Initial Pregnancy Rate Research Articles

P-545 Early pregnancy outcomes associated with age and AMH following Next-Generation Sequencing(NGS)-based Preimplantation Genetic Testing for Aneuploidy(PGT-A) in single euploid embryo transfers for advanced age patients

Abstract Study question Does age and AMH impact early pregnancy outcomes in euploid single embryo transfers using NGS-based PGT-A for individuals aged 35 and above? Summary answer In this NGS-based PGT-A study, no significant differences in early pregnancy rates were observed based on age or AMH among individuals aged 35 and above. What is known already A recent meta-analysis suggest a decline in pregnancy rates with advancing age in the context of euploid embryo transfer following PGT-A. However, it’s crucial to acknowledge variations in PGT-A techniques across studies. While individuals with low AMH levels may encounter challenges in obtaining euploid embryos, the precise impact of AMH on pregnancy rates post-embryo acquisition remains unclear. Study design, size, duration A retrospective cohort study was conducted on 428 cycles of single euploid embryo transfers in patients aged 35 and above, performed at the single infertility center from January to December 2022. Participants/materials, setting, methods The study examined frozen-thawed embryo transfer cycles in patients aged 35 and above, analyzing early pregnancy outcomes in single euploid embryo transfers. Participants were grouped by age(35-38, 38-42, 42 and above) and AMH levels(<1, 1-3, >3 ng/ml). NGS-based PGT-A conducted embryo biopsy. The analysis aimed to detect differences in initial pregnancy rates among age and AMH subgroups. The differences between groups were compared using Pearson’s chi-squared test. Main results and the role of chance The average age at transfer was 39.05 ± 2.53 years, with a mean AMH of 2.60 ± 1.86(ng/ml). The mean number of prior frozen embryo transfer (FET) cycles was 1.9 ± 2.04, with 119 patients (27.8%) having undergone three or more transfer cycles. Transfer cycles comprised 363 (84.8%) artificial cycles and 65 (15.2%) ovulatory cycles, with an average endometrial thickness of 8.95 ± 1.73 mm at the time of transfer. The β-hCG positive rate was 57.7% (247/428), and the clinical pregnancy rate was 53.5% (229/428). When analyzing positive b-hCG results by age, rates were 59.9% (82/137) for ages 35-38, 55.5% (121/218) for ages 38-42, and 60.3% (44/73) for ages 42 and above (p = 0.641). In terms of AMH, β-hCG positive rates were 52.1% (37/71) for <1, 59.1% (120/203) for 1-3, and 58.0% (83/143) for ≥3, with no statistically significant differences (p = 0.584). Clinical pregnancy rates were 54.0% for ages 35-38, 51.8% for ages 38-42, and 57.5% for ages 42 and above. For AMH, rates were 50.7% for <1, 53.2% for 1-3, and 54.5% for ≥3, with no statistically significant differences (p = 0.869). Limitations, reasons for caution Being a retrospective study analyzing patients undergoing embryo transfer, our study overlooks variables related to oocyte retrieval. Given the challenges of obtaining euploid embryos in advanced age, further investigation is warranted to explore the probability and duration of embryo acquisition in this population. Wider implications of the findings In this study, utilizing NGS-based PGT-A after generating euploid embryos, no significant differences in early pregnancy outcomes were observed for women aged 35 and older, considering age and AMH levels. The study suggests further research is needed to explore if NGS-based PGT-A can address limitations of traditional chromosomal testing methods. Trial registration number not applicable

Single closed-tube quantitative real-time PCR assay with dual-labelled probes for improved sex determination of equine embryos

