Stage Embryos Research Articles

Clinical pregnancy rate for frozen embryo transfer with HRT: a randomized controlled pilot study comparing 1 week versus 2 weeks of oestradiol priming

Research questionDoes a frozen-embryo transfer in an artificially-prepared endometrium (FET-HRT) cycle yield similar clinical pregnancy rate with 7 days of oestrogen priming compared to 14 days?DesignThis is a single-centre, randomized, controlled, open-label pilot study. All FET-HRT cycles were performed in a tertiary centre between October 2018 and January 2021. Overall, 160 patients were randomized, with a 1:1 allocation, into two groups of 80 patients each: group A (7 days of E2 prior to P4 supplementation) and group B (14 days of E2 prior to P4 supplementation). Both groups received single blastocyst stage embryos on the 6th day of vaginal P4 administration. The primary outcome was the feasibility of such strategy assessed as clinical pregnancy rate, secondary outcomes were biochemical pregnancy rate, miscarriage rate, live birth rate and serum hormone levels on the day of FET. Chemical pregnancy was assessed by an hCG blood test 12 days after FET and clinical pregnancy was confirmed by transvaginal ultrasound at 7 weeks.ResultsThe analysis included 160 patients who were randomly assigned to either group A or group B on the seventh day of their FET-HRT cycle if the measured endometrial thickness was above 6.5 mm. Following screening failures and of drop-outs, 144 patients were finally included both in group A (75 patients) or group B (69 patients). Demographic characteristics for both groups were comparable. The biochemical pregnancy rate was 42.5% and 48.8% for group A and group B, respectively (p 0.526). Regarding the clinical pregnancy rate at 7 weeks, no statistical difference was observed (36.3% vs 46.3% for group A and group B, respectively, p = 0.261). The secondary outcomes of the study (biochemical pregnancy, miscarriage, and live birth rate) were comparable between the two groups for IIT analysis, as well as the P4 values on the day of FET.ConclusionsIn a frozen embryo transfer cycle, performed with artificial preparation of the endometrium, 7 versus 14 days of oestrogen priming are comparable, in terms of clinical pregnancy rate; the advantages of a seven-day protocol include the shorter time to pregnancy, reduced exposure to oestrogens, and more flexibility of scheduling and programming, and less probability to recruit a follicle and have a spontaneous LH surge. It is important to keep in mind that this study was designed as a pilot trial with a limited study population as such it was underpowered to determine the superiority of an intervention over another; larger-scale RCTs are warranted to confirm our preliminary results.Trial registrationClinical trial number: NCT03930706.

When is warmer better? Disentangling within‐ and between‐generation effects of thermal history on early survival

Abstract Understanding the fitness consequences of thermal history is necessary to predict organismal responses to global warming. This is especially challenging for ectotherms with complex life cycles, where distinct life stages can differ in thermal sensitivity, acclimate to different thermal environments and accrue responses to acclimation within and between generations. Although acclimation is often hypothesized to benefit organisms by helping them (or their offspring) to compensate for negative impacts of environmental change, mixed support for this hypothesis highlights the need to assess alternatives. Assessments that explicitly dissect responses across life stages and generations, however, remain limited. We assess alternative hypotheses of acclimation (none, beneficial, colder‐is‐better and warmer‐is‐better) within and between generations of a marine tubeworm whose vulnerability to warming rests on survival at early planktonic stages (gametes, embryos and larvae). First, we acclimate parents, gametes and embryos to ambient and projected warmer temperatures (17°C and 22°C) factorially by life stage. Next, we rear offspring with differing acclimation histories to the end of larval development at test temperatures from 10°C to 28°C (lower and upper survival limits respectively). Last, we estimate thermal survival curves for development, and compare them among thermal histories. We show that survival curves are most responsive to parental acclimation followed by acclimation at embryogenesis, but are buffered against acclimation at fertilization. Moreover, curves respond independently to acclimation within and between generations, and respond largely as predicted by the warmer‐is‐better hypothesis, despite the semblance of beneficial acclimation after successive doses of warmer temperature. Our study demonstrates the varied nature of thermal acclimation and the importance of considering how responses aggregate across complex life cycles when predicting vulnerability to warming. Read the free Plain Language Summary for this article on the Journal blog.

Age-related advanced glycation end-product accumulation impairs mitochondrial regulation after vitrification†.

