Sydney IVF Research Articles

P-231 The effect of warming blastocysts in Vitrolife media previously vitrified in Cook media on reproductive potential and clinical outcome

Abstract Study question Does the use of a different manufacturer’s media for vitrification and warming of blastocysts affect pregnancy, miscarriage and live birth rates. Summary answer Warming blastocysts in Vitrolife media, previously vitrified in Cook media, does not significantly affect the pregnancy or live birth rate. What is known already Current good practice is to vitrify and warm blastocysts in media manufactured by the same company. This is due to the optimisation of the protocols and media composition intending to maintain high survival rates and associated pregnancy rates. However, circumstances can arise, such as supply and production issues, whereby it is not possible to source the same media for warming vitrified blastocysts as the media they were vitrified in. Study design, size, duration This retrospective cohort study was conducted at TFP-Oxford Fertility. Frozen embryo transfer (FET) cycles included in the study were performed between 2018 and 2022. Embryos that had undergone genetic testing were excluded from the study. The mean women's age was 35 in the control group (Cook warming media) and 36 in the study group (Vitrolife warming media). The average number of embryos transferred was 1.06 vs 1.17 in control vs study group. Participants/materials, setting, methods For the present study 817 patients underwent FET between 2016 and 2018 whereby blastocysts were vitrified in cook media (Sydney IVF Blastocyst Vitrification Kit, K-SIBV-5000) and warmed in cook media (Sydney IVF Blastocyst Warming Kit, K-SIBW-5000). In comparison 704 patients underwent a FET between 2018 and 2022 whereby blastocysts were vitrified in cook media and warmed in vitrolife warming media (Rapidwarm™ Blast, 10120). The pregnancy, clinical pregnancy and live birth rates were compared. Main results and the role of chance Fisher’s exact test was used to calculate the p value and a level of 0.05 was used for significance. The results showed that there was no significant difference in the pregnancy rate, clinical pregnancy rate or live birth rate for blastocysts warmed in Vitrolife media vs Cook media (59.0% vs 57.0% p = 0.56, 47.2% vs 43.9% p = 0.44, 39.3% vs 37.0% p = 0.72) Limitations, reasons for caution This is a retrospective study at a single centre. The number of previous treatment cycles per couple was not included in the study. The study only looked at a comparison of Cook and Vitrolife warming media, no other commercially available media was tested. Wider implications of the findings Although good practice to vitrify and warm blastocysts in the same manufacturer media, our results show that the reproductive potential and clinical outcome may not be adversely affected by using a different manufacturer’s media. However, composition of the media and protocol should be considered and further studies should be conducted. Trial registration number not applicable

P-084 Novel Single-Step Microfluidic Chamber Outperforms Density Gradient Centrifugation in Post-Selection Motility And DNA Integrity

