P–682 Serum progesterone level as prognostic factor in frozen-thawed embryo transfer cycles: effect of selected threshold on gestational results. Systematic review, stratified meta-analysis and meta-regression

Study question Is there an optimum progesterone threshold level below which gestational results are significantly worse in frozen embryo transfer cycles (FET) with hormone replacement therapy (HRT)? Summary answer Low serum progesterone during luteal phase of HRT-FET cycles impairs substantially its gestational outcomes, regardless of threshold level, origin of oocytes and euploidy of embryos. What is known already HRT for endometrial preparation in FET or oocyte donation cycles is widely used. Oestrogen doses are usually patient-tailored varying upon endometrial thickness, whereas the optimal level of progesterone exposure has not been defined. Various studies have found a negative association between serum progesterone levels measured during luteal phase and FET results in terms of pregnancy and miscarriage rates. Most likely there is an optimal level below which results are worse but a standard threshold level is yet to be established, as in almost every study a different threshold has been found. Study design, size, duration Systematic review and stratified meta-analysis with meta-regression following PRISMA guidelines. An electronic search of MEDLINE, EMBASE, Web of Science, Cochrane Gynaecology and Fertility Specialised Register of Controlled Trials and ClinicalTrials.gov was conducted from inception to January 2021. The aim was to identify prospective or retrospective cohort studies measuring serum progesterone levels around frozen embryo transfer date in HRT cycles. A combination of the following key search terms was used: “progesterone”, “serum”, “frozen embryo”, “transfer”, “frozen-thawed”. Participants/materials, setting, methods Studies analyzing association of luteal serum progesterone with FET-HRT outcomes were included. Risk of bias within studies was assessed using the Newcastle-Ottawa Scale (NOS). Clinical/ongoing pregnancy and miscarriage rates (C/OPR,MR) were considered as primary and secondary outcomes respectively. Odds Ratios with 95% Confidence Interval (OR,95%CI) were calculated applying a random effects model meta-analysis. Heterogeneity was assessed using the I2 statistic. A meta-regression was conducted to examine the association of the effect with the threshold level. Main results and the role of chance The systematic search retrieved 792 studies, 494 after duplicates removal of which 343 were screened and 51 assessed for eligibility. 12 studies, reporting 14 threshold levels, were included in the meta-analysis involving 5009 HRT-FET cycles. Two of them were prospective cohort studies while the rest were retrospective. 10 of them have been published in peer review journals and two were conference abstracts. Quality of studies assessed with NOS varied between 5 and 9. The progesterone threshold ranged from 5.0 to 21.94 ng/ml. Low progesterone levels were associated with less C/OPR (OR: 0.52; 95% CI: 0.40 to 0.66; 11 studies, 5009 cycles). Low progesterone was also associated with high MR (OR: 2.01; 95% CI: 1.57 to 2.58; 9 studies, 2560 pregnancies). These effects showed remarkable consistency in specific sub-analyses considering separately studies with progesterone thresholds up to or above 10 mg/mL, and studies carried out in cycles using oocyte donation, autologous oocytes and embryo aneuploidies screening. Meta-regression did not identify significant association between size effect and progesterone threshold, regarding neither C/OPR (regression coefficient: 0.02; CI 95%: –0.02 to 0.06; p: 0.28) nor MR (regression coefficient: 0.11; CI 95%: –0.13 to 0.36; p: 0.32). Limitations, reasons for caution High degree of clinical and statistical heterogeneity was found due to different routes and doses of progesterone administration, date of progesterone analyses and variety of thresholds as well as high diversity of embryo origin. Despite sensibility analysis by embryo origin any of these sources of heterogeneity can preclude the results. Wider implications of the findings: Despite low progesterone levels are significantly associated to lower gestational results, and a threshold of 10 ng/ml constitutes the median value of our distribution, high quality prospective studies are needed to validate the prognostic value of progesterone levels and to establish an standardised threshold level for clinical application. Trial registration number not required

The Origin of the Cyathea delgadii Sternb. Somatic Embryos Is Determined by the Developmental State of Donor Tissue and Mutual Balance of Selected Metabolites

Somatic embryogenesis is the formation of a plant embryo from a cell other than the product of gametic fusion. The need to recognize the determinants of somatic cell fate has prompted investigations on how endogenous factors of donor tissues can determine the pattern of somatic embryo origin. The undertaking of this study was enabled by the newly developed experimental system of somatic embryogenesis of the tree fern Cyathea delgadii Sternb., in which the embryos are produced in hormone-free medium. The contents of 89 endogenous compounds (such as sugars, auxins, cytokinins, gibberellins, stress-related hormones, phenolic acids, polyamines, and amino acids) and cytomorphological features were compared between two types of explants giving rise to somatic embryos of unicellular or multicellular origin. We found that a large content of maltose, 1-kestose, abscisic acid, biologically active gibberellins, and phenolic acids was characteristic for single-cell somatic embryo formation pattern. In contrast, high levels of starch, callose, kinetin riboside, arginine, and ethylene promoted their multicellular origin. Networks for visualization of the relations between studied compounds were constructed based on the data obtained from analyses of a Pearson correlation coefficient heatmap. Our findings present for the first time detailed features of donor tissue that can play an important role in the somatic-to-embryogenic transition and the somatic embryo origin.

