Embryo Transfer Technique Research Articles

Revival of an efficient patient and operator friendly embryo transfer technique: UMbET (ultrasound measurement before embryo transfer)

Revival of an efficient patient and operator friendly embryo transfer technique: UMbET (ultrasound measurement before embryo transfer)

Neospora caninum is not transmissible via embryo transfer, but affects the quality of embryos in dairy cows

The aim of this study was to evaluate embryo transfer in cattle as a pathway of transmitting neosporosis and to quantitatively and qualitatively compare the embryo production from seropositive and seronegative donors. Superovulatory treatments were performed on eight Girolando donors (four seropositive and four seronegative for N. caninum), resulting in the recovery of ninety-one structures (embryos and non-fertilized structures). Embryos collected and classified as viable were transferred to seronegative recipients and analyzed by PCR to identify N. caninum. No difference was observed in the number of structures collected from seropositive and seronegative donors (40 and 51; P = 0.64). There was a significant difference in the percentages of freezable (25 % and 74 %, P = 0.04) and transferable (27.5 % and 78.4 %, P = 0.003) embryos. The recipients had similar pregnancy rates (60 % and 57.1 %, P = 0.97) and pregnancy loss (50 % and 43.8 %, P = 0.58). There was no change in the serological status of the recipients, and no protozoan DNA was identified in none of the samples. It was concluded that the embryo transfer technique is safe regarding the transmission of neosporosis; however, the quality of embryo production was compromised from donors seropositive for N. caninum.

Transcriptomic Analysis of Vitrified-Warmed vs. Fresh Mouse Blastocysts: Cryo-Induced Physiological Mechanisms and Implantation Impact.

Blastocyst vitrification has significantly improved embryo transfer methods, leading to higher implantation success rates and better pregnancy outcomes in subsequent frozen embryo transfer cycles. This study aimed to simulate the transcriptional changes caused by vitrifying human blastocysts using mouse blastocysts as a model and to further investigate these changes' effects. Utilizing a human vitrification protocol, we implanted both vitrified and fresh embryos into mice. We observed the implantation success rates and performed transcriptomic analysis on the blastocysts. To validate the results from messenger RNA sequencing, we conducted reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) to measure the expression levels of specific genes. Based on mRNA profiling, we predicted the microRNAs responsible for the regulation and used qPCR basic microRNA assays for validation. Our observations revealed a higher implantation success rate for vitrified embryos than fresh embryos. Transcriptomic analysis showed that vitrified-warmed blastocysts exhibited differentially expressed genes (DEGs) primarily associated with thermogenesis, chemical carcinogenesis-reactive oxygen species, oxidative phosphorylation, immune response, and MAPK-related signaling pathways. RT-qPCR confirmed increased expression of genes such as Cdk6 and Nfat2, and decreased expression of genes such as Dkk3 and Mapk10. Additionally, gene-microRNA interaction predictions and microRNA expression analysis identified twelve microRNAs with expression patterns consistent with the predicted results, suggesting potential roles in uterine epithelial cell adhesion, trophectoderm development, invasive capacity, and immune responses. Our findings suggest that vitrification induces transcriptomic changes in mouse blastocysts, and even small changes in gene expression can enhance implantation success. These results highlight the importance of understanding the molecular mechanisms underlying vitrification to optimize embryo transfer techniques and improve pregnancy outcomes.

Survival and Development of in Vivo Produced Boran and Boran* Holstein Cross Embryos