In addition to fulfilling many breeders’ curiosity, equine embryonic sex determination can have a profound commercial impact. However, the application of currently described assays for equine embryonic sexing has rendered variable diagnosis and validation rates, with sensitivity being the main problem. In addition, while pregnancy results of in vivo-flushed equine embryos following a needle aspiration biopsy equal those of non-biopsied embryos, the effect on in vitro-produced embryos is unknown. Here, we aimed to develop a highly sensitive and specific assay for equine sex determination that can be directly performed on few embryonic cells, and to test the effect of a needle aspiration biopsy on the viability of the in vitro-produced embryo. To this end, a multiplex quantitative real-time PCR (qPCR) assay with dual-labelled probes was designed to allow the simultaneous generation of both male-specific and control fragments in a single closed-tube reaction, avoiding potential sample loss or contamination. To improve sensitivity, multicopy and polymeric genes were chosen to be specifically amplified, i.e., eight copies of Y-chromosomal ETSTY5 as male-specific and four autosomal UBC monomers as control fragment. Specificity was enhanced by the equine-specific character of ETSTY5 and by using dual-labelled probes. The assay was optimised with equine male and female genomic DNA and demonstrated a 100% accuracy and a >95% qPCR efficiency down to 10 pg of DNA. The assay was subsequently applied to determine the sex of 44 in vitro-produced embryos, collecting trophectoderm biopsies by means of a needle aspiration biopsy and herniating cells. Of all trophectoderm biopsies and herniating cell samples (n = 54), 87% could be diagnosed. Assay results were validated on a second sample obtained from the biopsied embryo (n = 18) or, by ultrasound-based sex determination of the foetus (n = 7) following the transfer of the biopsied embryo to a recipient mare, with about half of the embryos being fillies and colts. The needle aspiration biopsy procedure did not impair initial pregnancy rate or early pregnancy losses as compared to non-biopsied embryos. In conclusion, we report a safe, reliable, fast, and cost-effective assay for equine sex determination which was validated for the sex determination of in vitro-produced embryos based on few embryonic cells, and needle aspiration biopsy did not impair the embryo's viability. The assay and safe biopsy strategy hold potential for other applications.

A drop in serum progesterone from oocyte pick-up +3 days to +5 days in fresh blastocyst transfer, using hCG-trigger and standard luteal support, is associated with lower ongoing pregnancy rates.

A drop in serum progesterone from oocyte pick-up +3 days to +5 days in fresh blastocyst transfer, using hCG-trigger and standard luteal support, is associated with lower ongoing pregnancy rates.

1 Effect of cytokine-supplemented maturation medium on bovine somatic cell nuclear transfer embryo development

Invitro maturation is an important process in the invitro production of embryos. It has been shown recently that 3 cytokines: fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1), increase the efficiency of IVM, blastocyst production, and invivo development following somatic cell nuclear transfer (SCNT) in pigs (Yuan et al. 2017 PNAS 114, E5796-E5804). This study was designed to assess the effect of these cytokines on IVM in bovine oocytes, their consecutive development to blastocyst, and pregnancy rate when used in SCNT. Cumulus–oocyte complexes (COCs) were retrieved from abattoir-derived ovaries, matured for 21h in either standard maturation medium [TCM-199 (Gibco/Life Technologies), 10% fetal bovine serum, 0.5µgmL−1 FSH, 5µgmL−1 LH, 100UmL−1 penicillin/streptomycin] or maturation medium supplemented with 20ngmL−1 human LIF, 20ngmL−1 IGF1, and 40ngmL−1 FGF2. After IVM, the first polar body and metaphase plate were removed from MII oocytes. Fibroblast cells were injected in the perivitelline space and fused with enucleated oocytes in 0.28M sorbitol fusion medium (0.1mM calcium, 0.5mM magnesium, 0.5mM HEPES, 1gmL−1 bovine serum albumin) by a single pulse of 1.75 kV/cm for 22ms. Reconstructed embryos were activated by exposure to 5μM ionomycin for 5min, followed by 4h of incubation in 2mM 6-(dimethylamino)purine and 10μgmL−1 cycloheximide. Cleavage and blastocyst rates were assessed at Day 2 and Day 7, respectively. Blastocysts were transferred to oestrus synchronized recipients and initial pregnancy rates assessed at Day 40 after embryo transfer. Statistical analysis was performed using one-way ANOVA or chi-squared test. Data are presented as mean±s.e.m. The MII rate was significantly higher in maturation medium supplemented with cytokines compared with control medium (80.2±2.33%, n=885 vs. 66.8±1.82%, n=822; P<0.05, 7 replicates, one-way ANOVA). No statistical difference was found in the cleavage rate of SCNT embryos between treatment and control groups (94.2±1.34%, n=259 vs. 90.9±1.22%, n=208; P>0.05, 8 replicates, one-way ANOVA), respectively. However, a significant increase in blastocyst rate was observed in the treatment group compared with the control group (40.6±5.1%, n=446 vs. 24.3±2.9%, n=300; P<0.05, 8 replicates, one-way ANOVA). SCNT embryos derived from the treatment group also resulted in a significant increase in initial pregnancy rates (50.3±20.9%, n=48 vs. 29.0±20.6%, n=31; P<0.05, 4 replicates, chi-squared). Full-term pregnancy rates are pending. In conclusion, addition of FGF2, LIF, and IGF1 to maturation medium improves bovine IVM and SCNT blastocyst development and initial pregnancy rates. The effect on full-term pregnancy success is yet to be determined. This research was supported by UAES project (1343) and RFBR (18-29-07089).