Abstract Study question Does sperm selection by a novel single-step microfluidic chamber offer effective selection of motile sperm without DNA damage compared with Density Gradient Centrifugation (DGC)? Summary answer The novel single-step microfluidic chamber is a good alternative to conventional DGC, increasing DNA integrity and progressive sperm motility in recovered sperm populations. What is known already High levels of sperm DNA fragmentation (SDF) can have negative impacts on reproductive outcomes such as clinical pregnancy and miscarriage rates. Microfluidic sperm selection technologies have been developed to select sperm with lower level of SDF and higher levels of motility to improve Assisted Reproductive technology (ART) outcomes. While these methods have achieved variable success, very few have proven clinically relevant and fail to perform selection in a robust and simple methodology that can be adopted in clinics. Study design, size, duration A prospective cohort study including 21 donated semen samples processed in 2021. Each sample was split to perform DGC and microfluidic sperm selections side-by-side. Laboratory outcomes were evaluated using semen parameters pre and post processing and included concentration, motility, and DNA fragmentation index (DFI). Participants/materials, setting, methods The study was performed using samples donated at the Department of Obstetrics and Gynaecology, Monash University, Melbourne, Australia. DGC (10 minutes at 300g and 5 minutes wash at 300g) and microfluidic sperm selection (one-step sperm selection;15-minute incubation for selection) were performed with Sydney IVF 80/40 gradients and gamete buffers, respectively. DFI was assessed by chromatin sperm dispersion assay (SCD). Results were analysed by Friedman’s test and differences were considered significant when p-value < 0.05. Main results and the role of chance For the unprocessed raw semen, the starting DFI values varied from 3.0 to 23.3% with an average of 11.38% ± 5.51 and motility varied between 25.3% and 86.7% with an average of 56.42% ± 18.57. The device consistently outperformed DGC in all 21 samples for DFI (0.94% ± 1.12 vs 4.87% ±3.69, P = 0.0012) and in 20 of the 21 samples for motility post-processing DGC (90.88% ± 6.8 vs 73.58% ± 14.97, P = 0.0061). The average concentrations of the raw, DGC, and microfluidic sperm samples were 94.5, 74.12, and 3.55 million sperm/mL, respectively. Thus, both DGC and the microfluidic device showed improvements in SDF, motility, and concentration over the raw sample. However, the microfluidic device significantly outperformed DGC in terms of percentage SDF and motility, indicating its potential for clinical implementation. Limitations, reasons for caution This cohort study did not include samples from infertile patients. Furthermore, the effect of each sperm selection methods on reproductive outcomes was not a part of the study. A larger number of samples across a range of clinically infertile samples is required to fully characterise the microfluidic devices efficacy. Wider implications of the findings The microfluidic device we report here has the potential to become a new tool to improve the efficacy and consistency of sperm selections. When compared to DGC it offers improved sperm motility and DNA integrity from a platform that is simple to use and less skill-based than conventional methods. Trial registration number not applicable

Influence of Extracellular Vesicles of the Follicular Fluid on Morphofunctional Characteristics of Human Sperm.

We studied the effect of extracellular vesicles of the follicular fluid on morphofunctional characteristics of human spermatozoa using CASA (computer-assisted sperm analysis) analytical system. The vesicles were obtained by sequential centrifugation at different rotational speeds and frozen at -80°C in the Sydney IVF Gamete Buffer medium. The sperm fraction was isolated from the seminal fluid of 21 patients aged 27-36 years by differential centrifugation in a density gradient. The precipitate was suspended in Sydney IVF Gamete Buffer to a concentration of 106/ml and incubated with vesicles (1:2) at 37°C in a CO2 incubator for 30 min and 1 h. Sperm fraction incubated without vesicles served as the control. After incubation, some sperm samples were centrifuged at 700g for 5 min and fixed in 2.5% glutaraldehyde in 0.1 M buffer for transmission electron microscopy. After 30-min and 1-h incubation, the progressive and total sperm motility improved, the curvilinear and linear velocity of spermatozoa did not change significantly. Incubation with vesicles significantly changed the trajectory of sperm movement, which can attest to an increase in their hyperactivation and, probably, fertilizing capacity. Analysis of the effect of extracellular vesicles of follicular fluid on sperm motility will help to improve the effectiveness of assisted reproductive technology programs with male infertility factor by improving sperm characteristics in patients with asthenozoospermia and increasing the fertilizing ability of the sperm.

In vitro maturation of Mus musculus mice oocytes after hyperosmotic shock induced by vitrification solutions.

Objective:To evaluate in vitro oocyte maturation rates in embryonic culture medium after induction by hyperosmotic shock caused by exposure to vitrification solutions.Methods:Bilateral oophorectomy was performed on 20 prepubescent female mice (Swiss). Immature (Prophase I) oocytes (N = 400) were obtained by ovarian dissection, divided into 4 groups, and transferred to culture dishes containing fertilization medium (Sydney IVF Fertilization Medium, Cook® Medical). The control group (CG) did not receive treatment, the test groups (G1, G2, G3) were treated with vitrification solution - 2 (VI-2: 14 M sucrose + ethylene glycol and dimethyl sulfoxide) for 30 seconds and subsequently: G1: 30 seconds in devitrification solution - 2 (DV-2: 0.5M sucrose); G2: 60 seconds DV-2; G3: 60 seconds DV-1(1M sucrose) and 180 seconds DV-2. All groups were cultivated for 24 hours in an incubator at 37ºC and 5% CO2 (Thermo model 3110). After this period, we checked their maturation status.Results:Oocytes exposed to VI-2, DV-1 and DV-2 (G3) showed the highest rate of competence in resuming meiosis and reaching the MII stage; however, there was no statistically significant difference (G3 = 50.5% - 49/97; CG = 27.8% - 10/30).Conclusions:Oocyte exposure to vitrification solutions, in order to cause osmotic shock, did not interfere with the resumption of meiosis in mice oocytes.

