Transferable Quality Embryos Research Articles

Stress and anxiety on embryo transfer day do not affect in vitro fertilization (IVF) outcome

To evaluate whether stress and/or anxiety, at the moment of embryo transfer, affect IVF outcome. Prospective cohort study. Eighty five women undergoing IVF/ICSI (intracytoplasmic sperm injection) treatment, up to 35 years-old and with good prognosis for conception (between 5 and 20 oocytes recovered, sperm collected by masturbation and at least one good quality embryo for transfer) were selected. As measuring instruments for stress we used Lipp Inventory of Stress and a salivary cortisol assay. To evaluate anxiety we used IDATE State Anxiety Inventory. Inventories were applied a few minutes before embryo transfer. Saliva samples for cortisol assay were collected right after embryo transfer. Statistical analysis of salivary cortisol results was done using generalized linear model with distribution gamma and link identity. The analysis of anxiety and stress inventories were done using student test for means of two populations with equal variance and chi-squared test, respectively. Five patients (5,8%) presented with high anxiety levels. Sixteen patients (18,8%) presented stress symptoms and 15 patients (17,6%) had salivary cortisol levels above reference values. However, no statistical differences were found in salivary cortisol averages between patients who did and did not get pregnant (P=0,846). No statistical differences were found between the state of anxiety and the occurrence of pregnancy (P=0,695), nor between the presence of stress and the occurrence of pregnancy (P=1,000). Anxiety and stress, at the moment of embryo transfer, do not seem to interfere on pregnancy rates in IVF/ICSI cycles. These results could help patients to overcome the feeling of guilt related to stress and anxiety, very common in these treatments.

311 SUPEROVULATION OF NORTH AMERICAN BISON WITH TWO INJECTIONS OF FOLLICLE-STIMULATING HORMONE

Few studies have examined superovulation of North American Bison. In cattle, ovarian superstimulation is usually achieved with 6 to 8 injections of FSH at half-day intervals. However, handling bison repeatedly stresses the animals, which could adversely affect their reproductive potential, as well as pose a risk of injury to the bison and handlers. To limit the number of times the bison were handled, we tested a two-injection superovulation scheme using sodium hyaluronate (MAP-5, Bioniche Animal Health Inc., Belleville, Ontario, Canada) in the FSH diluent, which serves to slow the absorption of FSH when given IM. We hypothesised that the two-injection superstimulation protocol would result in recovery of more embryos on average than a single-embryo recovery protocol. Although a traditional superovulation scheme with 6 to 8 injections of FSH would have been a better comparison, our goal was to handle the bison minimally. Eight female bison ranging in age from 5 to 11 years were used as embryo donors. For superovulation, females with a corpus luteum (CL) were given 25 mg of prostaglandin F2α (PGF, Lutalyse, Pfizer Animal Health, New York, NY, USA) IM followed by 266 mg of FSH (Folltropin V in MAP-5 diluent, i.m., Bioniche Animal Health Inc.) 12 days later (or 9 days after presumed oestrus 3 days post-PGF). Forty-eight hours after the first FSH injection, 134 mg of FSH IM and 25 mg of PGF IM were given. Two days later females were put in a pen with a bison bull for natural breeding. Seven days after assumed oestrus, embryos were recovered nonsurgically. Although the situation is not clear in bison, there is evidence in cattle that superovulated cycles influence embryo collections in subsequent cycles. Consequently, females were randomly assigned to a superovulation or single-embryo recovery treatment for each cycle; however, consecutive superovulation protocols were never conducted without a short oestrous cycle in between. Superovulated females were given PGF at embryo recovery after superovulation, followed by PGF 12 to 14 days later, and bred off the assumed oestrus of the second PGF injection. Embryos were collected from females 4 times (2 superovulation and 2 single embryo cycles, except one bison that was superovulated once). Data were analysed using a one-tailed t-test. Superovulation resulted in greater mean numbers of palpable CL (3.7; P < 0.001), embryos collected (1.8; P < 0.05), and transferable quality embryos (0.8; P < 0.05) compared with the single-embryo recovery protocol (mean palpable CL, 1.0; embryos collected, 0.5; transferable embryos, 0.2). Notably, the bison breeding season is July to September and occasionally animals breed in October; this experiment was conducted in October and November. Thirteen transferable embryos were nonsurgically transferred to recipients; 6 pregnancies were established, but 5 were resorbing by 2 months of gestation; 1 healthy calf was carried to term. Two injections of FSH with a long-acting diluent can be used to increase the number of embryos recovered from bison compared with a single-embryo recovery scheme.

The Effect of Local Injury to the Endometrium for Implantation and Pregnancy Rates in ICSI –ET Cycles with Implantation Failure: a randomised controlled study

To assess the effect of local injury to the endometrium for implantation, pregnancy and live birth rates in ICSI –ET cycles with recurrent implantation failure. The study was prospectively designed and performed in a university hospital. A group of 100 women, who failed to conceive during one or more cycles of IVF and embryo transfer (ET), was treated with a long protocol for controlled ovarian hyperstimulation. The IVF treatment and ET were preceded by repeated endometrial biopsies, in a randomly selected 50 of a total of 100 women. Age of the women, days of stimulation, total dose of gonadotropins, E2 concentrations and endometrial thickness on hCG day, total MII stage oocytes, the total number of high quality embryos for transfer, the day of transfer and the number of embryos transferred were similar for both groups. Transfer of a similar number of embryos (138 and 141 in the experimental and control patients, respectively) resulted in rates of implantation (34.67 % vs. 30.88 %; p= 0,1384) clinical pregnancy (60 % vs. 34 %; p= 0.009), and live births per ET (44 % vs. 24 %; p= 0.03), which were higher in the experimental group as compared to controls. These results suggest that IVF treatment that is preceded by local injury to the endometrium improved the rates of embryo implantation, clinical pregnancy, and live births in asiste reproductive technique.