Vitrification is an important assisted reproductive technology, although it induces mitochondrial dysfunction in embryos. Herein, we aimed to investigate whether age-associated accumulation of advanced glycation end-products (AGEs) in oocytes impairs the recovery of embryos from cryopreservation-induced mitochondrial dysfunction/damage. Mouse eight-cell stage embryos developed in vitro were vitrified and warmed and incubated up to the blastocyst stage. AGE levels in oocytes were higher in both aged mice and AGE accumulation mouse models (MGO-mice) than those in young and control mice. In addition, the level of SIRT1 upregulation was lower for embryos of aged and MGO-mice than that for embryos of young and control mice. The highest mitochondrial DNA (mtDNA) content was detected in blastocysts derived from vitrified embryos of aged and MGO-mice. The spent culture medium of blastocysts derived from both aged and MGO-mice contained higher mtDNA content than that of the blastocysts derived from young and control mice. EX527 increased mtDNA content in the spent culture medium of vitrified embryos derived from young mice. In addition, p62 aggregate levels were higher in vitrified embryos of control mice than those in vitrified embryos of MGO-mice. The SIRT1 activator, resveratrol, increased p62 aggregation levels in vitrified embryos derived from young and aged mice, whereas vitrification did not affect p62 aggregation levels in embryos from aged mice. Therefore, age-associated AGE accumulation induces decreased responsive SIRT1 upregulation following vitrified-warmed treatment and impairs mitochondrial quality control activity in vitrified embryos.

Effect of non-permeable cryoprotectant sucrose on the development of spotted knifejaw (Oplegnathus punctatus) embryos

In this study, the toxicity of sucrose to Oplegnathus punctatus embryos was evaluated. Embryos at the 4–6 somite, tail-bud, heart formation, and heart-beating stages were exposed to 0, 0.5, 1,1.5, 2, 2.5, or 3 M sucrose for 1 h. Survival rates of embryos at the tail-bud, heart formation, and heart-beating stages after rehydration for 1 h were not affected by treatment with 2 M sucrose (the maximum concentration). Embryos at the tail-bud, heart formation, and heart-beating stages were exposed to 2 M sucrose for 0, 30, 60, 90, 120, 150, or 180 min. Long-term developmental indicators, including rates of survival, hatching, swimming, and malformation, were evaluated for 4 days after rehydration. Based on the survival rates 10 min after rehydration, the longest tolerance time for embryos at the three stages was 120 min. Based on long-term developmental indicators, the longest tolerance times were 60 min at the tail-bud, 60 min at the heart formation stage and 30 min at the heart beating stage. The malformation rates increased as the treatment time increased. The malformation rates were 100% when embryos were exposed to sucrose for ≥120 min. Malformation was divided into larval and embryonic abnormality. As the exposure time increased for tail-bud stage embryos, the rate of larval malformation increased. Treatment at heart formation and heart-beating stages resulted in higher rates of failure to hatch at exposure time. Based on these results, toxicity tests of non-permeable cryoprotectant in embryos requires the observation of development for at least 2 days after rehydration. Based on long-term observation, it was concluded that dehydration before freezing was not the direct cause of larvae deformity that hatched from frozen-thawing embryo. These results provide a reference for the singly use of representative non-permeable cryoprotectant sucrose.

The effect of the egg-predator Carcinonemertes conanobrieni on the reproductive performance of the Caribbean spiny lobster Panulirus argus

BackgroundThe Caribbean spiny lobster Panulirus argus is heavily fished throughout its Greater Caribbean and Gulf of Mexico distribution, suggesting a heightened susceptibility to a fisheries collapse. In 2017, a nemertean worm, Carcinonemertes conanobrieni was described from ovigerous females of P. argus in Florida, USA. A year later, the presence of the same egg predator was recorded along the southern Caribbean coast (Colombia). The effect of this egg predator on the reproductive performance, including fecundity, embryo mortality, and reproductive output, of its host is unknown. This study tested whether C. conanobrieni affects embryo mortality, fecundity, and reproductive output in brooding females of P. argus.ResultsArtisan fishers caught 90 ovigerous lobsters near Pueblo Viejo, Magdalena, Colombia. Each ovigerous female was examined for the presence/absence of the egg predator. Lobster egg mortality (%), fecundity (nº eggs female−1), and reproductive output (%) were estimated. Prevalence of C. conanobrieni in the studied population was 87.78%. The mean intensity of C. conanobrieni (all life stages) in the population was 11.68 (± 1.98) egg predators per brood mass sample. Infected females brooding late-stage embryos exhibited lower fecundity, lower reproductive performance values, and higher embryo mortality compared to infected females brooding early-stage embryos. Embryo stage and worm infection level negatively impacted fecundity and reproductive output. Worm infection level and the number of adult nemertean worms also negatively affected embryo mortality.ConclusionsThese results demonstrate an adverse effect of C. conanobrieni on the reproductive performance of P. argus. The interactive impact of this egg predator, natural stressors, and anthropogenic stressors on individual P. argus reproductive performance could facilitate losses at large-scale fisheries levels.