Effect of warming method on embryo quality in a simplified equine embryo vitrification system

Equine embryo vitrification is still not a well-established technique in equine practice. Notably, little work has been done on the effect of the warming system on viability of vitrified embryos. Our goal was to evaluate the effect of warming without cryoprotectants on in vitro - produced (IVP) embryo viability in culture, quality assessment parameters, and pregnancy after transfer. Equine IVP blastocysts were vitrified using commercial embryo vitrification media and a semi-closed vitrification device. In Exp. 1, we evaluated two warming temperatures (room temperature, RT, ∼22 °C; and 38 °C) for each of three warming systems: commercial warming solution (Kit); commercial embryo holding medium (EHM) with decreasing concentrations of sucrose (EHM + SS); or EHM alone without added sucrose. Embryos (n = 9 to 14 per treatment) were cultured in vitro for 24 h, stained with DAPI, TUNEL, and fluorophore-labelled phalloidin, and evaluated for nucleus number, mitotic rate, apoptotic rate, and actin filament distribution. In Exp. 2, to survey embryo viability in vivo, vitrified IVP blastocysts were shipped to an embryo transfer facility, then warmed immediately before transfer to recipient mares, using the warming treatments associated with the nominally best (Kit-RT, Kit-38, EHM-RT) and poorest (EHM + SS-38) assessed embryo quality in Exp. 1 (n = 7 to 8 per treatment). Subsequently, IVP blastocysts produced as part of our clinical program were vitrified and shipped, then warmed in embryo holding medium at an embryo transfer facility before transfer to recipient mares; fresh IVP embryos were shipped and transferred as controls. In Exp. 1, embryos increased significantly in diameter after culture (P < 0.01), with no difference among treatments. There was no difference (P > 0.05) in the number of viable nuclei, apoptotic rate, or microfilament distribution among treatments, or between vitrified-warmed and Control embryos. The mitotic rate was higher (P = 0.021) for Kit-RT (3.6%) when compared with the other treatment groups (1.5–2.0%). In Exp. 2, there was no difference (P > 0.05) in initial pregnancy (71.4–87.5%) or heartbeat (57.1%–85.7%) rates among warming treatments. In the clinical trial, there was no difference (P > 0.05) between vitrified-warmed and Control embryos in initial pregnancy (90.9% and 66.6%, respectively) or heartbeat (81.8% and 66.6%, respectively) rates. These results indicate that a semi-closed vitrification system using commercially-available media, and incorporating warming in the field in a single step using commercial embryo holding medium without cryoprotectants, can provide high pregnancy rates with IVP equine embryos.

Survival, re-expansion, and pregnancy outcome following vitrification of dromedary camel cloned blastocysts: A possible role of vitrification in improving clone pregnancy rate by weeding out poor competent embryos

There is a clinical demand for efficient cryopreservation of cloned camel embryos with considerable logistic and economic advantage. Vitrification of in vivo derived embryos has been reported in camels, but there is no study on vitrification of cloned embryos. Moreover, whether characteristic differences between cloned and in vivo derived embryos imply different vitrification requirement is unresolved. Here, we compared survival, re-expansion and pregnancy rates of cloned embryos vitrified using two commercial vitrification kits (Cryotec and Kitazato), developed basically for human embryos, and a vitrification protocol developed for in vivo camel embryos (CVP). Cloned embryos responded dynamically to vitrification-warming steps in commercial kits, with a flat shrinkage in the final vitrification solution and a quick re-expansion to the original volume immediately after transferring to the isotonic warming solution. Contrarily, full shrinkage was not observed in CVP method, and majority of embryos were still collapsed post-warming. The immediate re-expansion was highly associated and predictive of higher survival and total cell number, and also better redox state of embryos vitrified by Cryotec and Kitazato kits compared to CVP method. Importantly, while 30% blastomere loss, verified by differential dye exclusion test, was tolerated in vitrified embryos, >50% blastomeres loss in non-expanded blastocysts implied the minimal essential cell survival rate for blastocoelic cavity re-expansion in vitrified cloned camel blastocysts, irrespective of vitrification method. A protocol-based exposure of embryos to cryoprotectants indicated that cryoprotectant toxicity, per se, may not be involved in lower cryosurvival of embryos in CVP vs. Cryotec and Kitazato. The initial pregnancy rates were numerically higher in Cryotec and Kitazato frozen transfers compared to fresh transfer (56.3, 60 and 33.3%, respectively), and importantly, a higher percentage of established pregnancies in vitrified groups passed the critical 3 months period of early embryonic loss compared to sibling fresh clone pregnancies (50, 40, and 10%, respectively). Results confirmed the suitability of Cryotec and Kitazato kits for vitrification of cloned camel embryos and that vitrification may improve pregnancy outcome by weeding out poor competent embryos.