The clinical need for a method of identification of embryos destined to become a blastocyst in assisted reproductive technology cycles

To provide a rationale for continuation of efforts to improve the outcome of Assisted Reproductive Technology outcomes, thereby increasing the likelihood of the live birth of healthy neonates. Description of rationale and a framework leading to improvement in Assisted Reproductive Technology outcomes. The opportunity for improvement in the success rate for Assisted Reproductive Technology outcome is predicated on selection of the highest quality embryo(s) for transfer. However, such approaches must be balanced by a limit to the number of embryos transferred so as to reduce the risk for multiple births and particularly higher order multiple gestations. Blastocyst transfer offers one such successful approach, but is confounded by suggestions of an increased risk of both pregnancy complications and epigenetic disorders. There is a need for development of approaches which, individually or in combination, may assist in the early detection of embryos destined to develop into blastocysts.

228 DOMINANT FOLLICLE ABLATION IMPROVES SUPEROVULATORY RESPONSE AND EMBRYO YIELD IN BUFFALOES SUBJECTED TO INTEGRATED SINGLE-OVULATION EMBRYO TRANSFER - MULTIPLE-OVULATION EMBRYO TRANSFER PROGRAM

Effect of the removal of inhibitory consequences of a dominant follicle (DF) on the recruitment, growth and ovulation of follicles in cyclic buffaloes induced to superovulate, was evaluated. Eight elite, multiparous, donor Murrah buffaloes, 80 to 120 days postpartum, were included in an integrated single-ovulation embryo transfer–multiple-ovulation embryo transfer (SOET-MOET) schedule. Superovulation with FSH (Folltropin® NIH-FSH-P1, Bioniche Animal Health, Belleville, ON, Canada) was preceded by nonsurgical single embryo (SOET) recovery on Day 5 or 6 after prostaglandin F2α (Lutalyse®, Pfizer, Madison, NJ, USA)-induced oestrus and AI (Day 0). For MOET, Folltropin was administered from Day 9 p.m. through to Day 13 p.m. in a twice-daily descending dose schedule (0–5, 5–4, 4–3, 3–2, 2–1 mL; total dose of 580 mg of NIH-FSH-P1). A dose of 25 mg of Lutalyse was administered on Day 12 a.m. and p.m. Donors were inseminated with proven semen on Day 14 a.m. and p.m. and Day 15 a.m. Nonsurgical embryo collection was carried out on Day 19 or 20 followed by administration of Lutalyse on Day 25. Following sexual rest for one complete oestrous cycle, Day 5 or 6 SOET was repeated. On Day 9, ultrasound-guided follicle aspiration was used to ablate the DF and the second MOET treatment was initiated at the same time. Mean diameter of DF at the start of superovulation treatment on Day 9 was similar between the first (12.5 ± 0.66 mm) and second (12.2 ± 0.33 mm) MOET cycles. At AI, mean number (9.6 ± 1.43 vs 13.4 ± 1.87) of follicles >9 mm were higher (P < 0.05) in the second (ablation) MOET cycle, though their mean diameters were similar (11.7 ± 0.21 vs 12.3 ± 0.21 mm; P = 0.17). On the day of ova/embryo collection, almost similar numbers of large follicles (4.1 ± 0.93 and 4.4 ± 0.84), with mean diameters of 12.9 ± 0.74 and 13.4 ± 0.83 mm, were observed for the first and second MOET, respectively. Mean (± SEM) numbers of corpora lutea (3.9 ± 0.81 and 4.9 ± 0.61; P = 0.19) and transferable quality embryos (0.6 ± 0.37 and 1.6 ± 0.37; P = 0.077) were counted in the first and second (ablation) MOET cycles, respectively. These preliminary findings suggest that ablation of DF before start of superovulatory treatment may be beneficial in improving the yield of transferable embryos in a buffalo MOET program. Director, CIRB, is acknowledged for the experimental facilities.

269 THE USE OF A NEW EXTENDER FOR STORING FRESH BOVINE SEMEN FOR LONG PERIODS

Processing bovine semen in fresh long life extender for use over 3 to 4 days after collection is a widely used technique in New Zealand (Shannon and Vishwanath 1995 Anim. Reprod. Sci. 39, 1-10). Advantages include greater use of valuable sires, transport without liquid nitrogen, and the possibility of more efficient use of sexed sorted semen. The new extender (Ext. A) also has the advantage of containing no egg yolk. This study compares this new long-life extender (Ext. A) with an existing product (Ext. B) and frozen/thawed semen. Semen from 12 different bulls was diluted to a concentration of 8 × 106 mL-1 and gradually cooled to 16°C. All samples were held at ambient temperature in the dark and motility was evaluated over a storage period of 4 days comparing the extenders. In this part of the trial Ext. A maintained motility better than Ext. B (P = 0.001) during the 4-day storage period (24 h: 90 v. 70%; 96 h: 85 v. 50%). The second part of the trial compared the conception rates (CR) in cows from the use of fresh long-term-extended semen and frozen/thawed semen. On 19 farms, 8546 cows were inseminated with fresh semen stored for 1 to 3 days and 7280 cows were inseminated with frozen semen. The overall CR at 7 to 8 weeks for the 19 farms was 73.7%. On 18 farms within the same farming group, 8498 cows received frozen semen and the CR was 71.1%. Pregnancy results were 2.6% (P = 0.001) higher CR at scanning in herds where fresh semen was used compared with the farms where only frozen/thawed semen was used (73.7 v. 71.1%). In the third part of the trial, semen from 4 different bulls were extended to 1 × 106 mL-1 in Ext. A and held at ambient temperature for 6 days prior to use for IVF. Our lab standard frozen/thawed bull semen was used as a control. Table 1 shows that semen held at ambient temperature in Ext. A for 6 days produced a similar percentage of transferable quality embryos to our IVP control frozen/thawed semen (26.9 v. 25.7%). We conclude that preserved bovine semen in fresh long-life extender for several days offers some advantages in AI and IVP programs compared with frozen semen. Table 1.Fresh semen extender (Ext.A) compared with frozen semen in IVP We appreciate the assistance of Liberty Genetics Ltd.

