oocytes in the ovary.

Identifying real differences in live birth rates between HMG and rFSH in IVF

Fertility treatment strives for the delivery of a healthy live birth. Human menopausal gonadotrophin (HMG) and recombinant FSH (rFSH) are the two types of gonadotrophin currently used for ovarian stimulation in assisted reproduction treatments. Although both HMG and rFSH have been shown to be effective, a number of studies have examined whether a potential difference in clinical benefit or outcome exists between treatments. Unlike rFSH preparations, HMG contains both FSH and LH activity (in the form of LH and human chorionic gonadotrophin, which are short- and long-acting, respectively). The beneficial effect of exogenous LH activity has been investigated in the Menotrophin versus Recombinant FSH in-vitro Fertilisation Trial (MERiT), which revealed differences in embryo quality and endometrial receptivity between rFSH and highly purified HMG. Current evidence suggests that HMG provides significantly higher live birth rates than rFSH in women undergoing ovarian stimulation for in-vitro fertilization/intracytoplasmic sperm injection cycles using long gonadotrophin-releasing hormone agonist protocol. Further studies will continue to provide data with which to expand these findings and optimize the chances of achieving a live birth following assisted reproduction treatment.

Cessation of Gonadotropin-Releasing Hormone Antagonist on Triggering Day: An Alternative Method for Flexible Multiple-Dose Protocol

This study was performed to analyze retrospectively outcomes of stimulated in vitro fertilization (IVF) cycles where the gonadotropin-releasing hormone (GnRH) antagonist was omitted on ovulation triggering day. A total of 92 consecutive IVF cycles were included in 65 women who are undergoing ovarian stimulation with recombinant FSH. A GnRH antagonist, cetrorelix 0.25 mg/day, was started when leading follicle reached 14 mm in diameter until the day of hCG administration (Group A, 66 cycles) or until the day before hCG administration (Group B, 26 cycles). The duration of ovarian stimulation, total dose of gonadotropins, serum estradiol levels on hCG administration day, and the number of oocytes retrieved were not significantly different between the two groups. The total dose of GnRH antagonist was significantly lower in Group B compared to Group A (2.7±0.8 vs. 3.2±0.9 ampoules). There was no premature luteinization in the subjects. The proportion of mature oocytes (71.4% vs. 61.7%) and fertilization rate of mature (86.3±19.7% vs. 71.8±31.7%) was significantly higher in Group B. There were no significant differences in embryo quality and clinical pregnancy rates. Our results suggest that cessation of the GnRH antagonist on the day of hCG administration during a flexible multiple-dose protocol could reduce the total dose of GnRH antagonist without compromising IVF results.

The effect of strain of Holstein–Friesian cow on size of ovarian structures, periovulatory circulating steroid concentrations, and embryo quality following superovulation

When managed under grass-based systems of production, the New Zealand (NZ) strain of Holstein–Friesian cow has superior reproductive performance compared to the North American (NA) strain despite having similar solids-corrected milk (SCM) yields. This study compared the ontogeny of early pregnancy events in NZ and NA cows. Ten NZ and 10 NA cows were submitted to a superovulation protocol on three occasions. Blood samples were collected daily from every cow from days −3 to +7 relative to a synchronized oestrus during each superovulation protocol. Pre-ovulatory oestradiol concentrations, follicle diameter, post-ovulatory progesterone concentrations, corpus luteum (CL) diameter, and circulating insulin-like growth factor-I concentrations did not differ between the two strains. Uteri were non-surgically flushed 7 days post-AI, embryos were isolated and graded. The proportion of transferable embryos recovered was higher ( P < 0.01) in the NZ cows compared with the NA cows. A greater ( P = 0.01) proportion of the recovered structures were at the blastocyst stage in the NZ cows. Peak SCM yield and body condition score (BCS) at the time of peak SCM yield were not different between strains. However, during the experimental period the NA cows maintained significantly higher daily SCM yields, whereas the NZ cows replenished significantly greater levels of BCS. The results indicate that differences in periovulatory steroid concentrations and size of ovarian structures do not explain the differences in embryo quality between the two strains. However, strain differences in nutrient partitioning from the time of peak SCM yield through late lactation may provide the key signals responsible for superior embryo quality in NZ cows.

Asian Ethnicity Associated With Decreased Live Birth Rate After Blastocyst Transfer

Background: Ethnic differences in ART outcomes have been reported but are poorly understood. Data from a national registry showed that Asian patients had a lower pregnancy rate than caucasian patients, even when controlling for baseline characteristics. Earlier studies do not control for differences in embryo quality which may account for differences in implantation. Patients undergoing embryo transfer on day 5 have more information available about embryo quality than patients with cleavage-stage embryo transfers.