One of biotechnology technique that is frequently used to enhance the number of animals with superior genetic ability and high productivity is embryo transfer. Embryos can be obtained in vivo or in vitro, and they can be frozen and then thawed before being delivered to the recipient animals. Conception rates are influenced by a number of variables, including the quality and developmental stage of the embryo, the location of the embryo's deposit in the uterus, the degree of difficulty of the transfer, whether to use a fresh or frozen embryo, the operator's experience, the corpus luteum's quality, whether to use a heifer or a cow, and the time of year the transfer occurs. The fertility of domestic animals is severely impacted by early embryonic death. For this reason, this study was carried out to gather data on early embryonic development that is normal, the amount and timing of embryonic mortality, potential endogenous and exogenous causes of embryonic loss, and to develop strategies to lower embryonic mortality. A total of 40 embryos (20 fresh and 20 frozen, 26 quality grade 1 and 14 quality grade 2, 29 compact Morula stage and 11 early Blastocyst stage) were transferred to 40 recipient cows (22 Boran and 18 H-B cross) with different body condition score. Return to heat was used as method of pregnancy diagnosis and all recipients were followed around day 14 post embryo transfer and 14 animals were shown heat sign, the rest 26 animals were suspected for pregnancy (65%). Using ultrasound, a pregnancy diagnosis was made on day 45, and 20 recipient animals were found to be 50% positive for PD. On day 60, PD positive animals were re-examined with ultrasound and only 10 were confirmed as PD positive (25%). The PD negative on day 45 and day 60 were suspected to be early embryonic mortality. Other pregnancy loss occurred in this study was abortion nearly after five month of pregnancy. It is not doubtful that, the technique of embryo transfer is utilized to increase the reproductive rates of important female animals. However, it needs proper management for both donor and recipient animals. Therefore, for the successful application of the technology optimum level of feeding both quantity and quality, health management and conducive environment should be fulfilled for all animals.

The use of an embryo transfer simulator to compare transfer techniques and pregnancy outcomes among physicians

To evaluate the association between embryo transfer techniques and pregnancy outcomes. This is a prospective observational study with a retrospective cohort. University Clinic. Patients underwent embryo transfers between 2015 and2020. Fourteen physicians performed 25 mock embryo transfers on the embryo transfer simulator and completed a questionnaire assessing preferred embryo transfer techniques. Quantitative performance metrics on the embryo transfer simulator were measured. Individual physician embryo transfer success rates were retrospectively collected from all fresh and cryopreserved embryo transfers between January 1, 2015, and January 1, 2020. Associations between embryo transfer techniques (preferred technique and simulator performance metrics) and each physician's historical patient pregnancy outcomes were assessed. Associations between embryo transfer techniques and live births were assessed. There were significant differences in embryo transfer techniques between physicians, including touches to the fundus, distance to the fundus, duration of embryo transfer, duration of the complete procedure, time spent navigating the cervical canal, velocity of embryo expulsion, time waited after embryo expulsion, and total score on the embryo transfer simulator. After controlling for confounders and multiple transfers per physician, the duration of embryo transfer was significantly associated with live birth, with longer durations associated with decreased live birth rates. Shorter placement distance to the fundus and higher velocity of embryo expulsion were both significantly associated with higher rates of ectopic pregnancy. This study revealed significant differences in transfer techniques among physicians. The use of the embryo transfer simulator for physicians in practice can elucidate differences and create opportunities for data-driven improvement in embryo transfer success rates.

Factors influencing ovum pick-up technique results in cattle

The Ovum Pick-Up (OPU) technique, which is used in in vitro embryo production (IVP) to retrieve immature oocytes from live donor animals, is one of the most important biotechnological procedures used in cattle breeding. The most important advantage of this technology is that it allows for the reproducible retrieval of immature oocytes from living donor animals. It is particularly useful in dairy cattle breeding to address infertility issues and boost the production of superior animals with high genetic value. The OPU technique offers several advantages, including its applicability to cows ranging from six-month-old calves to the first three months of pregnancy, its effectiveness in animals with genital tract infection or acyclic cattle, and its ability to yield a higher number of embryos within the same period compared to the Multiple Ovulation and Embryo Transfer (MOET) technique. Understanding and improving the technical and biological factors influencing the OPU procedure is necessary to increase and optimize donor animal use in IVP and the number of quality oocytes obtained. This review aims to examine the specifics of the OPU approach and the factors influencing its performance in light of contemporary literature, as well as to propose fresh ideas to researchers.

Первый в России опыт эмбриотрансфера у домашней собаки: методика эксперимента и перспективы клинического применения

The study was conducted on six physiologically and sexually mature females of Labrador Retriever breed. Algorithms of synchronization of sexual cycles in dogs were developed, optimal terms of embryo ejection were revealed taking into account species specifics of oogenesis and ovulation process. The method of embryo evaluation and the technique of embryo transfer to the recipient were modified for dogs. On the basis of the conducted research, a holistic technique of embryotransfer in the domestic dog was developed. This method was practically applied, resulting in the first successful procedure of embryotransfer in a dog in Russia with subsequent confirmation of its reliability by genetic testing.