7 THE EFFECT OF A NOVEL RECOMBINANT PROTEASE TREATMENT ON BOVINE SPERM FERTILIZING CAPACITY AND EMBRYO DEVELOPMENT IN VITRO

As described in the IETS Manual (Stringfellow and Seidel, 1995), and endorsed by the OIE, trypsin can be used (for specific pathogens and livestock) to effectively remove certain infectious agents from in vivo-derived embryos for international transport. Because of the multimillion-dollar AI industry for livestock, the OIE has encouraged more research in developing similar decontamination techniques for semen as an added safeguard to animal quarantine for the prevention of disease transmission. Most or all of the earlier studies on embryos used a porcine pancreatic-derived trypsin. Because of more stringent guidelines from international regulatory agencies on the use of animal products, several serine protease recombinants are now available. Previous experiments comparing the porcine pancreatic extract with a recombinant bovine sequence trypsin developed in corn resulted in no statistical difference in cleavage or morula/blastocyst rates. (Mattson et al. 2008 Theriogenology 69, 724–727). An additional in vivo study treating bovine sperm with a yeast-derived human-sequence trypsin resulted in significantly more transferable-quality embryos after the AI of superovulated cows as compared with sperm not treated with trypsin (Blevins et al. 2008 Reprod. Fertil. Dev. 20, 84). The goal of this experiment was to examine the in vitro development of bovine embryos produced from sperm treated with a recombinant trypsin found in a commercially available density gradient centrifugation (DGC) product (Bovipure, Nidacon, Sweden) compared with DGC without trypsin. Oocyte aspiration, maturation, fertilization, and embryo culture were performed using standard methods in 5 replications (n = 2220 oocytes). Semen was collected and pooled from 2 Bos taurus bulls and frozen in an egg-yolk cryodiluent (Biladyl, Minitube). The semen was processed using Bovipure DGC composed of 2 mL of 40% colloid of silane-coated silica particles containing either a yeast-derived human sequence recombinant trypsin containing no animal by-products (n = 1126 oocytes) or the same colloid without trypsin as the control group (n = 1094 oocytes). Both 40% concentrations were layered over 2 mL of an 80% concentration of the same colloid without any additives. The density gradients were centrifuged at 300g for 20 min, after which time the pellets were washed in 5 mL of prewarmed TL Hepes solution (Cambrex) and centrifuged at 500g for 10 min. The resulting sperm pellets were then resuspended in a volume calculated to provide 1 × 106 sperm mL–1, to be used for in vitro inseminations. Results were compared using a 2-tailed unpaired t-test. Cleavage rates for the trypsin-treated sperm (n = 969, 35.8%) and the control (n = 950, 44.3%) groups were not statistically different (P = 0.20). Although more embryos reached the morula to blastocyst stages in the control group (n = 421, 61.0%) than in the trypsinized group (n = 347, 54.7%), these differences also were not statistically significant (P = 0.85). In conclusion, trypsinized Bovipure DGC of sperm before insemination showed no detrimental effects on IVF-derived bovine embryo development.

48 TREATMENT OF CLONED BOVINE EMBRYOS WITH HISTONE DEACETYLASE INHIBITORS INCREASES IN VITRO DEVELOPMENT BUT NOT IN VIVO CLONING EFFICIENCY