Case study

The objective of this field study was to measure the effect of implementing a simple systematic reproduction management program in dairy herds with reproductive performance near or below typical in the industry. Thirty-nine herds across Canada that had annual herd 21-day pregnancy rates (PR) between 8 and 15%, and did not have a systematic reproduction program, were enrolled in a program that consisted of: enrollment of cows that were not inseminated by 70 days-in-milk into a timed insemination program (Ovsynch); enrollment of cows diagnosed not pregnant on Ovsynch; and a change to biweekly veterinary visits for reproductive management. Annual herd PR was compared one year after implementation of the program. On average, the systematic management program was associated with a mean increase in PR of 3.6 percentage points (95% confidence interval, 2.5 to 4. 7; P&lt;0.0001), accounting for initial insemination and conception rates, herd size and method of pregnancy diagnosis. Assuming a milk price of $23/cwt to reflect Canadian milk price net of purchase of quota, economic analyses indicated that herds that increased PR by at least two points were estimated to have an annual net profit improvement between $20 and $250 CDN per cow, depending on the initial PR and the magnitude of the increase in PR. Overall, 72 to 77% of herds that implemented the program were estimated to have a net economic benefit.

The effect of foot-and-mouth disease vaccination on early pregnancy loss in beef heifers in Argentina

The threat of Foot and Mouth Disease (FMD) in South America has global economic implications and retaining a FMD Free status under the World Organization for Animal Health (OIE) remains a top priority. In Argentina the Servicio Nacional de Sanidad y Calidad Agroalimentaria (SENASA), the national service of agri-food health and quality, requires cattle located in the Pampean region of the Salado River basin to receive two foot-and-mouth disease (FMD) vaccinations per year, which results in one vaccination coinciding with beef cattle breeding season. While the vaccination program remains necessary, there is a growing concern amongst food animal veterinarians, that the overlap of FMD vaccination with the first 35 days of the breeding season is associated with early pregnancy loss (EPL). To address this concern, a preliminary randomized controlled trial t study was conducted to investigate the risk ratio (RR) of EPL in vaccinated, pregnant Aberdeen Angus heifers. Initially (Day 0), 858 heifers underwent fixed time-AI (FTAI). Subsequently, on day 33, following pregnancy diagnosis by transrectal ultrasonography pregnant heifers (n = 311) were randomly allocated to two treatment groups. Group 1 (162 animals) received an inactivated oil emulsion FMD vaccine, and Group 2 (149 animals) received a saline injection (control). On day 51 (18 days post vaccination), pregnancy status was re-evaluated by ultrasonography. The initial pregnancy rate (PR) on Day 33 was 58% (498/858 animals). On Day 51 (18 days post vaccination), PR in Group 1 was 96.3% (156/162 animals), and in Group 2 (control) was 98.6% (147/149 animals). The EPL in Group 1 was 3.7% (6/162 animals) and in Group 2 was 1.3% (2/149 animals). The RR of EPL in Group 1, compared to Group 2, was 2.8 (95% confidence interval: 0.6–13, p-value: 0.20). With such a wide range in confidence intervals and a p value of 0.20 a larger prospective study would be necessary to establish an unequivocally statistically significant link between heifer vaccination 33 days post FTAI and an increased risk of EPL.

Is Aspiration of Blastocoelic Fluid Essential to Vitrify Large Equine Embryos?