Steps forward in embryo transfer technique: a retrospective study comparing direct versus afterload catheters at different time frames.

To assess whether embryo transfer (ET) technique can influence the clinical pregnancy rate (CPR) and its correlation with the embryo transfer difficulty. This single center retrospective cohort analysis of fresh and frozen single blastocyst transfers performed between January 2016 and December 2021 included fresh and frozen single blastocyst transfers performed during the study timeframe. Direct technique was the only one used from January 2016 to September 2017. From September 2017 to March 2019, the choice between the two techniques was given by randomization, due to a clinical trial recruitment. From April 2019, only the afterload technique was used. Preimplantation genetic testing cycles and gamete donation procedures and cycles performed with external gametes or embryos were excluded. CPR was the primary outcome, while difficult transfer rate the secondary one. Univariate and multivariate logistic regressions were performed. During the period, 8,189 transfers were performed. CPR of the afterload group resulted significantly higher compared to the direct group (44.69% versus 41.65%, OR 1.13, 95% CI 1.02-1.25, p = 0.017) and the rate of difficult transfers two-thirds lower (9.06% versus 26.85%, OR 0.27, 95% CI 0.24-0.31, p < 0.001). Our study demonstrated that CPR is significantly affected by the ET technique. In particular, with the afterload protocol, both CPR and easy transfer rates increased. http://clinicaltrials.gov registration number: NCT05364528, retrospectively registered on 3rd of May 2022.

Four-dimensional ultrasound guided embryo transfers improve live birth rates when compared to the clinical touch technique: a randomised controlled trial

Most aspects of in-vitro fertilisation (IVF) have changed dramatically since introduction, but embryo transfer (ET) technique remains largely unaltered. We aimed to determine whether four-dimensional ultrasound guided embryo transfers (4D UGET) could improve pregnancy rates when compared with clinical touch technique (CTT). This was a single centre open labelled randomised controlled trial in a tertiary fertility centre in the UK. 320 women were randomised on the day of single ET. The primary outcome was clinical pregnancy rate (CPR), secondary outcomes included live birth rate (LBR), biochemical pregnancy rate (BPR), miscarriage, pregnancy of unknown location (PUL) and ectopic pregnancy. 4D-UGET resulted in significantly higher CPR [50% vs 36% p = 0.02, OR 1.78 (1.12–2.84)] and LBR [41% vs 28%, p = 0.02, OR 1.77 (1.09–2.87)] when compared to CTT technique. Miscarriage (p = 0.49), PUL (p = 0.14) and ectopic pregnancy (p = 0.96) were similar between the two groups. LBR, from this trial, are significantly higher than the current UK average (41% vs 24%). 4D UGET allows for superior imaging of the uterine cavity, whilst tailoring the embryo deposition point specifically to the patient. Further RCTs are required to determine if these results can be replicated in other units and whether 4D UGET is superior to 2D UGET.

Role of Dydrogesterone for Luteal Phase Support in Assisted Reproduction.

Clinical outcomes of in vitro fertilization (IVF) have significantly improved over the years with the advent of the frozen-thawed embryo transfer (FET) technique. Ovarian hyperstimulation during IVF cycles causes luteal phase deficiency, a condition of insufficient progesterone. Intramuscular or vaginal progesterone and dydrogesterone are commonly used for luteal phase support in FET. Oral dydrogesterone has a higher bioavailability than progesterone and has high specificity for progesterone receptors. Though micronized vaginal progesterone has been the preferred option, recent data suggest that oral dydrogesterone might be an alternative therapeutic option for luteal phase support to improve clinical outcomes of IVF cycles. Dydrogesterone has a good safety profile and is well tolerated. Its efficacy has been evaluated in several clinical studies and demonstrated to be non-inferior to micronized vaginal progesterone in large-scale clinical trials. Oral dydrogesterone may potentially become a preferred drug for luteal phase support in millions of women undergoing IVF.