Previous studies in the mouse have shown treatment of somatic cell nuclear transfer (SCNT) embryos with histone deacetylase inhibitors (HDACi) to significantly increase cloning efficiency (Kishigami S et al. 2006 BBRC 340, 183–189; van Thuan N 2007 Asian Reproductive Biology Society 4, 9 abst). Increasing histone acetylation may open donor chromatin allowing better access for oocyte cytoplasmic factors to facilitate reprogramming. Here, we determined the effect of two HDACi, Trichostatin A (TSA), and scriptaid (Sigma-Aldrich, Castle Hill, NSW, Australia), on bovine cloning efficiency. Zona-free SCNT was performed with serum starved fibroblasts fused to enucleated MII-arrested IVM oocytes. After 4 h, reconstructs were activated with 5 μm ionomycin and 2 mm 6-dimethylaminopurine (DMAP) and cultured individually in 5 μL drops of AgResearch synthetic oviduct fluid (SOF) medium. Treatment with HDACi commenced concomitant with the 4 h DMAP incubation and continued in SOF for the remainder of the treatment period; totalling either 18 or 48 h post activation (hpa). TSA concentrations examined were: 0, 5, 50, and 500 nm, with all treatments containing 0.5% DMSO (n = 1121). Following TSA treatment, increased histone (H) acetylation at lysine (K) of H4K5 was confirmed by semi-quantitative immunofluorescence at the eight-cell stage. Scriptaid concentrations examined were: 0, 5, 50, 250, and 1000 nm, with all treatments containing 0.5% DMSO during DMAP and 0.1% DMSO during IVC (n = 1059). In vitro development on Day 7 was expressed in terms of transferable quality embryos as a percentage of reconstructs cultured. Data were analyzed using a generalized linear model with binomial variation and logit link. Embryos from selected treatments were transferred singularly to recipient cows on Day 7 with pregnancy data analyzed using Fisher’s exact test. Day 7 in vitro development was significantly greater with 5 nm TSA treatment for 18 hpa compared to controls (47.1% v. 34.5%; P < 0.02). Treatment of embryos with TSA for 48 hpa had no effect at any concentration tested. In contrast, scriptaid treatment for 18 hpa had no effect in vitro, while exposure for 48 hpa at 1000 nm significantly increased the development of transferable quality embryos compared to 0 nm (44.0% v. 32.4%; P < 0.005). There was no significant difference in embryo survival rates at D150 of gestation between embryos treated with 0 or 5 nm TSA for 18 hpa (8/48 v. 10/48; 16.7% v. 20.8%). However, in vivo development at Day 150 of gestation following treatment of embryos with 1000 nm scriptaid for 48 hpa was significantly lower compared to controls (1/37 v. 6/31; 2.7% v. 19.4%; P < 0.05). Contrary to the mouse, TSA or scriptaid treatment as used in this study did not increase cloning efficiency in cattle. The use of various HDACi either alone or in combination with DNA demethylating agents may still prove beneficial for reprogramming following nuclear transfer. Supported by FRST C10X0303.

Effect of patient's age on the criteria for day 5 embryo transfers

OBJECTIVE: Criteria are used to determine when patients should have a Day 3 (D3) or Day 5 (D5) embryo transfer (ET) with most dependent on the no. of embryos available on D3. Patients who meet the criteria have a D5 ET allowing for the transfer of potentially better quality embryos. Our program's criteria for a D5 transfer has been ≥ 4 good quality embryos at ≥7-cell stage on D3, and has been used regardless of the age of the patient. The objective of this study was to determine the effect of patient age on D5 ET results.DESIGN: Retrospective data analysis.MATERIALS AND METHODS: Data was analyzed on IVF patients < 41 years of age with ≥ 4 embryos on D3 who received an ET from 2006 and 2007. Patients were divided into two age groups: ≤ 34 years and patients 35 to 40 years. Patients were again divided into 1) those that did not meet the criteria for extended culture and had a D3 ET and 2) those that met the criteria and underwent a D5 ET. The variables analyzed included no. of embryos, no. embryos transferred, implantation (imp.) and clinical pregnancy rate (PR).Tabled 1Day 3 TransfersDay 5 TransfersPMean No. 2PN6.59.50.0001Mean No. Embryos Transferred3.12.10.0001Implantation (%)66/300 (22)33/112 (29)NSClinical Pregnancy (%)47/96 (48)21/52 (40)NS Open table in a new tab CONCLUSIONS: Our study found patients ≥ 35 years may not benefit from a D5 transfer using our current criteria. This may be due to the lower number of embryos transferred on D5. Rather than increasing the number of embryos that are transferred on D5 and risk increasing the rate of multiple pregnancies, our program has amended our D5 ET protocol for patients ≥ 35 to be ≥ 6 good quality embryos on the morning of D3. To date, this change has resulted in an increased rate of both imp.and PR by allowing us to select better quality embryos for transfer on both Days 3 and 5. OBJECTIVE: Criteria are used to determine when patients should have a Day 3 (D3) or Day 5 (D5) embryo transfer (ET) with most dependent on the no. of embryos available on D3. Patients who meet the criteria have a D5 ET allowing for the transfer of potentially better quality embryos. Our program's criteria for a D5 transfer has been ≥ 4 good quality embryos at ≥7-cell stage on D3, and has been used regardless of the age of the patient. The objective of this study was to determine the effect of patient age on D5 ET results. DESIGN: Retrospective data analysis. MATERIALS AND METHODS: Data was analyzed on IVF patients < 41 years of age with ≥ 4 embryos on D3 who received an ET from 2006 and 2007. Patients were divided into two age groups: ≤ 34 years and patients 35 to 40 years. Patients were again divided into 1) those that did not meet the criteria for extended culture and had a D3 ET and 2) those that met the criteria and underwent a D5 ET. The variables analyzed included no. of embryos, no. embryos transferred, implantation (imp.) and clinical pregnancy rate (PR). CONCLUSIONS: Our study found patients ≥ 35 years may not benefit from a D5 transfer using our current criteria. This may be due to the lower number of embryos transferred on D5. Rather than increasing the number of embryos that are transferred on D5 and risk increasing the rate of multiple pregnancies, our program has amended our D5 ET protocol for patients ≥ 35 to be ≥ 6 good quality embryos on the morning of D3. To date, this change has resulted in an increased rate of both imp.and PR by allowing us to select better quality embryos for transfer on both Days 3 and 5.