Equine embryo vitrification is still not a well-established technique in equine practice. Notably, little work has been done on the effect of the warming system on viability of vitrified embryos. Our goal was to evaluate the effect of warming without cryoprotectants on in vitro - produced (IVP) embryo viability in culture, quality assessment parameters, and pregnancy after transfer. Equine IVP blastocysts were vitrified using commercial embryo vitrification media and a semi-closed vitrification device. In Exp. 1, we evaluated two warming temperatures (room temperature, RT, ∼22 °C; and 38 °C) for each of three warming systems: commercial warming solution (Kit); commercial embryo holding medium (EHM) with decreasing concentrations of sucrose (EHM + SS); or EHM alone without added sucrose. Embryos (n = 9 to 14 per treatment) were cultured in vitro for 24 h, stained with DAPI, TUNEL, and fluorophore-labelled phalloidin, and evaluated for nucleus number, mitotic rate, apoptotic rate, and actin filament distribution. In Exp. 2, to survey embryo viability in vivo, vitrified IVP blastocysts were shipped to an embryo transfer facility, then warmed immediately before transfer to recipient mares, using the warming treatments associated with the nominally best (Kit-RT, Kit-38, EHM-RT) and poorest (EHM + SS-38) assessed embryo quality in Exp. 1 (n = 7 to 8 per treatment). Subsequently, IVP blastocysts produced as part of our clinical program were vitrified and shipped, then warmed in embryo holding medium at an embryo transfer facility before transfer to recipient mares; fresh IVP embryos were shipped and transferred as controls. In Exp. 1, embryos increased significantly in diameter after culture (P < 0.01), with no difference among treatments. There was no difference (P > 0.05) in the number of viable nuclei, apoptotic rate, or microfilament distribution among treatments, or between vitrified-warmed and Control embryos. The mitotic rate was higher (P = 0.021) for Kit-RT (3.6%) when compared with the other treatment groups (1.5–2.0%). In Exp. 2, there was no difference (P > 0.05) in initial pregnancy (71.4–87.5%) or heartbeat (57.1%–85.7%) rates among warming treatments. In the clinical trial, there was no difference (P > 0.05) between vitrified-warmed and Control embryos in initial pregnancy (90.9% and 66.6%, respectively) or heartbeat (81.8% and 66.6%, respectively) rates. These results indicate that a semi-closed vitrification system using commercially-available media, and incorporating warming in the field in a single step using commercial embryo holding medium without cryoprotectants, can provide high pregnancy rates with IVP equine embryos.

Growth and reproductive performance of yearling beef heifers implanted with Revalor G in the Nebraska Sandhills

ABSTRACT Crossbred beef heifers (n = 3,242), approximately 12 mo of age, were managed at 3 locations in the Nebraska Sandhills. Heifers were randomly assigned (May 1) to be implanted with 40 mg of trenbolone acetate plus 8 mg of estradiol (IMP; Revalor G, Merck Animal Health, Summit, NJ) or not implanted (control; CON). Heifers (238 ± 2 kg) grazed native Sandhills range for the duration of the trial (164 ± 4 d). Eighty-two ± 2 d following trial initiation, heifers were synchronized for estrus and received AI followed with clean-up bulls as part of a 25-d breeding season. Body weight was measured at the beginning and end of the trial. Pregnancy detection occurred 45 d following bull removal. During the second breeding season heifers were supplemented with 0.45 kg of a 32% CP supplement 15 d before and 15 d after placement with bulls for a 56-d breeding season. Implanted heifers gained more and were heavier ( P P P = 0.02; 93 vs. 96 ± 2%, IMP vs. CON, respectively). Implanting beef heifers with trenbolone acetate plus estradiol at approximately 12 mo of age increased ADG and summer BW gain; however, it decreased initial and subsequent pregnancy rate compared with heifers not implanted.

Preimplantation development of cloned canine embryos recovered by hysterectomy or surgical uterine flushing and subsequent pregnancy outcomes

Dog cloning offers a substantial potential because of the advancements in assisted reproductive technology and development of the human disease model in line with the transgenic technique. However, little is known about the development of the canine cloned embryo during the preimplantation period. The aim of this study was to investigate the most efficient method and time for collecting cloned canine preimplantation embryos and to ascertain the developmental timeline of cloned canine embryos. Two hundred cloned embryos were created and transferred into 11 surrogates. The preimplantation stage cloned embryos were then collected on Days 7, 8, and 9 using an ovariohysterectomy or the Foley balloon catheter method. The recovery rate of reconstructed embryos was 63.6% and 60.6% for the ovariohysterectomy and Foley balloon catheter methods, respectively. Although significant differences were observed in the early developmental stages (one-cell and 16-cell stages), no significant difference was observed in the blastocyst stage. Significantly higher blastocyst rate was observed when the embryos were collected on Day 8 (11.4%) than on Day 7 (0.0%; P < 0.05). At the proximal uterine horn on Day 7, no embryos at any stage were found, whereas on Days 8 and 9, blastocysts were found. We have observed a 63% initial pregnancy rate at 25 to 30 days after embryo transfer and a 50% full-term pregnancy rate, whereas 6.3% of the puppies were born, and 5.5% were born live among the total transferred embryos. Our results suggest that cloned embryos can develop to blastocysts by Day 8, and full-term pregnancy can be achieved after embryo transfer in canine.