A prospective study, using sibling oocytes, examining the effect of 30 seconds versus 90 minutes gamete co-incubation in IVF

Traditionally oocytes have been exposed to sperm overnight, for 16-20 h. This long period of co-incubation, however, has been shown to create problems with high levels of reactive oxygen species (ROS), which may affect embryo viability and cause hardening of the zona pellucida. Recently, a positive effect of reducing the co-incubation time to 90-120 min was reported. The objective of this study was to evaluate whether a further reduction of the co-incubation period could benefit the outcome of IVF. In this prospective study, 777 sibling oocytes from 81 women undergoing IVF were divided via alternate allocation to co-incubation for either 30 s (ultrashort co-incubation) (group A) or for 90 min (standard co-incubation) (group B). Endpoints were normal fertilization (two-pronuclear, 2PN), polyspermy (>2PN), embryo quality (EQ), clinical pregnancy (CP) and implantation (IR). The normal fertilization rates of the two groups were comparable: group A 58.6% versus group B 58.0%. Significantly lower rates of polyspermy were seen in group A compared to group B (2.8 versus 7.2%, P = 0.008). No statistically significant differences in EQ, CP or IR were seen. This is the first study demonstrating the achievement of good fertilization rates in IVF with ultrashort co-incubation. Significantly lower rates of polyspermy were seen in the group with ultrashort compared to the standard co-incubation group. Further studies are, however, needed in order to evaluate whether ultrashort co-incubation has any effect on the outcome of IVF.

Developmental Potential of Early-Cleaved Embryos Associated With Higher Rates of Blastocyst Formation and Utilization for Cryopreservation

The most important question for every embryologist is which method is reliable to predict the most implantation competent embryo for transfer. Early cleavage of embryos to the 2-cell stage is defined as cleavage occurring between 25-27 h post-insemination. Recent studies indicate that embryos with a high degree of implantation competence can be determined at the onset of embryonic development as early as the zygote stage. Embryos that cleave faster, have better morphology, less fragmentation, and reach the blastocyst stage more often. Furthermore, early cleaved embryos give rise to better blastocyst quality. To prove the high implantation competence of early cleaved embryos we associated and proved their “high quality” with further embryonic development such as progression to the blastocyst stage. Furthermore, using more stringent criteria to substantiate the quality of these embryos, we tracked their suitability and utilization for cryopreservation as a landmark for their final quality assessment. A retrospective analysis examining 1000 early cleaving embryos for their developmental potential up to blastocyst formation and utilization for cryopreservation. 5235 normal fertilized oocytes (2pn stage) from 387 patients (average age of 32.5) with equal or more than 8 normal fertilized oocytes were included in this study. Oocytes had undergone ICSI and were cultured in Sage Cleavage media and changed over on day 3 in to Sage Blastocyst media. ICSI was routinely performed between 1 p.m. and 2 p.m. (Day 0). On the following day (Day 1) between 3 p.m. and 4 p.m., the embryos were examined to see if early cleavage to the 2-cell stage had occurred. Embryos that had cleaved were marked as “early cleavers” (EC), whereas embryos that had not cleaved at this time were designated as “non early cleavers” (NEC). Finally, we examined and compared the subsequent progression of the EC and NEC embryos to the blastocyst stage and their suitability for cryopreservation. Chi square test was used for statistical analysis of the data. The mean age of both patient populations was 32.5 years. About 19% (1000/5235 normal fertilized oocytes) cleaved to the 2-cell stage between 25-27 h post-insemination. Furthermore, EC developed at a significantly higher percentage to the blastocyst stage compared with NEC (53.0% vs. 17.0%, p<.001). The higher quality of early-cleaved embryos compared with NEC was also reflected in the utilization rate for cryopreservation (60.6% vs. 36.3%, p<.001). Tabled 1 Our data confirms significant differences in embryo quality between early and non early-cleaved embryos. Furthermore, early-cleaved embryos progressed to the blastocyst stage at a higher rate, and formed higher quality blastocysts suitable for cryopreservation. Blastocyst quality is always associated with the success of blastocyst transfer. Finally, early cleavage is a embryonic marker of normality/quality of an embryo and ends with a high portion of good quality blastocyst.

The incidence of apoptosis after IVF with morphologically abnormal bovine spermatozoa

Normal embryonic development depends on the maintenance of a population of normal healthy cells within each embryo. The aim of this study was to use a combination of apoptotic measures to assess differences in embryo quality after IVF with semen samples with high percentages of abnormal spermatozoa. Semen samples were obtained and cryopreserved from four Holstein bulls before (5 day prior) and after (2 week-post-insult; 2 week-PI and day 20; 3 week-PI) a scrotal insulation period of 48 h (day 0). The swim-up sperm separation method was used. The post-thaw morphology revealed a decrease ( P < 0.01) in the percentages of normal spermatozoa in the 3 week-PI samples in comparison with the pre-insult samples for Bulls I and III (74–22.3 and 67.7–0.5%, respectively). The percentage of vacuolated spermatozoa increased significantly for Bull II. After 18 h of sperm-oocyte co-incubation, zygotes were cultured and subpopulations were removed from culture at day 8 and subjected to either the TUNEL or caspase assay. On day 8, caspase intensity increased significantly for both Bull I (217 ± 147) and Bull III (229 ± 98) for the 3 week-PI embryo groups compared to the equivalent embryo groups for Bull II (98 ± 115) and Bull IV (90 ± 111). In conclusion, the inability to consistently measure apoptosis with TUNEL alone complicated the assessment of differences in embryo quality. Thus, it is uncertain exactly when during early pre-implantation development the differences in embryo quality are first manifest. Despite discrepancies, our results clearly indicated a difference in the embryo quality between embryos obtained after IVF with semen samples from bulls that had an intense response to scrotal insulation.