Technical Note: Effect of density gradient centrifugation with trypsin on the in vivo fertilising capability of bovine spermatozoa

The present study investigated the effect of a novel density gradient centrifugation (DGC) treatment using recombinant trypsin on the in vivo fertilising capability of bovine spermatozoa compared with a standard method. In Trial 1, semen collected from Boran and Ankole (Bos indicus) bulls was treated either with a silane-coated silica particle colloid formulated for humans with a recombinant trypsin or processed using a standard method (dilution in an egg yolk-based diluent). Semen processed by the two methods was used to artificially inseminate (AI) superovulated cattle. Day 7 embryos were flushed and assessed for fertilisation rates and embryo quality. Trial 2 used a trypsinised silane-coated silica particle colloid formulated specifically for bovine semen. Trial 1 resulted in significantly higher fertilisation rates using the trypsinised human DGC treatment than cows inseminated using the standard method (75.2% v. 67%, respectively; P < 0.01), but the numbers of transferable-quality Day 7 embryos did not differ between the two groups (P > 0.05). Results for Trial 2 indicated that cows inseminated with the trypsinised bovine DGC treatment had significantly increased fertilisation rates compared with the standard method (88.4% v. 63.1%, respectively; P < 0.01) and had significantly higher numbers of transferable-quality embryos (70.3% v. 51.8%, respectively; P < 0.01). In summary, bovine sperm treatment before AI by DGC and recombinant trypsin increases fertilisation rates and can result in more transferable-quality embryos compared with standard methods.

Male factor infertility, low fertilisation rate following ICSI and low number of high‐quality embryos are associated with high order recurrent implantation failure in young IVF patientsa

The aim of this study was to characterise the clinical and laboratory parameters in young in vitro fertilisation(IVF) patients with high order recurrent implantation failure (RIF). The first 3 cycles (n = 141 cycles) of 47 consecutive (age <35 years) couples with RIF (> or =6 IVF cycles) were compared with 252 consecutive cycles of 152 couples,treated during the same period, who conceived within the first 3 IVF cycles. The incidence of male factor infertility or of combined male and female factors was significantly higher in the study group than the control group (male factor: 78.7 versus 42.5%, p= 0.002, OR = 3.2 (95% CI: 1.56.8)). Lower fertilisation rates were noted for the intracytoplasmic sperm injection (ICSI)-treated oocytes of the study group compared with the control group. The availability of high quality embryos for transfer was significantly lower in the RIF group. On multivariate logistic regression analysis, the significantly different variables between the groups were cause of infertility (male factor or combined male and female factors) (p = 0.03),fertilisation rate (p = 0.038), and semen concentration (p = 0.05). CONCLUSIONS; Young IVF patients with high order RIF are characterised by male factor infertility, lower fertilisation rate with ICSI, and fewer high quality embryos available for transfer.

Are IVF patients with an increased body mass index less likely to have good quality embryos?

To determine whether a rising BMI has a negative effect on embryo quality based on the amount of embryos available for cryopreservation after selection for transfer. Each IVF cycle will ideally result in a cohort of 1–2 good quality embryos for transfer with excess embryos for cryopreservation. Factors that may influence embryo quality include the amount of stimulating drug administered as well as the length of time the patient is exposed to these medications. Patients with a high BMI require both increased dosage and increased time on medication because of the impaired response of the ovaries to gonadotropin therapy. The purpose of this study was to test the hypothesis that there will be a gradual decline in number of embryos cryopreserved as BMI increased. Retrospective analysis of patients undergoing IVF/ICSI from January 2004–December 2006. All PGD, donor egg, patients with a BMI > 40.0, age > 42 and patients with embryos cryopreserved for reasons other than good quality after ET were excluded. 1870 patients were analyzed between January 2004 and December of 2006. BMI was defined as: underweight <20, normal 20–24.9, overweight 25–29.9, obese class I 30–34.9 and obese class II 35–39.9 (WHO criteria). Chi square was used for statistical analysis. The number of good quality embryos available for cryopreservation after ET selection did not gradually decrease as patients BMI increases. Although not significant, there was a decrease from the obese class I category (28%) to obese class II (19%). The number of 7–8 cell embryos on day 3 did not decrease with a rising BMI. There was a significant decrease from the obese class I to obese class II (P<0.01). Also, a significant decrease in implantation rate from the normal weight group to the overweight group (P<0.001). TableBMI and embryo quality<19.920–24.925–29.930–34.935–39.9% embryos cryopreserved2926252819% 7–8 cell383639∗4034∗∗% Implantation rate252415♦1512% SAB44446∗ P≤0.025 ∗∗P≤0.01♦P≤0.001. Open table in a new tab ∗ P≤0.025 ∗∗P≤0.01♦P≤0.001. There was a significant decrease in the % of 7–8 cells from obese class I to obese class II, although, there was no significance in the amount of cryopreservation, suggesting that embryo quality was not negatively effected by a rise in BMI. A rise in BMI was correlated with decreased implantation potential and increased pregnancy loss. Further studies are needed to determine the cause of pregnancy loss and implantation failure in obese patients.