Genome-wide DNA methylation and transcriptome analyses reveal the key gene for wool type variation in sheep

BackgroundWool fibers are valuable materials for textile industry. Typical wool fibers are divided into medullated and non-medullated types, with the former generated from primary wool follicles and the latter by either primary or secondary wool follicles. The medullated wool is a common wool type in the ancestors of fine wool sheep before breeding. The fine wool sheep have a non-medullated coat. However, the critical period determining the type of wool follicles is the embryonic stage, which limits the phenotypic observation and variant contrast, making both selection and studies of wool type variation fairly difficult.ResultsDuring the breeding of a modern fine (MF) wool sheep population with multiple-ovulation and embryo transfer technique, we serendipitously discovered lambs with ancestral-like coarse (ALC) wool. Whole-genome resequencing confirmed ALC wool lambs as a variant type from the MF wool population. We mapped the significantly associated methylation locus on chromosome 4 by using whole genome bisulfite sequencing signals, and in turn identified the SOSTDC1 gene as exons hypermethylated in ALC wool lambs compare to their half/full sibling MF wool lambs. Transcriptome sequencing found that SOSTDC1 was expressed dozens of times more in ALC wool lamb skin than that of MF and was at the top of all differentially expressed genes. An analogy with the transcriptome of coarse/fine wool breeds revealed that differentially expressed genes and enriched pathways at postnatal lamb stage in ALC/MF were highly similar to those at the embryonic stage in the former. Further experiments validated that the SOSTDC1 gene was specifically highly expressed in the nucleus of the dermal papilla of primary wool follicles.ConclusionIn this study, we conducted genome-wide differential methylation site association analysis on differential wool type trait, and located the only CpG locus that strongly associated with primary wool follicle development. Combined with transcriptome analysis, SOSTDC1 was identified as the only gene at this locus that was specifically overexpressed in the primary wool follicle stem cells of ALC wool lamb skin. The discovery of this key gene and its epigenetic regulation contributes to understanding the domestication and breeding of fine wool sheep.

Inducing Pseudopregnancy in Female Mice Without the Need for Vasectomized Males Prior to Non-Surgical Embryo Transfer or Artificial Insemination.

For successfully maintaining pregnancy with embryo transfer or artificial insemination,female recipient mice must be induced into a pseudopregnant state. Female mice are traditionally paired overnight with vasectomized males, and the following morning, the presence of a copulation plug is assessed. To increase the efficiency of producing pseudopregnant females, a cervical manipulation technique has been standardized to be used in combination with non-surgical embryo transfer or artificial insemination techniques in mice. The blunt end of a small plastic rod is inserted vaginally to contact the cervix and is vibrated for 30 s by contact with a trimmer. The procedure is quick and does not require anesthesia or analgesia. This technique increases the reliability and predictability of producing pseudopregnant females and entirely eliminates the requirement for vasectomized males. For CD1 mice, the efficiency of pseudopregnancy induction using cervical manipulation was 83% for females in estrus (N = 76) but only 38% of females in estrus were plugged by vasectomized males (N = 24). Artificial insemination in CD1 mice was performed by estrus synchronization with hormones, cervical manipulation, and the uterine transfer of sperm. Artificial insemination recipients receiving cervical manipulation (N = 76) had a pregnancy rate of 72% and an average litter size of 8.3 pups. This method can also be used to produce pseudopregnant females for non-surgical embryo transfer. Therefore, inducing pseudopregnancy by cervical manipulation is a convenient and efficient alternative to mating with a vasectomized male when performing non-surgical assisted reproduction techniques. Using cervical manipulation provides 3Rs (replacement, reduction, and refinement) benefits for assisted reproduction techniques by reducing the number of animals required and eliminating the necessity for surgically altered males.

P-755 Timeless importance of the embryo transfer technique: a real-life retrospective study comparing direct versus afterload catheters