Influence of somatic cell donor breed on reproductive performance and comparison of prenatal growth in cloned canines

Using in vivo–flushed oocytes from a homogenous dog population and subsequent embryo transfer after nuclear transfer, we studied the effects of donor cells collected from 10 different breeds on cloning efficiency and perinatal development of resulted cloned puppies. The breeds were categorized into four groups according to their body weight: small (≤9 kg), medium (>9–20 kg), large (>20–40 kg), and ultra large (>40 kg). A total of 1611 cloned embryos were transferred into 454 surrogate bitches for production of cloned puppies. No statistically significant differences were observed for initial pregnancy rates at Day 30 of embryo transfer for the donor cells originated from different breeds. However, full-term pregnancy rates were 16.5%, 11.0%, 10.0%, and 7.1% for the donor cells originated from ultra-large breed, large, medium, and small breeds, respectively, where pregnancy rate in the ultra-large group was significantly higher compared with the small breeds (P < 0.01). Perinatal mortality until weaning was significantly higher in small breeds (33.3%) compared with medium, large, or ultra-large breeds where no mortality was observed. The mean birth weight of cloned pups significantly increased proportional to breed size. The highest litter size was examined in ultra-large breeds. There was no correlation between the number of embryo transferred and litter size. Taken together, the efficiency of somatic cell cloning and fetal survival after embryo transfer may be affected significantly by selecting the appropriate genotype.

Effect of IVF laboratory air quality on pregnancy success

OBJECTIVE: To determine if embryo quality and pregnancy success was increased following construction of an IVF laboratory incorporating features designed to improve ambient air quality compared to a previous laboratory. DESIGN: Retrospective analysis of IVF database. MATERIALS AND METHODS: Fertilization rates, number of transferable embryos, and initial pregnancy rates for all first IVF and ICSI patient cycles ages <35 were compared for the 36 months before and 15 months after occupation of a new IVF laboratory incorporating features designed by a consultant (Alpha Environmental Inc.) to improve ambient air quality. These features included isolation of the IVF lab, retrieval room and transfer room, increased number of fresh air exchanges and positive pressure throughout, avoiding use of building materials that release volatile organic compounds (VOCs), and both chemical and particulate filtration of outside air. The old laboratory had fewer fresh air exchanges, relied on standard hospital fresh air, and after 3/18/2007 had a free standing air filtration unit with HEPA and activated carbon filter designed for use in IVF laboratories (zIVF-AIRe 100C). RESULTS: Outcomes were compared over three time periods: I Old lab 1-1-06 to 1-18-07 n=102, II Old lab 3-19-07 to 12-31-08 n=120, III New lab 1-1-09 to 3-15-10 n=102. There were no significant differences in diagnosis, age, or ratio ICSI/IVF cycles. The total number of oocytes and number of embryos were greater in I and II. The percent of retrievals without embryo transfer was less and the implantation rate and clinical pregnancy rate were significantly higher in III.Tabled 1ParameterIIIIIII+II vs III p.Fertilization rate.74.73.81.09Total embryos/zygote.95.96.96.09No transfer %8.85.81.0.02Clinical pregnancies %44.146.463.4.02Implantation rate.34.40.47.00 Open table in a new tab CONCLUSION: Ambient air quality may affect gamete and embryo quality at critical times when either are outside the controlled environment of the incubator.

144 PREGNANCY RETENTION OF BOVINE RECIPIENTS FOLLOWING TRANSFER OF EMBRYOS EXPOSED TO A PROSTAGLANDIN2ALPHA RECEPTOR ANTAGONIST DURING COLLECTION