7 EFFECT OF DENSITY GRADIENT CENTRIFUGATION WITH TRYPSIN ON THE FERTILIZING CAPABILITY OF BOVINE SPERM

The goal of this research was to investigate the effect of a novel density gradient centrifugation (DGC) treatment on the fertilizing capability of bovine sperm as compared to a standard method. Domestic bull (Bos taurus) semen was used for AI and the production of embryos from in vivo-matured bovine oocytes. In 2004, a preliminary study compared the novel semen treatment using a trypsinized PVP-coated silica particle suspension (Percoll; Sigma, St. Louis, MO, USA) to a standard method (4� dilution with an egg yolk diluent) on the fertilizing capacity in vivo of bovine sperm (de la Rey et al. 2005 J. Reprod. Fertil. 17, 242 abst). Although not statistically significant (P = 0.69), there were more transferable quality embryos recovered from cows inseminated using the treated sperm method v. control (58.9 v. 43%, respectively). In this report we provide the results of two additional trials utilizing the novel semen treatment and substituting Percoll with a silane-coated silica particle medium containing a recombinant trypsin (r-protease). In the second trial (2005), semen samples collected from three bulls were processed by DGC: 2 mL of 40% PureSperm (NidaCon International AB, M�lndal, Sweden) containing recombinant trypsin (TrypLE Select, Gibco/Invitrogen, Carlsbad, CA, USA), which overlaid 2 mL of 80% PureSperm containing 10 µg mL–1 soy-based protease inhibitor (Sigma), was overlaid with the semen sample using a novel centrifuge tube insert (ProInsert, Nidacon) and then centrifuged at 300g for 20 min. The sperm pellets were recovered and washed (500g for 10 min) in 10 mL pre-warmed TL-HEPES medium (Cambrex Corp., East Rutherford, NJ, USA). The washed sperm pellets were then resuspended in the same total volume of pre-warmed Biladyl� A (Minit�b, Tiefenbach, Germany) as the standard method and used to AI a total of 42 (control) and 47 (treatment) superovulated cows three times at 12 h intervals. Day 7 embryos were recovered and assessed for stage and morphological quality. In Trial 3 (2006), semen samples collected from three bulls were processed by DGC containing r-protease and a soybean protease inhibitor (BoviPure Pro, NidaCon), media specifically formulated for domestic bull semen. Sperm pellets were washed in 10 mL BoviWash medium (NidaCon). The washed sperm pellet was resuspended in the same total volume of pre-warmed Biladyl A as the standard method and used to AI a total of 23 (control) and 25 (treatment) superovulated cows and embryos evaluated as in Trial 2. The results between control and treated groups were compared using the Mann-Whitney (Wixcoxon rank sum) test. Trial 2 using PureSperm tended to result in higher fertilization rates than for cows inseminated using the standard method (75.2% v. 67%, respectively) but the results were not statistically significant (P = 0.63). Results for Trial 3 indicated that cows inseminated with BoviPure Pro-treated sperm had significantly increased fertilization rates as compared to the standard method (88.4% v. 63.1%, respectively; P = 0.02) and had higher numbers of transferable quality embryos (70.3% v. 51.8%, respectively; P = 0.38). In summary, BoviPure Pro sperm treatment before AI significantly increases fertilization rates and can result in as much as an 18.5% increase in transferable quality embryos as compared to standard methods.

Birth after double cryopreservation of human oocytes at metaphase II and pronuclear stages

To document the possibility to double-cryopreserve human metaphase II and pronuclear oocytes. Case report. University-based IVF center. A couple with primary andrological infertility. Pronuclear stage oocytes derived after sperm injection into frozen-thawed metaphase II oocytes were cryopreserved again for later transfer. Post-thaw survival rates and pregnancy. Five metaphase II oocytes survived the freeze-thaw procedure. Three of five oocytes presented with two pronuclei after sperm injection and were cryopreserved again. Post-thawing, all three oocytes developed into good quality embryos for transfer. A singleton pregnancy was achieved, resulting in the birth of a healthy girl. Double-cryopreservation of oocytes at metaphase II and at pronuclear stage is a treatment option in human-assisted reproduction.

Comparison of Preimplantation Genetic Diagnosis-Aneuploidy Screening (PGD-AS) With 5 Chromosome Versus 9/10 Chromosome Panels

The purpose of this study was to evaluate the percent of embryos found to be normal using a 5 chromosome panel (PGD5) vs. a 9/10 chromosome panel (PGD9/10) out of total number of embryos biopsied in IVF cycles employing PGD-AS, and the effect of using PGD 5 vs. PGD 9/10 on pregnancy rates. A retrospective analysis of 268 patients undergoing IVF with PGD-AS from September 2003 to May 2004. The medical records were reviewed for age, number of embryos biopsied, number of normal embryos per PGD-AS, number of embryos transferred, and pregnancy outcome including twinning/higher order multiple pregnancies (HOMP) for 4 physicians with similar stimulation protocols. 51 patients met inclusion criteria. Student t-test for two samples and chi-square test were used for statistical analyses. The data for the two groups is shown in Table 1. The mean age, number of total embryos biopsied, and number of embryos transferred were similar between the two groups. The PGD5 group had a higher absolute number and percent of normal embryos diagnosed by PGD (p<0.005, p<0.0001). The pregnancy rate was higher in the PGD5 group, but this did not reach significance. The patients able to conceive (PGD5 or PGD9/10) had a significantly higher number of normal embryos as diagnosed by PGD (4.0 ± 2.2 vs. 2.0 ± 0.97, p<0.0005) and a higher number of embryos transferred (2.5 ± 0.7 vs. 1.9 ± 0.7, p<0.005) as compared to those unable to conceive. One set of quadruplets and two sets of twins were noted in the PGD5 group and one set of quadruplets and three sets of twins in the PGD 9/10 group. Table 1 Tabled 1 A trend towards higher pregnancy rates was noted in the PGD5 group. This may indicate that with an increased number of chromosomes analyzed there is a greater decrease in specificity. A higher number of false-positive abnormal embryos may result in fewer embryos available for transfer. This may lead to transfer of only the remaining available embryos (9/10 chromosome panel) vs. providing a choice to select the best quality embryos for transfer (5 chromosome panel). The comparison of pregnant to non-pregnant women showed a higher number of normal embryos available on day of transfer and a higher number of embryos transferred (perhaps with higher quality as well). The implantation rate was higher in the PGD5 group. This confirms the higher quality of embryos transferred using the 5 chromosome panel vs. the 9/10 chromosome panel. The twinning /HOMP rate was similar in the PGD5 vs. the PGD9/10 group, failing to show an adverse effect of increase in number of embryos transferred. Therefore, PGD using a 9/10 chromosome panel offers little added value over a 5 chromosome panel with a potential adverse effect on pregnancy rates.