Abstract Study question Can the embryo transfer (ET) technique influence clinical pregnancy rate (CPR)? Summary answer Our study demonstrated that CPR is significantly affected by the ET technique. In particular, with the afterload technique both CPR and easy transfer rates increased. What is known already Despite it having widely demonstrated that the occurrence of difficult ETs reduces the success of assisted reproductive technology (ART) cycles and that afterload technique has showed a decrease in the number of difficult transfers compared to the direct one, few data are available assessing pregnancy outcomes in the two different techniques. The present study represents the progression of a published prospective randomized controlled trial (RCT) performed in the same setting on a smaller population comparing direct and afterload techniques on the difficult transfer rate, but not designed to demonstrate differences in terms of CPR. Study design, size, duration Single center retrospective cohort analysis of 8,189 fresh and frozen single blastocyst transfers performed between January 2016 and December 2021 in a tertiary University affiliated fertility center. Of the total, 2,000 ETs were performed with the direct technique and 6,189 with the afterload one. All fresh single day-5 and single day-5 and day-6 frozen blastocyst transfers performed during the study timeframe were included. Participants/materials, setting, methods Direct technique was the only one used from January 2016 to September 2017. In the clinical trial recruitment period, the choice was given by randomization. From April 2019, only the afterload technique was used: the operator never had chance to choose the technique. Preimplantation genetic testing cycles, gamete donation and day-7 blastocyst transfers were excluded. CPR was the primary outcome, while difficult transfer rate the secondary one. Univariate and multivariate logistic regression have been performed. Main results and the role of chance During the period, 8,189 single blastocyst transfers were performed. Afterload ET showed a percentage of difficult transfers which was almost one third compared to that of the direct ET, being respectively 9.06% and 26.85% (OR 0.27, 95% CI 0.24-0.31p &amp;lt;0.001). Overall, the CPR between the two groups was significantly different: 44.69% in afterload ET and 41.65% in direct ET (OR 1.13, 95% CI 1.02-1.25, p = 0.017). As for the interaction between the two techniques and difficult transfer on CPR, only the direct difficult ET resulted in a significantly lower CPR with an OR of 0.62 (95%CI 0.49-0.77, p &amp;lt; 0.001). Lastly, among the 15 operators that performed the ETs, a range of difficult transfers from 3.8% to 45.4 % in the direct group and 0.8% to 20.5% in the afterload group was found (p &amp;lt; 0.001). Limitations, reasons for caution The main limitations of the study are the difference in sample sizes between the two groups, the wide range of time considered, and the 15 operators included. A further possible reason is the high prevalence of difficult transfer, probably due to the broad definition we used. Wider implications of the findings The study points out how in embryo transfers the use of the afterload technique instead of the direct one significantly improves clinical pregnancy rate and easy transfer rate. Since there is a limited knowledge on the embryo transfer technique impact, our findings aim to increase the effectiveness of embryo transfers. Trial registration number NCT05364528.

Embryo transfer technique affects pregnancy rates in recipient mares

Embryo transfer in horses started in the 1970′s and has evolved since then in several technical and biological areas that may influence the overall outcome. The transfer method evolved from surgical to non-surgical (NSET), with pregnancy rates for NSET related closely to operator skills. Recently, high pregnancy rates have been reported by non-specialized operators using the cervical forceps method (Cuervo Arango et al., Veterinary Record. 2018,183;10,1-5). The aim of this retrospective study was to compare pregnancy and early embryonic loss rates between conventional and cervical forceps transfer techniques (Wilsher and Allen, Equine Vet. Educ. 2004. 16; 1, 39-44), operated by two veterinarians with different degrees of experience. During the last 12 breeding seasons in a commercial equine embryo transfer program in Chile, 345 embryos were transferred by two operators with low / moderate (n=86) or high experience (n=259), using a conventional manual technique and 228 embryos were transferred by the same two operators (n=81) and (n=147) respectively, using a bovine cervix forceps/Polansky speculum technique. Embryo flushes were performed at 8 or 9 days after ovulation on 102 donor mares aged 4 to 24 years old and transferred to recipient mares (n=387) aged 3 to 12 years old. Ovulation synchrony ranged from -1 to +4 days when comparing the recipient to the donor. No medications were used during embryo transfer and procedures were done at the samefacilities. Data were analyzed by a binary logistic regression model in which pregnancy and embryo loss were treated as dependent variables, and the experience of the operator and the ET technique as independent variables. Pregnancy rates at first diagnosis (12 to 14 days post ovulation) and embryo loss rate until 60 days of gestation were compared between and within operator groups. Overall pregnancy rates were higher (P<0.001) by the cervical forceps / speculum embryo transfer technique (195/228; 85%) than with the conventional technique (236/345; 65%) in both groups combined. Early embryonic loss rate did not differ (P>0.1) between the groups (6.0 vs. 4.6%, respectively). For the conventional technique, there was a significant operator effect (P=0.015) on the pregnancy rates at the first diagnosis (57 vs. 72%); no operator effect (P>0.1) was observed when using cervical forceps (84 vs. 86 %). These results agree with the report of Cuervo-Arango et al. (2018). We also confirmed that simple bovine cervical forceps work as well as the equine specific forceps reported by Wilsher and Allen (2004). In conclusion, the cervical forceps technique yielded higher pregnancy rates, with no effect of the operator's experience compared to the conventional technique in which the skill and experience of the operator is crucial and needs time and losses to achieve.