Pathogenic and Transmission Potential of Wildtype and Chicken Embryo Origin (CEO) Vaccine Revertant Infectious Laryngotracheitis Virus

Infectious laryngotracheitis (ILT) is an infectious upper respiratory tract disease that impacts the poultry industry worldwide. ILT is caused by an alphaherpesvirus commonly referred to as infectious laryngotracheitis virus (ILTV). Vaccination with live attenuated vaccines is practiced regularly for the control of ILT. However, extensive and improper use of live attenuated vaccines is related to vaccine viruses reverting to virulence. An increase in mortality and pathogenicity has been attributed to these vaccine revertant viruses. Recent studies characterized Canadian ILTV strains originating from ILT outbreaks as related to live attenuated vaccine virus revertants. However, information is scarce on the pathogenicity and transmission potential of these Canadian isolates. Hence, in this study, the pathogenicity and transmission potential of two wildtype ILTVs and a chicken embryo origin (CEO) vaccine revertant ILTV of Canadian origin were evaluated. To this end, 3-week-old specific pathogen-free chickens were experimentally infected with each of the ILTV isolates and compared to uninfected controls. Additionally, naïve chickens were exposed to the experimentally infected chickens to mimic naturally occurring infection. Pathogenicity of each of these ILTV isolates was evaluated by the severity of clinical signs, weight loss, mortality, and lesions observed at the necropsy. The transmission potential was evaluated by quantification of ILTV genome loads in oropharyngeal and cloacal swabs and tissue samples of the experimentally infected and contact-exposed chickens, as well as in the capacity to produce ILT in contact-exposed chickens. We observed that the CEO vaccine revertant ILTV isolate induced severe disease in comparison to the two wildtype ILTV isolates used in this study. According to ILTV genome load data, CEO vaccine revertant ILTV isolate was successfully transmitted to naïve contact-exposed chickens in comparison to the tested wildtype ILTV isolates. Overall, the Canadian origin CEO vaccine revertant ILTV isolate possesses higher virulence, and dissemination potential, when compared to the wildtype ILTV isolates used in this study. These findings have serious implications in ILT control in chickens.

24 Lipid composition of fresh or frozen sexed bovine blastocysts produced invivo or invitro

Currently, invitro embryo production (IVP) is successfully applied commercially in cattle. However, the high sensitivity of embryos to cryopreservation compared with invivo-derived (IVD) embryos still impairs the dissemination of this biotechnology. Reduced cryotolerance is frequently associated with lipid accumulation in the cytoplasm mainly due to invitro culture conditions. The objective of this study was to evaluate the lipid content of fresh and frozen sexed bovine grade 1 IVP or IVD embryos. The same 8 Holstein heifers were used in a Latin square design for both IVP and IVD embryo production. Zygotes were cultured in synthetic oviductal fluid (SOF) supplemented with 1% oestrus cow serum. The same bull was used for IVP and IVD. All expanded Day 7 blastocysts (n=40 IVP and 40 IVD) were biopsied and sexed. Half of the embryos (n=20 in each group) was slow frozen (1.5M ethylene glycol, 0.1m sucrose) and thawed before lipid extraction. Remaining embryos underwent lipid extraction in the fresh state. Briefly, the liposoluble fraction of the embryos was extracted according to the Bligh and Dyer method using chloroform and methanol. Liquid chromatography–high-resolution mass spectrometry (LC-HRMS) analysis was performed and operated in positive ionization mode. Lipids with variance intensities greater than 30% in quality control samples were removed as well as those identified as background noise. Partial least square discriminant analysis (PLS-DA) was used to show the relationship between variance in the data and difference among embryo origin (IVP vs. IVD), state before extraction (fresh vs. frozen), and sex of the embryos (male vs. female). The differentially lipid species groups were identified using Wilcoxon test, and considered significantly different when P<0.05. LC-HRMS analysis allowed us to identify 75 lipids. PLS-DA showed that embryo origin (IVP vs. IVD) and state before extraction (fresh vs. frozen) can be determined by LC-HRMS profiles by group in PLS-DA plot, despite slight overlaps. Sex of the embryos did not allow us to differentiate the lipid profile. However, 15 lipids varied significantly between male and female IVD, predominantly triglycerides (TG), whereas no lipid varied between the sexes in the IVP homologues. Moreover, 26 lipids varied significantly between IVP and IVD fresh embryos with enrichment of IVP embryos in TG, phosphatidyl choline, cholesteryl ester, and less diglyceride and lysophospholipid (LP) compared with IVD embryos. The comparison of the lipid profiles before and after freezing for IVP embryos showed that only 7 lipids varied significantly between fresh and frozen states with a decrease in LP for the frozen embryos. For the invivo counterparts, 13 lipids varied significantly, including the same LP as those identified for IVP embryos in the same way. Our results showed that the embryonic lipid profile is mainly affected by IVP and slow freezing protocols and, to a lesser extent, by sex. Further studies are needed to improve IVP protocols and optimize the cryotolerance of IVP embryos in cattle.