Postthaw Embryo Compaction is Predictive of the Success of Frozen-Thawed Transfer

Although cryopreservation of supernumerary embryos has increased the overall success rate in human IVF-ET, the survival and implantation rate of frozen-thawed embryos is directly related to the quality of embryos at the time of freezing. Overnight incubation improves selection of good quality embryos for transfer. Studies showed that there was a ∼45% implantation rate for day 4 embryos after fresh morula ET. This study is designed to investigate the predictors of postthaw embryo compaction and the relationship to the outcome of frozen-thawed embryo transfer (FET) cycles.

330 UTILIZING PRE-IMPLANTATION GENETIC DIAGNOSIS AND OPU-IVP-ET TO GENERATE MULTIPLE PROGENY OF PREDETERMINED GENOTYPE FROM CLONED TRANSGENIC HEIFERS

Advanced reproductive technologies, comprising ovum pickup (OPU), in vitro embryo production (IVP), and embryo transfer (ET), were used to rapidly generate multiple F1 offspring from nine hemizygous founder-cloned transgenic (TG) heifers that highly express casein protein in their milk (Brophy B et al. 2003 Nat. Biotech. 21, 157–62). Moreover, embryo biopsy and genotyping were used to determine gender and presence or absence of the additional casein genes prior to ET. This minimized the number of surplus animals that would otherwise be generated from the breeding program. The aim was to generate 10 TG female (TG-F), 10 non-TG female (non-TG-F), two TG male (TG-M), and two non-TG male (non-TG-M) offspring. Some hemizygous TG bulls were required to enable the generation of homozygous transgenic animals in the next generation, while non-TG cattle would serve as controls in future studies. Transvaginal OPU was performed on non-stimulated TG heifers twice weekly for 6 weeks. Selected oocytes were matured, then fertilized in vitro (Day 0) with sperm from a conventional sire, and cultured to Day 6 (Thompson JG et al. 2000 J. Reprod. Fert. 118, 47–55). Transferable quality embryos were biopsied and cultured singularly for another 20 h, by which time genotyping results were available. On Day 7, individual embryos of the desired genotype were identified and transferred nonsurgically to synchronized recipients. Two biopsy methods were used, dependent on the stage of the embryo. For blastocysts, 10–12 trophectoderm cells were biopsied using an ultra-sharp splitting blade mounted on a micromanipulator (Herr CM et al. 1991 Theriogenology 35, 45–54). For morulae, the zonae pellucidae were enzymatically removed and two blastomeres per embryo aspirated with a blunt 30-μm micropipette (Chrenek P et al. 2001 Theriogenology 55, 1071–81). Aliquots either from the lysed blastocyst biopsy samples or following primer extension pre-amplification of DNA (Zhang L et al. 1992 Proc. Natl. Acad. Sci. USA 89, 5847–51) from blastomere samples were used to determine sex and casein transgene status using multiplex PCR. Following the OPU program, the TG heifers were artificially inseminated with semen from the same sire as used for IVF; eight calved (89%). Data were compared using Fisher's exact test. The mean number of transferable Day 6 embryos produced per donor per OPU session was 1.3. Pregnancy rates following OPU-IVP-ET on Day 35 were 27/62 (44%) with a calving rate of 29%. Nine TG-F, five non-TG-F, three TG-M, and one non-TG-M were delivered vaginally at term. All calves born were of the predicted genotype. There was no significant difference in embryonic survival to term either between TG and non-TG embryos transferred, or between biopsy methods. One OPU calf died shortly after birth; the other 17 survived to weaning, as did all the AI calves. The results show that pre-implantation genetic diagnosis combined with OPU-IVP-ET can be successfully used to produce small scale-up herds from founder TG females.

186 A NOVEL METHOD FOR ELIMINATING PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS FROM BOAR SEMEN AND ITS EFFECTS ON EMBRYO DEVELOPMENT