Do serum progesterone levels on the day prior to Day 5 Embryo transfer influence pregnancy outcome in artificial frozen-thaw embryo transfer cycle?

Background: Progesterone (PG) is an essential hormone in the process of implantation and pregnancy maintenance. Frozen-thawed embryo transfer (FET) is being performed worldwide. This study was designed to investigate whether serum progesterone levels on the day prior to day 5 frozen-thawed embryo transfer (FET) to hormonally prepared endometrium correlates with pregnancy outcomes. Materials and Methods: A single center longitudinal observational study was conducted at an infertility center over a period of three months from October 2022 to December 2022. The study participants were the patients attending the infertility center who satisfied the inclusion and exclusion criteria. A total of 30 participants were included in this study. Serum progesterone levels were measured on the day of starting the progesterone (P0) and on day four (P4), the day prior to embryo transfer. Data analysis was conducted using SPSS version 21. Results: The mean (SD) age in those who had positive pregnancy outcome was 34.22 (1.26) years and in those who had negative pregnancy outcomes was 39.92 (2.11) years and the difference was significant (P = 0.001). The mean (SD) serum progesterone levels in those who had positive pregnancy outcome at day 0 was found to be 0.61 (1.19) ng/ml and in those who had negative pregnancy outcomes was 0.28 (0.27) ng/ml but, the difference was not significant (P = 0.347). The mean (SD) serum progesterone levels in those who had positive pregnancy outcome at day 4 was found to be 10.84 (8.13) ng/ml and in those who had negative pregnancy outcomes was 8.13 (3.81) ng/ml and the difference was significant (P = 0.039). Conclusion: Pregnancy loss increases with age and serum progesterone measurements at day 4 prior to embryo transfer could be used to assess pregnancy outcomes in frozen-thawed embryo transfer (FET) technique of infertility treatment. Monitoring of serum PG levels can be done for diagnosing potential luteal support defects before performing FET with artificial cycle (AC).

Post-Thaw Day 5 Blastocyst Culture Time Prior to Transfer Does Not Affect Assisted Reproduction Technology (ART) Outcomes in Frozen-Thawed Embryo Transfer Cycles.

The frozen embryo transfer (FET) technique has been progressively used more worldwide due to improved culture conditions, as well as enhanced survival rates after vitrification. However, little is known about the effect of the post-thaw blastocyst culture duration prior to transfer on live birth rate in FET cycles. In this retrospective observational study, we evaluated the influence of two distinct post-thaw blastocyst culture spans (2-4 h versus 20-22 h) on clinical pregnancy and live birth rate. A total of n = 1927 frozen-warmed cycles were included in the analysis. Among those, n = 885 warmed blastocysts were cultured for 2-4 h, and n = 1029 were kept in culture for 20-22 h prior to transfer; the remaining blastocysts did not survive the warming protocol. We observed no significant differences in live birth and clinical pregnancy rates between the two groups. The blastocyst morphological evaluation at transfer improved following the longer culture time. No differences between the two groups were found also for gestational and neonatal outcomes. This work shows that different post-thaw embryo culture timings do not negatively impact pregnancy outcomes. Overall, these results are important in the context of the embryological laboratory in order to better organize the workflow and avoid unnecessary timing-related workload.