Efficiency of assisted hatching of the cryopreserved–melted embryos

Purpose . To measure outcomes of clinical research of efficiency of assisted hatching of cryopreserved embryos. Materials and methods. Patients who had un successful cycles IVF/ICSI with transfer of fresh embryos have been selected for participation in the research between 2014 and 2016 years. Patients were distributed in a random way for participation in the experiment and control groups. Results of embryos transfer of one or two cryopreserved and melted embryos were considered only. Embryos were cryopreserved at a stage of blastocyst, 5 days after extraction of oocytes by method of vitrification. Melting procedure was conducted in the morning of a day of embryos transfer following the instructions of the vitrification medium producer Cryotech (Japan). Assisted hatching was conducted with use of micropipettes of Holding Pipette Cook Medical (Australia) and Assisted Hatching/Zona Drilling Pipette Cook Medical (Australia). The treated embryos were cultivated up to a repeated estimation of morphology of embryos before transfer. Transfer of embryos has been conducted by a standard method with the use of catheter for non-invasive transfer of embryo Sydney IVF Cook Medical (Australia). The quantity of the transferred embryos varied from one to two. Results . 100 cryopreserved embryos were transferred which have been distributed in a random way either to the group with the assisted hatching or to the control group (without assisted hatching). A number of parameters of patients from both groups was analyzed, i.e. age of the patient at the time of melting of embryos, duration of infertility, causes of infertility, quantity of previous unsuccessful cycles IVF/ICSI. Any essential differences between patients within two groups based on the aforementioned parameters were not revealed. Also, there were no essential differences in number of the melted embryos, survival rate of embryos, quantity of the embryos transferred to patients. However, at the same time, parameters of positive results of tests on HCG (human chorionic gonadotropin) (42 % against 27 %) and quantity of clinical pregnancy (35 % against 24 %) were statistically higher in the group with assisted hatching comparatively to the control group (P < 0.004; P < 0.001 accordingly). Conclusions . Implantation of the transfered embryos and number of clinical pregnancies were statistically improved due to selection of the optimum freezing conditions and subsequent cultivation and conducting of procedure of mechanical incision of ZP.

Culture conditions affect Ca2+ release in artificially activated mouse and human oocytes.

Inconsistent fertilisation and pregnancy rates have been reported by different laboratories after application of ionomycin as a clinical method of assisted oocyte activation (AOA) to overcome fertilisation failure. Using both mouse and human oocytes, in the present study we investigated the effects of ionomycin and Ca2+ concentrations on the pattern of Ca2+ release and embryonic developmental potential. In the mouse, application of 5μM ionomycin in potassium simplex optimisation medium (KSOM) or 10µM ionomycin in Ca2+-free KSOM significantly reduced the Ca2+ flux and resulted in failure of blastocyst formation compared with 10μM ionomycin in KSOM. Increasing the Ca2+ concentration up to three- or sixfold did not benefit mouse embryonic developmental potential. Similarly, 10μM ionomycin-induced rise in Ca2+ in human oocytes increased with increasing total calcium concentrations in the commercial medium. Remarkably, we observed significantly reduced mouse embryo development when performing AOA over a period of 10min in Quinn's AdvantageTM Fertilisation medium (Cooper Surgical) and IVFTM medium (Vitrolife) compared with Sydney IVF COOK cleavage medium (Cook Ireland), using the same sequential culture system from the post-activation stage to blastocyst formation stage in different AOA groups. In conclusion, concentrations of both ionomycin and Ca2+ in culture media used during AOA can have significant effects on Ca2+ release and further embryonic developmental potential.