Porcine reproductive and respiratory syndrome virus (PRRSv) is known to cause venereal transmission of the disease via natural or artificial breeding and this constitutes a significant risk to AI programs in modern swine production. The objectives of this study were to determine the effectiveness of a novel density gradient centrifugation method incorporating trypsin to eliminate PRRSv from infected semen and to evaluate its effects on sperm viability and embryo development. Exp. 1: To assess the efficacy of the procedure on eliminating PRRSv, semen was collected from 21 infected boars. Concentrated sperm (1 mL) was layered on three Percoll (Sigma, St. Louis, MO, USA) density gradient columns, top to bottom: 1 mL 30%, 2 mL 45% with or without 0.25% trypsin (trypsin-treated and control, respectively), and 2 mL 90% with or without 10 μg/mL soy-based trypsin inhibitor (Sigma), and centrifuged (700g for 30 min). Trypsin-treated and control sperm were submitted for RT-PCR analysis pre- and post-treatment. Exp.2: To evaluate the effect on sperm quality, semen samples (n = 10) were collected from non-infected boars and processed as described in Exp. 1. Sperm motility, viability, and acrosomal integrity were evaluated at 0 and 2 h post-treatment. Exp. 3.1: To assess the in vitro fertilizing capability of trypsin-treated sperm, in vitro-matured porcine oocytes (n = 64) were inseminated, and cleavage (48 h post-insemination (PI)) and blastulation (144 h PI) rates were compared to those of oocytes (n = 63) inseminated with control sperm. Exp. 3.2: Trypsin-treated or control sperm (3 × 109/dose) were used to AI sows (n = 10). In vivo-generated embryos were surgically recovered 4–6 d post-AI, and embryo number, stage, and morphological quality were recorded. Data were analyzed using ANOVA and differences were considered significant at P &lt; 0.05. Sperm quality parameters are expressed as means ± SEM. Results showed that the procedure (with and without trypsin) was effective for eliminating PRRSv from infected boar semen. There were no differences at 0 or 2 h post-treatment between the control and the trypsin-treated boar sperm in motility (76 ± 4.9 and 56 ± 8.7 vs. 75 ± 4.4 and 48 ± 8.3%, respectively), viability (87 ± 2.6 and 75 ± 6.2 vs. 81 ± 3.2 and 80 ± 3.7%, respectively), and acrosomal integrity (96 ± 2.7 and 98.8 ± 0.8 vs. 98 ± 1.3 and 99 ± 0.4%, respectively). There was no difference between the control and trypsin-treated sperm used for IVF on cleavage (82 vs. 89%, respectively) and blastulation (20 vs. 32%, respectively) rates. There were significantly more transferable-quality embryos recovered from sows inseminated with trypsin-treated as compared to control sperm: 54/63 (85.7%) vs. 35/72 (48.6%), respectively. In conclusion, the novel trypsin gradient treatment was effective in eliminating PRRSv without detrimentally affecting sperm quality and has the potential to increase the numbers of transferable-quality embryos produced.

184 THE EFFECT OF A NOVEL SEMEN DISINFECTION TREATMENT ON THE VIABILITY AND FERTILIZING CAPACITY IN VIVO OF BOVINE SPERMATOZOA

The goal of this investigation was to determine the effect of a novel procedure, consisting of an initial incubation in antibiotic cocktail followed by centrifugation through a trypsinized Percoll gradient, on the viability and fertilizing capacity in vivo of bovine sperm as compared to standard processing methods. Exp 1: Semen collected by electroejaculation from 12 bulls were aliquoted into four treatment groups: (1) control (standard method) using Biladyl A (Minitübe, Tiefenbach, Germany) at a 1:4 (semen:diluent) dilution; (2) antibiotic cocktail (gentamicin, spectinomycin, lincomycin, tylosin, and kanamycin at 250, 300, 150, 200, and 1000 μg/mL, respectively) at a 1:9 (semen:cocktail) dilution and incubation (38°C) for 15 min; (3) trypsinized Percoll (Sigma, St. Louis MO, USA) gradient treatment by layering 1 mL semen on the top of a 90, 45, and 30% Percoll (bottom to top) gradient, with the 90 and 30% segments of the column containing 10 μg/mL soy-based trypsin inactivator (Sigma) and the 45% segment containing 0.25% trypsin (Sigma), and then centrifuging at 700g for 30 min; and (4) a combination of treatments 2 and 3 by concentrating the sperm by gentle centrifugation (300g for 10 min) after incubation in antibiotic cocktail, and then layering 1 mL of the sperm suspension on the Percoll gradients. In treatments 2–4, the treated sperm pellets were resuspended in Biladyl A (1:4), and all treatments were refrigerated (4°C) and examined at 0, 24, 48, and 72 hours for progressive motility, viability, and acrosomal integrity. The results were analyzed statistically using the Student's t-test (P &lt; 0.05). Exp 2: Semen samples collected from two bulls were treated with either the control method or the combination antibiotic cocktail and trypsinized Percoll methods (1 and 4 above), and then used to AI a total of 5 (control) and 6 (combined treatment) superovulated cows three times at 12-h intervals. Day 7 embryos were recovered and assessed for stage and morphological quality. The results for Exp 1 indicated that sperm treated with either the trypsinized Percoll alone or in combination with the antibiotic cocktail had significantly higher (P &lt; 0.05) progressive motility than the control at 0 (87.5 and 89.2 vs. 75.4%), 24 (87.1 and 87.9 vs. 73.2%), 48 (87.1 and 86.7 vs. 70.7%), and 72 h (82.1 and 80.4 vs. 64.6%, all respectively) post-treatment. Likewise, treated sperm had significantly greater viable and acrosome-intact sperm than the control at 0 to 72 h post-treatment. Although not statistically significant, there were more transferable-quality embryos recovered from cows inseminated using sperm treated with the combined method as compared to the control (58.9 vs. 43%, respectively). In conclusion, the trypsinized Percoll gradient method for processing bovine semen (alone or in combination with antibiotic cocktail) improved the quality of bovine sperm refrigerated for up to 72 h and had no detrimental affect on the number of transferable-quality embryos collected after AI.

The outcome of in vitro fertilization (IVF) treatment in patients who electively inseminate ≤ 4 oocytes

The option of cryopreserving excess embryos allows the opportunity to inseminate all oocytes retrieved, thereby increasing the probability of obtaining a large number of embryos. This offers the advantage of being able to select the best quality embryos for transfer, from a large embryo cohort pool, potentially enhancing the probability of conception. In our program, patients who elect not to cryopreserve, have the number of eggs inseminated limited to ≤ 4. This results in a limited number of embryos, which prevents the selection of the best quality embryos for transfer from a large embryo cohort pool.