PSX-B-21 Equine Embryo Viability Based on Morphokinetics

Abstract The objective of this study was to analyze equine embryo morphokinetics and whether or not they were indicative of survivability upon successful implantation into the uterus of a recipient female. In equine reproduction, fertility is a lowly heritable trait that has an exceedingly large financial impact on the ability to grow genetic lines. In order to increase pregnancy rates, producers must have access to accurate decision-making tools. Ten (n = 10) fertilized equine embryos collected via flushing by licensed veterinarian, Dr. Gregg Veneklasen, DVM in Canyon, TX. Flushed embryos were filmed for a thirty-five (35) second observation period with an Iolight Microscope and then processed through video motion magnification (VMM) software Lambda Vue, that amplified embryo activity 300x their normal movement. These VMM embryos were then assessed for time specific morphological changes using Image J software. Embryo morphological dimension shifts previously unseen under a simple microscope became humanly perceptible and measurable. For morula stage embryos, measurements of significance were taken on the inner cell mass (ICM), total embryo area, trophoblast to ICM distance, and finally the vertical, diagonal, and horizontal axis of the sub-zonal distance. Protocols were developed for embryos in the blastocyst stages, where measurements were taken on the vertical, diagonal, and horizontal axis of the total embryo, area of the embryo, notation of general ICM location, and trophoblast thickness around the embryo face. All measurements were taken in microns every five seconds for the thirty-five second recording period. Findings presented that embryos with a greater ICM, total embryo area, and sub-zonal vertical, diagonal, and horizontal distance established pregnancies as frequently as embryos with less momentous differences in distance (P &amp;lt; 0.1). This research has shown the relationship between morphokinetics and pregnancy had no significance of determining pregnancy viability. This resulted in enhanced prediction of embryo transfer techniques based on traditional grading, making pregnancy rates more predictable and consistent.

Evidence and consensus on technical aspects of embryo transfer.

BACKGROUNDUltrasound-guided embryo transfer (US-GET) is a widely performed procedure, but standards for the best practice are not available.OBJECTIVE AND RATIONALEThis document aims to provide an overview of technical aspects of US-GET after considering the published data and including the preparation for the embryo transfer (ET) procedure, the actual procedure, the post-procedure care, associated pathologies, complications and risks, quality assurance and practitioners’ performance.SEARCH METHODSA literature search for evidence on key aspects of the ET procedure was carried out from database inception to November 2021. Selected papers (n = 359) relevant to the topic were analysed by the authors. The following key points were considered in the papers: whether ultrasound (US) practice standards were explained, to what extent the ET technique was described and whether complications or incidents and how to prevent such events were reported. In the end, 89 papers could be used to support the recommendations in this document, which focused on transabdominal US-GET.OUTCOMESThe relevant papers found in the literature search were included in the current document and described according to the topic in three main sections: requirements and preparations prior to ET, the ET procedure and training and competence for ET. Recommendations are provided on preparations prior to ET, equipment and materials, ET technique, possible risks and complications, training and competence. Specific aspects of the laboratory procedures are covered, in particular the different loading techniques and their potential impact on the final outcomes. Potential future developments and research priorities regarding the ET technique are also outlined.LIMITATIONS, REASONS FOR CAUTIONMany topics were not covered in the literature review and some recommendations were based on expert opinions and are not necessarily evidence based.WIDER IMPLICATIONSET is the last procedural step in an ART treatment and is a crucial step towards achieving a pregnancy and live birth. The current paper set out to bring together the recent developments considering all aspects of ET, especially emphasizing US quality imaging. There are still many questions needing answers, and these can be subject of future research.STUDY FUNDING/COMPETING INTEREST(S)No funding. A.D.A. has received royalties from CRC Press and personal honorarium from Cook, Ferring and Cooper Surgical. The other co-authors have no conflicts of interest to declare that are relevant to the content of this article.

P-765 Association between oocyte retrieval technique and number of oocytes retrieved