Unconditioned commercial embryo culture media contain a large variety of non-declared proteins: a comprehensive proteomics analysis.

Which non-declared proteins (proteins not listed on the composition list of the product data sheet) are present in unconditioned commercial embryo culture media? A total of 110 non-declared proteins were identified in unconditioned media and between 6 and 8 of these were quantifiable and therefore represent the majority of the total protein in the media samples. There are no data in the literature on what non-declared proteins are present in unconditioned (fresh media in which no embryos have been cultured) commercial embryo media. The following eight commercial embryo culture media were included in this study: G-1 PLUS and G-2 PLUS G5 Series from Vitrolife, Sydney IVF Cleavage Medium and Sydney IVF Blastocyst Medium from Cook Medical and EmbryoAssist, BlastAssist, Sequential Cleav and Sequential Blast from ORIGIO. Two batches were analyzed from each of the Sydney IVF media and one batch from each of the other media. All embryo culture media are supplemented by the manufacturers with purified human serum albumin (HSA 5 mg/ml). The purified HSA (HSA-solution from Vitrolife) and the recombinant human albumin supplement (G-MM from Vitrolife) were also analyzed. For protein quantification, media samples were in-solution digested with trypsin and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). For in-depth protein identification, media were albumin depleted, dialyzed and concentrated before sodium dodecyl sulfate polyacrylamide gel electrophoresis. The gel was cut into 14 slices followed by in-gel trypsin digestion, and analysis by LC-MS/MS. Proteins were further investigated using gene ontology (GO) terms analysis. Using advanced mass spectrometry and high confidence criteria for accepting proteins (P < 0.01), a total of 110 proteins other than HSA were identified. The average HSA content was found to be 94% (92-97%) of total protein. Other individual proteins accounted for up to 4.7% of the total protein. Analysis of purified HSA strongly suggests that these non-declared proteins are introduced to the media when the albumin is added. GO analysis showed that many of these proteins have roles in defence pathways, for example 18 were associated with the innate immune response and 17 with inflammatory responses. Eight proteins have been reported previously as secreted embryo proteins. For six of the commercial embryo culture media only one batch was analyzed. However, this does not affect the overall conclusions. The results showed that the HSA added to IVF media contained many other proteins and that the amount varies from batch to batch. These variations in protein profiles are problematic when attempting to identify proteins derived from the embryos. Therefore, when studying the embryo secretome and analyzing conditioned media with the aim of finding potential biomarkers that can distinguish normal and abnormal embryo development, it is important that the medium used in the experimental and control groups is from the same batch. Furthermore, the proteins present in unconditioned media could potentially influence embryonic development, gestation age, birthweight and perhaps have subsequent effects on health of the offspring. The study was supported by the Danish Agency for Science, Technology and Innovation. Research at the Fertility Clinic, Aarhus University Hospital is supported by an unrestricted grant from Merck Sharp & Dohme Corp and Ferring. The authors declare no conflicts of interest.

A time-lapse analysis comparing sequential culture CLEAVAGE MEDIUM SK02-10® from KITAZATO BIOPHARMA® and Sydney IVF cleavage medium k-icm-100® from COOK MEDICAL®

A time-lapse analysis comparing sequential culture CLEAVAGE MEDIUM SK02-10® from KITAZATO BIOPHARMA® and Sydney IVF cleavage medium k-icm-100® from COOK MEDICAL®

Mouse embryo co‐culture with autologous cumulus cells and fetal development post‐embryo transfer