Increasing efficiency and success of MOET continues to be a goal of researchers and practitioners. Although numerous studies report success in establishing pregnancies, fewer evaluate term development and report number of live calves born. In a previous study, Scenna FN et al. (2008 Reprod. Fertil. Dev. 20, 154) exposed embryos to 3 different medium treatments while being collected from superovulated beef donors on Day 7. Medium treatments consisted of a commercially available medium plus 1 mL of DMSO (Control), commercial medium plus 100 nm of AL-8810 (AL100), or a commercial medium plus 1000 nm of AL-8810 (AL1000). Embryos were evaluated for grade and stage according to IETS guidelines. Embryos (n = 1734 at 6 locations across 13 replicates) were transferred (fresh or frozen in ethylene glycol) by 4 experienced technicians. Pregnancy rates were determined by ultrasonography 28 to 35 days after transfer and were increased in recipients receiving embryos collected in media containing AL100 (65%) and AL1000 (60%) compared with Control (50%; P &lt; 0.05) as previously reported. As a continuum of this research interest, pregnancy retention rates (PRR) of recipient animals receiving embryos exposed to a prostaglandin F2α receptor antagonist (AL100 or AL1000) during collection or serving as nontreated controls were determined. Pregnancy retention rate was defined as the percentage of animals calving that were diagnosed pregnant at 28 to 35 days after transfer. From this pool of recipient animals, calving information was available to date on 494 confirmed pregnancies (presence of an embryo with a heartbeat). Data were obtained on 192 Control, 108 AL100, and 194 AL1000 recipients and analyzed using generalized mixed model analysis of variance (Glimmix) in SAS (SAS Institute, Cary, NC). Similar to initial pregnancy rates, PRR did not differ between AL100 and AL1000 (89 ± 0.04% and 90 ± 0.03%, respectively; P &gt; 0.10); therefore, these groups were combined (AL) for subsequent analysis of PRR. Regardless of treatment, overall PRR were 88 ± 0.01% for fresh and 85 ± 0.03% for frozen embryos (P &gt; 0.10). Additionally, gestation length (days) was similar between recipient animals receiving Control (282 ± 1.19), AL100 (281 ± 1.30) or AL1000 (281 ± 1.17; P &gt; 0.10). However, addition of AL to collection medium of embryos increased PRR compared with Control (90 ± 0.03% v. 83 ± 0.03%, respectively; P = 0.06). Currently, an interaction was not noted between freezing method and media treatment (P &gt; 0.10). Although collection of data continues, addition of a prostaglandin F2α receptor antagonist to the collection medium of embryos improves PRR of recipient animals without altering gestation length.

Outcome of ICSI with Morphologically Selected Spermatozoa

In ICSI, sperm is selected based on its motility and shape under 200–400× magnification. However, sperm morphology, especially the existence of vacuoles, may relate to sperm nuclear DNA fragmentation. Bartoov et al. reported the application of sperm selection under high (1,000–6,000×) magnification, which they referred to as IMSI (intracytoplasmic morphologically-selected sperm injection). In our initial trials, the fertilization, blastocyst development, pregnancy and miscarriage rates of 115 single blastocyst transfer cycles with IMSI or ICSI were 84.0% vs. 75.5%, 47.5% vs. 43.7%, 50.0% vs. 47.4% and 10.0% vs. 11.0%, respectively. Further studies are required to establish the systems and clarify the indications for IMSI.

Poor outcome in oocyte donation after elective transfer of a single cleavage-stage embryo in Turner syndrome patients

In this retrospective, matched study we evaluated the outcome of 31 oocyte donation (OD) cycles performed in 29 Turner syndrome (TS) patients involving the elective transfer of a single, fresh cleavage-stage (day 2 to 3) embryo. Due to the fact that ongoing pregnancy rate was statistically significantly lower (3.2% versus 22.5%) in TS patients when compared with matched, non-TS recipients, other strategies (such as single blastocyst transfer) should be evaluated that could enable better outcomes and at the same time avoid potential complications related to multiple pregnancies in this particularly high obstetric-risk group.

30 Years of IVF: What Does the Future Hold?

The publication of the first successful human birth after IVF-embryo transfer (IVF-ET) in 1978 revolutionized the infertility industry [1]. The original technique involved oocyte retrieval in a natural ovulatory cycle. The tools for retrieval and the culture medium for embryos were rather primitive compared with those used today, hence, initial pregnancy rates were low. In addition, despite careful monitoring, there were often times when the one egg could be released without being ‘caught’. This combination of factors resulted in low pregnancy rates [2,3]. The pervading thoughts at this time were that in a given cohort of antral follicles, some of the oocytes would be genetically normal whilst some would have meiosis errors, and it would be merely fortuitous if the follicle containing a genetically normal egg became dominant. Thus, stimulating multiple follicles with clomiphene citrate alone or with clomiphene and human menopausal gonadotropin, became the usual method for preparing oocyte retrieval [4]. An even greater recruitment of follicles could be achieved by the use of gonadotropin stimulation instead of clomiphene. This would not only allow more fresh embryos to be transfered, but also produce extra embryos that could be cryopreserved for future use [5]. Thus, one could try to maximize the success from one expensive oocyte harvest (cost of medications, monitoring, oocyte retrieval and anesthesia) by performing a much less expensive frozen-embryo transfer in case of failure to achieve a pregnancy with the fresh-embryo transfer [6]. Unfortunately, in approximately 20% of the cycles, the high serum estradiol levels generated by the expensive gonadotropins caused premature luteinization and, thus, cancellation of

Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation

The epigenetic status of a donor nucleus has an important effect on the developmental potential of embryos produced by somatic cell nuclear transfer (SCNT). In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Alicia/Basilea) into metaphase II oocytes and analyzed the levels of histone H3-lysine 9-lysine 14 acetylation (acH3K9/14) in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with blastomeres from in vivo fertilized or parthenogenetic embryos. The levels of acH3K9/14 were higher in RCCs than in RFFs (P<0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC cloned embryos induced a higher initial pregnancy rate as compared to RFF cloned embryos (40 vs 20%). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed, live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly increased the level of acH3K9/14 and the proportion of nuclear transfer embryos developing to blastocyst (49 vs 33% with non-treated RFF, P<0.05). The distribution of acH3K9/14 in either group of cloned embryos did not resemble that in in vivo fertilized embryos suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres from in vivo derived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and may be a useful epigenetic mark to predict efficiency of SCNT in rabbits.

102 RABBIT CLONING: HISTONE ACETYLATION STATUS OF DONOR CELLS AND CLONED EMBRYOS

Epigenetic status of the genome of a donor nucleus is likely to be associated with the developmental potential of cloned embryos produced by somatic cell nuclear transfer (SCNT). Prevention of epigenetic errors by manipulation of the epigenetic status of donor cells is expected to result in improvement of cloning efficiency. In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Ali/Bas) into metaphase II (MII) oocytes and analyzed the levels of histone H3K9 acetylation in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with one or two blastomeres from in vitro-fertilized or parthenogenetic embryos. Histone acetylation in donor cells and cloned embryos was detected by anti-acH3K9 antibody using Western immunoblot analysis or immunochemistry, respectively. Data were analyzed by chi-square (developmental rates) or Student-Newman-Keuls (histone acetylation) test. The levels of acetylated histone H3K9 were higher in RCCs than in RFFs (P &amp;lt; 0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC-cloned embryos induced a higher initial pregnancy rate as compared to RFF-cloned embryos (40% vs. 20%; P &amp;lt; 0.05). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed; a live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly (P &amp;lt; 0.05) increased the level of histone H3K9/14 acetylation and the proportion of nuclear transfer embryos developing to blastocyst (49% vs. 33% with non-treated RFF; P &amp;lt; 0.05). The distribution of signals for acH3K9 in either group of cloned embryos did not resemble that in in vivo-fertilized embryos, suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres from in vivo-derived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and can be a useful epigenetic mark to predict efficiency of SCNT rabbits. This work was supported by the Bayerische Forschungsstiftung and by Therapeutic Human Polyclonals, Inc.

Pregnancy Rates in Lactating Dairy Cows After Presynchronization of Estrous Cycles and Variations of the Ovsynch Protocol

Our objectives were to determine pregnancy rates after altering times of the second GnRH injection, insemination, or both in a combined Presynch + Ovsynch protocol, to accommodate once-daily lockup of dairy cows. Lactating dairy cows (n=665) from 2 dairy herds in northeastern Kansas were studied. Cows ranged from 24 to 44 d in milk (DIM) at the start of the Pre-synch protocol, which consisted of 2 injections of PGF2α 14 d apart, with the second injection given 12 d before initiating the Ovsynch protocol. Cows were blocked by lactation number and assigned randomly to 3 treatments consisting of variations of the Ovsynch protocol. Cows in 2 treatments received injections of GnRH 7 d before and 48h (G48) after the PGF2α injection. Timed AI (TAI) was conducted at the time of the second GnRH injection (G48 + TAI48) or 24h later (G48 + TAI72). Cows in the third treatment received the injections of GnRH 7 d before and at 72h after PGF2α and were inseminated at the time of the second GnRH injection (G72 + TAI72). Pregnancy was diagnosed weekly by palpation per rectum of uterine contents on d 40 or 41 after TAI. Pregnancy rates differed between herds, but they were consistently greater for G72 + TAI72 than for G48 + TAI48 and G72 + TAI72. Subsequent calving rates were consistent with differences in initial TAI pregnancy rates. Pregnancy loss was least for cows on the G72 + TAI72 treatment. Body condition scores (BCS) ranged from 1.0 to 4.0 when assessed on Monday of the breeding week. An interaction of BCS and herd was detected in which cows in herd 1 having poorer BCS (<2.25) had greater pregnancy rates than cows of greater BCS (≥2.25), whereas the reverse was true in herd 2 in which overall pregnancy rates were greater. We concluded that inseminating at 48 or 72h after PGF2α, when GnRH was administered at 48h after PGF2α, produced fewer pregnancies than inseminating and injecting GnRH at 72h after PGF2α for cows whose estrous cycles were synchronized before initiating this variant of the Ovsynch protocol.