Abstract Study question Is there an optimal oocyte retrieval (OR) technique to retrieve a maximum number of oocytes and mature oocytes (MII)? Summary answer While certain OR techniques were associated with higher egg to follicle ratios, this did not correlate with more MII oocytes. What is known already While there are multiple studies assessing embryo transfer technique and associated outcomes, as well as practice committee guidelines on performing embryo transfers, there are no data on optimal OR techniques and associated outcomes. Studies have compared laparoscopic, transabdominal, transvesicular and transvaginal OR techniques, and transvaginal OR has become the standard of care. However, there are no data on the preferred transvaginal OR technique for retrieving the most oocytes and MII oocytes per follicle cohort. Study design, size, duration This was a retrospective study where nine attending physicians completed a survey on OR techniques. Responses were confirmed by fellow trainees not involved in the study who had worked with each physician for at least one year. Number of oocytes/follicle cohort, MIIs/follicle cohort and MIIs/oocytes retrieved (%MII) were assessed for each attending’s technique. Data were stratified by number of follicles on ultrasound on day of trigger (&amp;lt;6, 6-10, &amp;gt;10). Participants/materials, setting, methods The parameters evaluated: spatial plane in which the probe was held, direction of retrieval, order of retrieval (by follicle size, or not), retrieval of both large and very small follicles, re-sticking follicles, reversing the probe to retrieve the opposite side, and curetting the follicles or not. The technique with the highest outcome ratio was the referent technique. Adjusted relative risks were calculated controlling for BMI and infertility diagnosis. Main results and the role of chance Physicians had different survey responses, resulting in nine techniques, despite eight physicians training at the same institution. Patient demographics were equivalent between techniques. For &amp;lt;6 follicles, three techniques resulted in significantly fewer oocyte/follicle (0.97 +/- 0.48, 0.95 +/- 0.66, and 0.90 +/- 0.41) compared to the top-performing technique (TPT) (1.11 +/- 0.55). There were no significant differences in MII/follicle or %MII. For 6-10 follicles, two techniques resulted in significantly fewer oocyte/follicle (0.95 +/- 0.39 and 0.93 +/- 0.35) compared to the TPT (1.06 +/- 0.42). A different technique had significantly higher %MII (0.77 +/- 0.19) compared to two other techniques (0.74 +/- 0.21 and 0.72 +/- 0.22) within the 6-10 follicle group. For &amp;gt;10 follicles, two techniques resulted in significantly fewer oocyte/follicle (1.01 +/- 0.42 and 1.07 +/- 0.40) compared to the TPT (1.15 +/- 0.41). These two techniques also resulted in fewer MII/follicle (0.75 +/- 0.33 and 0.81 +/- 0.34) compared to the same TPT (0.87 +/- 0.34). There were no significant differences in %MII for this group. There was no consistent TPT across follicle number groups or for all outcome variables. The parameters most associated with TPT were re-sticking and curetting follicles. Limitations, reasons for caution While statistically significant, some outcome ratios are similar with wide confidence intervals, limiting the clinical significance of these outcomes. We did not evaluate pregnancy and live birth rates so the results from our study cannot be directly correlated to IVF success. Wider implications of the findings There does not appear to be a clear TPT, even for patients with few follicles. Providers who perform OR in a similar fashion to physicians at our institution should feel confident that they obtain equivalent oocyte yields as others. Trial registration number Not Applicable

First report of mule-in-mule pregnancies with live births following embryo transfer

Many of the equid species can interbreed to produce viable but usually infertile offspring, with the mule (♂donkey [2n=62] × ♀mare [2n=64]) being the commonest of these hybrids. Despite its reduced ability to breed successfully, the mule has been shown capable of carrying and rearing horse and donkey foals using between-species embryo transfer (ET). These pregnancies have provided insight into the early development of the conceptus by highlighting the role of the uterine environment on endometrial cup development and lifespan (Allen et al., Reproduction, 1993); 98(1), 55-60). The aim of this preliminary study was to see if a mule (2n=63) was capable of carrying a mule pregnancy to term, which to the authors' knowledge has not previously been reported. Mule embryos were produced in vivo, by inseminating donor horse mares with fresh donkey semen. The uteri of donor mares were flushed on either Day 7.5 or 8 post-ovulation and three grade 1 expanded blastocysts and one grade 1 early blastocyst were recovered. In Argentina, two embryos were transferred using the Wilsher ET technique into each of two anestrous recipient muleson day 8 after initiating treatment with IM estradiol 17β for 3 days followed by long-acting progesterone every 7 days until day 100 of gestation. In Italy, two embryos were simultaneously transferred by a conventional ET technique into a cyclic recipient mule on day 5 after ovulation. The transfers resulted in two singleton pregnancies, in Argentina, and a twin pregnancy, which was manually reduced to a singleton, in Italy. Repeated ultrasound examinations showed normal conceptus and, subsequently, fetal development to term. Spontaneous delivery of three female mule foals occurred on Days 355, 342, and 335 of gestation, respectively and no placental abnormalities were recorded. No complications occurred during either parturition or peripartum, and the resulting mule fillies developed normally to weaning. To the best of the authors’ knowledge, this is the first report of mules foaling mules after embryo transfer. More studies are warranted to describe the hormonal profile of mule-in-mule pregnancy, especially equine Chorionic Gonadotrophin (eCG) which is known to be influenced by both maternal and fetal factors.