This study was conducted to examine the potential for implantation and sustainable fetal development of mouse embryos cultured from the pronuclear to blastocyst stage. Pronuclear embryos from ICR mice (Harlan Sprague-Dawley) were cultured in Sydney IVF sequential media (Cook) to the blastocyst stage in medium only or co-cultured with autologous cumulus cells. We also experimented with co-culture in 100 µL drops. Drop co-culture produced blastocyst formation rates with a mean of 47.0%, which was significantly higher (P < 0.05) compared to embryos cultured in identical culture conditions except without cumulus cells at 27.3%. Blastocysts obtained in vitro in Cook medium only and co-cultured in Cook medium with cumulus cells were transferred to pseudopregnant females of ICR strain. The day of blastocyst transfer into surrogate females was designated as post-transfer of blastocyst day 1 (PT 1). The implantation and fetal development was compared to embryo transfer of in vivo derived blastocysts, which served as controls. There were no statistical differences for implantation and fetal development rates for blastocysts cultured in vitro in either Cook medium only or co-culture in Cook medium with cumulus cells compared to in vivo-derived blastocysts. The advantage of the co-culture system is in generating more blastocysts available for transfer.

Perinatal outcome of twice-frozen-thawed embryo transfers: a clinical follow-up study

We evaluated our clinical data on refrozen-thawed ETs (92 cycles) and found that human embryos were capable of withstanding two freeze-thaw cycles, resulting in normal live births after transfer at a rate similar to that of primary frozen-thawed embryos. This is the first follow-up study to present perinatal outcomes of children born after embryo re-cryopreservation, and our results should encourage clinicians to explore the possibility of performing the refreezing procedure.

Application of two different synthetic sequential media for the human IVF-ET program: a prospective, randomized, and comparative study

ObjectiveSince IVF program was first established, various types of media and culture systems have been developed either in-house or commercially. The aim of this study was to compare the efficacy of in-house Maria Research Center (MRC) media to that of commercially available Sydney IVF media in human day 3 embryo transfer cycles.MethodsThree hundred sixty nine couples were included in this prospective, randomized, and comparative study. All couples undergoing IVF treatment at the Maria Fertility Hospital were randomly assigned to either Sydney IVF (n=178) or MRC (n=191) media.ResultsNo difference was observed between the MRC media and Sydney IVF media groups with respect to fertilization rate (74.4% vs. 75.5%). The clinical pregnancy and implantation rates of MRC media (47.1% and 20.0%, respectively) were also similar to those of Sydney IVF media (44.4% and 19.4%, respectively). However, the proportion of embryos with good quality on day 3 was significantly higher in the MRC media group than the Sydney IVF media group (50.2% vs. 43.2%) (p<0.05).ConclusionMRC media were as effective as Sydney IVF media for sustaining embryo development and pregnancy rates. The present study implies that MRC media can be a suitable alternative to commercially available media for human IVF-ET program.

Ovarian stimulation, in vitro fertilization, and effects of culture conditions on baboon preimplantation embryo development

To evaluate the effects of ovarian stimulation and intracytoplasmic sperm injection (ICSI)-induced fertilization and efficacy of various culture systems on in vitro development of baboon embryos. In vitro study, animal model. Research laboratory. Baboons in laboratory animal research facility. Baboons received FSH (75 IU daily) for 7 to 8 days and FSH/LH (75/75 IU daily) for 3 days, followed by hCG (2,000 IU). Oocytes were retrieved laparoscopically 36 hours after hCG. Intracytoplasmic sperm injection was performed on metaphase II (MII) oocytes. Fertilized embryos were placed into different culture conditions and feeder cell coculture. Embryo development was observed through the most advanced stages, including blastocyst formation. Oocytes retrieved, fertilization rates, multicell embryo rates, and blastocyst rates. Baboon oocytes (n = 1,924, from 49 cycles) were retrieved. Significant heterogeneity was seen in ovarian response to exogenous gonadotropins and subsequent oocyte maturation. The percentage of MII oocytes showed no significant difference among individual female baboons and stimulation cycles. Nearly two thirds of MII oocytes were successfully fertilized with ICSI. Blastocyst rates varied significantly among embryos in different treatments. Coculture with feeder cells in P1/Blast, Quinn's Advantage, and Sydney IVF media generated better blastocyst rates. We tested multiple media and feeder cell combinations to optimize culture conditions in baboon embryo culture and obtained a high blastocyst rate similar to those reported for rhesus monkey embryos cultured in vitro, but still lower than with assisted reproductive technologies in women.