Comparison of four media types during 3-day human IVF embryo culture

The aim of this study was to compare the effectiveness of human tubal fluid (HTF), G1.2®, Sage Cleavage® and Life Global® media for IVF outcome during 3-day culture of human embryos. A three-phase auto-controlled study was conducted in which IVF outcome was compared between (1) HTF and G1.2, (2) HTF and Cleavage, and (3) Cleavage and Life Global. In phase 1, no differences in embryo quality were observed between HTF and G1.2. However, embryos derived from intracytoplasmic sperm injection (ICSI) displayed significantly improved quality when grown in HTF versus G1.2. No differences in pregnancy and implantation rates were observed in cases where embryos transferred were grown exclusively in HTF or G1.2 media. In phase 2, embryo quality was significantly improved for embryos cultured in Cleavage versus HTF media ( P < 0.001). However, pregnancy, implantation and spontaneous abortion rates were similar between the two media. In phase 3, there were no differences in embryo quality, pregnancy, implantation, and spontaneous abortion rates between Cleavage and Life Global media. Overall, the data indicate that Life Global and Cleavage media yield similar results in a 3-day IVF culture programme. Cleavage medium is superior to HTF, as evidenced by significantly improved embryo quality ( P < 0.001). Meanwhile, HTF medium is superior to G1.2 for ICSI cases.

162 EMBRYO QUALITY AND COLOR IN HOLSTEIN FRIESIAN HEIFERS AND COWS IN RELATION TO SERUM PARAMETERS

Bovine embryo freezability is closely linked with quality and differs between breeds (Visintin JA et al. 2002 Theriogenology 57, 345–359). Dark embryos have a poor cryotolerance resulting in low pregnancy rates (Hill BR and Kuehner LF 1998 Theriogenology 49, 168), and are characterized by excessive accumulation of lipid droplets which in turn may be influenced by the biochemical composition of the embryonic environment. Earlier work revealed that Holstein Friesian (HF) cows yield significantly darker embryos compared to Belgian Blue beef cows (Leroy JLMR et al. 2004 Reprod. Fert. Dev. 16, 211). In this field examine we aimed to study the differences in embryo quality and color between HF cows (producing milk) and heifers (non-producing) in relation to four serum parameters that have previously been linked with embryo quality and fertility: urea, total protein (TP), total cholesterol (TC), and triglycerides (TG). Blood samples were collected from HF cows (n = 54) and HF heifers (n = 33) prior to embryo flushing on Day 7 after superovulation and subsequent insemination. Serum was stored frozen until assay with commercial photometric assays. Embryos were scored individually by the same operator for quality (excellent to poor: score 1 to 4) and color: light (L), medium (M), or dark (D) using a binocular stereomicroscope (40×). Independent Student's t-test and chi-square test were used when appropriate. Heifers and cows yielded on average (± SEM) 5.1 ± 0.82 and 6.1 ± 0.71 embryos per flushing, respectively. Significantly more cow embryos were classified as dark compared to heifer embryos (L, M, D: 20.4%, 55.5% and 24.1% vs. 70.8%, 27.4%, and 1.8%, for cow and heifer embryos, respectively) (P &lt; 0.05). Significantly more heifer embryos showed an excellent morphological quality (62.5% of heifer embryos compared to 13.1% of cow embryos) (P &lt; 0.05). Serum concentrations (± SEM) of urea (4.5 ± 0.2 vs. 2.8 ± 0.2 mM), TP (7.59 ± 0.07 vs. 6.57 ± 0.09 g/dL) and TC (183 ± 5.3 vs. 105 ± 3.8 mg/dL) were significantly higher and serum concentrations of TG (17.2 ± 0.6 vs. 23.8 ± 0.9 mg/dL) significantly lower in cows compared to heifers (P &lt; 0.05). In this study, embryo color (i.e. lipid content) and quality as well as urea, TP, TC, and TG serum concentrations were clearly influenced by the production parameter (cow = producing vs. heifer = non-producing). These findings imply that producing milk (parity = 0 or &gt;0) may be an important confounder. Therefore, this variable should be taken into account when studying multiple variables influencing embryo quality or color simultaneously. Further analyses are ongoing to reveal which factors at the cow level are affecting the embryo quality and color. This research was funded by IWT-Flanders (Grant no. 13236).