Derivation of three new human embryonic stem cell lines

Human embryonic stem cells are pluripotent cells capable of extensive self-renewal and differentiation to all cells of the embryo proper. Here, we describe the derivation and characterization of three Sydney IVF human embryonic stem cell lines not already reported elsewhere, designated SIVF001, SIVF002, and SIVF014. The cell lines display typical compact colony morphology of embryonic stem cells, have stable growth rates over more than 40 passages and are cytogenetically normal. Furthermore, the cell lines express pluripotency markers including Nanog, Oct4, SSEA3 and Tra-1-81, and are capable of generating teratoma cells derived from each of the three germ layers in immunodeficient mice. These experiments show that the cell lines constitute pluripotent stem cell lines.

Conjoined twins in a triplet pregnancy after intracytoplasmic sperm injection and blastocyst transfer: case report and review of the literature

To describe an exceptional case of conjoined twins in a triplet pregnancy after intracytoplasmic sperm injection (ICSI) and blastocyst transfer. Case report. University teaching hospital reproductive endocrinology department and infertility practice. A 34-year-old woman underwent ICSI and received two blastocysts transferred. Transvaginal ultrasonography performed sequentially during early pregnancy. Ultrasound images of the fetus in gestational sac. Two gestational sacs in the uterus were revealed at the 5th week. At the 8th week of gestation, a single fetus was seen in one sac, whereas thoracopagus conjoined twins was diagnosed in the other sac. At the 10th week, the conjoined twins had a spontaneous cardiac arrest confirmed by color Doppler. The subsequent course was uneventful, and a healthy child was born at 39th week. To date, a small number of cases of conjoined twins in IVF/ICSI pregnancies have been reported, in which most cases were treated with manipulations causing possible trauma in the zona pellucida. Our case is unique in that the transferred embryos were blastocysts that might have had additional damage on the zona pellucida from the longer culture.

Non-invasive and highly sensitive measurement of the respiratory activity of individual human embryos by scanning electrochemical microscopy

OBJECTIVE: Oxygen consumption is an ideal indicator of embryo quality. Recently we developed a novel cell respiration measuring system, scanning electrochemical microscopy (SECM). This technique is a useful method for evaluating embryo quality that correlates metabolic respiration with morphological quality, developmental potential, and pregnancy rate following embryo transfer in bovine. DESIGN: In this study, we employed SECM to accurately determine the oxygen consumption of single, identical human embryos at different developmental stages. MATERIALS AND METHODS: Surplus human embryos which remained unused upon completion of fertility treatment were used in this study. Their use in academic research was consented to by the patient. The embryos were cultured in Sydney IVF Cleavage Medium until Day 3, after which they were cultured in Sydney IVF Blastocyst Medium. Oxygen consumption by individual human embryos was quantified by a modified SECM measuring system. The microelectrode was scanned along the sample and the oxygen consumption rate was calculated with custom software based on spherical diffusion theory. Some embryo were examined using a transmission electron microscope. RESULTS: The oxygen consumption rates (×1014 mol s-1) of single embryos were low at 2-8-cell stages (0.51±0.05) but increased by the morula (0.61±0.11) and early blastocyst (0.72±0.06) stages. Later blastocysts exhibited even higher oxygen consumption rates (1.00±0.19). Ultrastructural studies revealed that most mitochondria in embryos up to the 8-cell stage were immature and had a spherical or ovoid shape. However, by the morula stage, mitochondria have elongated. This morphology is even more pronounced in mitochondria present in blastocysts with high oxygen consumption rates. CONCLUSIONS: SECM can be applied to measure the oxygen consumption of single human embryos at various developmental stages. The maturation of mitochondria correlated with the increase of oxygen consumption rate during the development of embryos. The development of mitochondria may be an important factor in embryo quality. The SECM technique may be a valuable tool for accurately assessing the quality of embryos as this approach provides objective criteria for evaluating embryos that complements morphological evaluation.

Culture of Mouse Zygotes Induces Epigenetic Changes in the Expression of the agouti viable yellow Allele in the Resulting Progeny.

It is well recognised that perturbation of fetal growth may influence postnatal homeostasis. Recent evidence indicates that the growth environment of the preimplantation embryo may also have long-term consequences for progeny. Epigenetic modification of the genome is one molecular mechanism that could explain a persistence of altered gene expression. This study aimed to assess whether culture of zygotes through to the blastocyst stage, followed by their transfer to recipient females, induced defined epigenetic changes in gene expression that persisted through to adulthood. The agouti viable yellow (Avy) allele is epigenetically sensitive, and is a widely used model for studying epigenetic phenomena. It has active and inactive epialleles that are hypo- and hyper-methylated, respectively. Progeny inheriting the allele in its active state have yellow fur; those with the inactive epiallele have an agouti coat. Some progeny are mosaics for the two epigenetic states and have a mottled coat color. Males (Avy/a C57BL/6) carrying the epiallele were mated to wildtype females (A/A FVB). The following treatments were applied: (1) zygotes were collected and cultured for 94 hours and the resulting blastocysts transferred to recipient females; (2) blastocysts were collected from females and then transferred to recipients without being subjected to embryo culture; and (3) pregnancy was allowed to proceed without intervention. Zygotes were cultured in Sydney IVF Media suite (Cook Womens Health, Australia), a widely used commercial sequential human embryo culture media formulation. Embryos were transferred to day 3 (post coitum) pseudopregnant white Swiss recipient females. The number, sex, genotype, and epi-phenotype of the resulting progeny were recorded. The culture of zygotes caused a significant shift in the expression of the epialleles in progeny compared to embryos that were transferred without culture (P=0.014), or those born following natural matings (P<0.001). Offspring that resulted from embryo culture are more likely to have active (hypomethyalted) epialleles, resulting in a higher proportion of adult progeny with yellow fur. The distribution of epialleles was not different (P>0.05) for the two control treatments not subjected to embryo culture. Embryo culture had no effect on the survival of embryos carrying the Avy allele compared to the wildtype allele. The study shows that culture of mouse zygotes through to the blastocyst stage changed the expected post-natal expression of an epigenetically sensitive allele. Embryo culture has been shown to cause many acute changes to the pattern of gene expression, and there are case reports of human ART progeny with abnormalities with an epigenetic basis. The results of this study provide evidence for the epigenetic basis for the long-term memory of preimplantation embryos growth environment, and for robust and readily accessible methodology for addressing the underlying molecular bases for this phenomenon.

429. Culture of mouse zygotes altered epigenetic states of the agouti viable yellow allele in the resulting progeny

Epigenetic modification of the genome is one potential mechanism for the persistence of altered gene expression. Recent evidence indicates that the growth environment of the preimplantation embryo may have long-term consequences in the gene expression of progeny (Mahsoudi et al. 2007; Watkins et al. 2008). This study assessed whether culture of zygotes induced persistent epigenetic changes in gene expression. The agouti viable yellow (Avy) allele is epigenetically sensitive, and is a widely used model for studying epigenetic phenomena. It has active (hypomethyalted) and inactive (hypermethylated) epialleles. Progeny with an active epiallele have yellow fur; those with an inactive epiallele have an agouti coat. Some progeny are mosaics for the two epigenetic states and have a mottled coat color. Males (Avy/a C57BL/6) were mated to wildtype females (A/A FVB). Embryo treatments were: (1) zygotes collected and cultured for 96 h in Sydney IVF Media suite and transferred to day 3 pseudopregnant Swiss females; (2) blastocysts collected and transferred to recipients without being subjected to culture; (3) naturally mated females left to give birth without interventioin. The number, sex, genotype, and epi-phenotype of the resulting progeny were recorded. The culture of zygotes caused a significant shift in the expression of the epialleles compared with embryos that were transferred without culture (P = 0.014), or those born following natural matings (P &lt; 0.001). Offspring that resulted from culture are more likely to have active epialleles, resulting in a higher proportion of progeny with yellow fur. The results of this study show that culture of mouse zygotes changed the expected expression of the Avy allele. This study provides evidence for the epigenetic basis for the long-term ‘memory’ of preimplantation embryo’s growth environment, and for robust and readily accessible methodology for addressing the underlying molecular bases for this phenomenon. (1) Mahsoudi B, Li A, O’Neill C. Assessment of the Long-Term and Transgenerational Consequences of Perturbing Preimplantation Embryo Development in Mice. Biol Reprod 2007; 77: 889–896. (2) Watkins AJ, Ursell E, Panton R, Papenbrock T, Hollis L, Cunningham C, Wilkins A, Perry VH, Sheth B, Kwong WY, Eckert JJ, Wild AE, Hanson MA, Osmond C, Fleming TP. Adaptive responses by mouse early emb.

In Vitro Development of Mouse Pronuclear Embryos to Blastocysts in Sequential Media With and Without Co-Culture of Autologous Cumulus Cells

The objective of this study was to use mouse embryos as a model system to investigate the effect of co-culture of cumulus cells in Sydney IVF sequential media (Cook) on embryo development, based on the hypothesis that feeder cells in co-culture with a sequential medium could work synergistically to further improve in vitro culture conditions for mammalian preimplantation embryos. The culture systems described here were evaluated by the ability to consistently produce high blastocyst formation rates and high cell number per blastocyst. The role of embryo-to-cell contact was assessed by using Transwell inserts with transparent microporous membranes. Pronuclear embryos of ICR mice were cultured to blastocysts in Cook sequential media, with and without mouse primary cultures of cumulus cells, and with or without inserts. Blastocyst formation rates and cell numbers of in vitro developing embryos in the different culture systems were compared to each other, and to in vivo derived blastocysts. Blastocyst formation rates for Cook medium only was 27.8% (without inserts) and 32.9% (with inserts), whereas Cook-Cumulus cells in identical culture systems was significantly higher at 45.8% (without inserts, P<0.05) and 55.6% (with inserts, P<0.05). When the embryos are suspended above the bottom of the well, for Cook medium significantly lower blastocyst formation rates were observed at 4.2% compared to Cook-Cumulus cells at 17.5% (P<0.05). Mean cell numbers of blastocysts obtained in all co-culture systems were significantly higher (P<0.05) compared to those developing in culture medium only. Although the putative mechanism is as yet unexplained, the improved blastocyst formation rates and cell numbers in co-culture when there is direct contact between the embryo and the cell monolayer suggest that the close proximity between the feeder cells and embryos is in part responsible for these effects.

A prospective randomized controlled trial comparing two embryo transfer catheters in an ART program

To compare the performance of the Cook Sydney IVF (SIVF) embryo transfer (ET) catheter and the Edwards-Wallace (EW) ET catheter. Prospective randomized controlled clinical trial with an intention-to-treat analysis. Two-center study. Four hundred consecutive women <40 years undergoing ET with two fresh embryos. Women were randomly allocated to undergo ET with either the EW or the SIVF catheter, with possible catheter change in case of insertion difficulties. Live birth and clinical pregnancy rates. Two hundred two women were allocated to the SIVF catheter and 198 to the EW catheter. No significant differences in the clinical pregnancy rates (odds ratio [OR] 0.99, 95% confidence interval [CI] 0.66-1.47) and live-birth rates (OR 1.09, 95% CI 0.72-1.65) were found. The EW catheter had to be changed more often than the SIVF catheter (OR 9.5, 95% CI 3.3-27.5) because of catheter insertion problems. The pregnancy and live birth rates were not significantly different with the two catheters, but catheter insertion failure was significantly more common with the EW catheter than with the SIVF catheter.

A study of two sequential culture media - Impact on embryo quality and pregnancy rates

Objective. A comparative study of embryo quality and pregnancy outcome between Sydney IVF medium and Quinn's Advantage sequential culture media. Design. A prospective randomised controlled trial and a retrospective study. Setting. In vitro fertilisation clinic in an academic research environment. Patients. All women South African Journal of Obstetrics and Gynaecology Vol. 13 (2) 2007: pp. 52-59