Daughter cells inherit YAP localization from mother cells in early preimplantation embryos.

The first stage of cell differentiation during mouse development is the differentiation into the trophectoderm and inner cell mass, which occurs during the 8-32-cell stages of preimplantation embryos. This differentiation is regulated by the Hippo signaling pathway. At the 32-cell stage, embryos establish a position-dependent distribution of the Hippo pathway coactivator, Yes-associated protein 1 (YAP, encoded by Yap1). The outer and inner cells showed nuclear and cytoplasmic localization of YAP, respectively. However, the process by which embryos establish position-dependent YAP localization remains elusive. Here, we established a YAP-reporter mouse line, Yap1mScarlet , and examined YAP-mScarlet protein dynamics during the 8-32-cell stages using live imaging. During mitosis, YAP-mScarlet diffused throughout the cells. YAP-mScarlet dynamics in daughter cells varied depending on the cell division patterns. YAP-mScarlet localization in daughter cells at the completion of cell division coincided with that in mother cells. Experimental manipulation of YAP-mScarlet localization in mother cells also altered its localization in daughter cells upon completion of cell division. In daughter cells, YAP-mScarlet localization gradually changed to the final pattern. In some divisions during the 8-16-cell stages, the cytoplasmic YAP-mScarlet localization preceded cell internalization. These results suggest that cell position is not a primary determinant of YAP localization and that the Hippo signaling status of the mother cell is inherited by the daughter cells, which likely contributes to the stabilization of the cell fate specification process beyond cell division.

Fads2b Plays a Dominant Role in ∆6/∆5 Desaturation Activities Compared with Fads2a in Common Carp (Cyprinus carpio).

Highly unsaturated fatty acids (HUFAs) are essential for mammalian health, development and growth. However, most mammals, including humans, are incapable of synthesizing n-6 and n-3 HUFAs. Fish can convert C18 unsaturated fatty acids into n-6 and n-3 HUFAs via fatty acid desaturase (Fads), in which Fads2 is a key enzyme in HUFA biosynthesis. The allo-tetraploid common carp theoretically encode two duplicated fads2 genes. The expression patterns and desaturase functions of these two homologous genes are still unknown. In this study, the full length of the fads2a and fads2b were identified in common carp (Cyprinus carpio). Expression analyses indicate that both genes were mainly expressed in the liver and the expression of fads2b is higher than fads2a at different developmental stages in carp embryos. Heterogenous expression and 3D docking analyses suggested that Fads2b demonstrated stronger ∆6 and ∆5 desaturase activities than Fads2a. The core promotor regions of fads2a and fads2b were characterized and found to have different potential transcriptional binding sites. These results revealed the same desaturase functions, but different activities of two homologues of fasd2 genes in common carp. The data showed that fads2b played a more important role in HUFA synthesis through both expression and functional analyses.

Effects of meiotic stage-specific oocyte vitrification on mouse oocyte quality and developmental competence.

Acquisition of germinal vesicle (GV) stage oocytes for fertility preservation (FP) offers several benefits over in vivo matured oocyte cryopreservation following ovarian stimulation, particularly for cancer patients necessitating immediate treatment. Two FP approaches for GV oocytes are available: vitrification before in vitro maturation (IVM) at the GV stage (GV-VI) or post-IVM at the metaphase II (MII) stage (MII-VI). The optimal method remains to be determined. In this study, mouse oocytes were collected without hormonal stimulation and vitrified either at the GV stage or the MII stage following IVM; non-vitrified in vitro matured MII oocytes served as the control (CON). The oocyte quality and developmental competence were assessed to obtain a better method for immediate FP. No significant differences in IVM and survival rates were observed among the three groups. Nevertheless, GV-VI oocytes exhibited inferior quality, including abnormal spindle arrangement, mitochondrial dysfunction, and early apoptosis, compared to MII-VI and CON oocytes. Oocyte vitrification at the GV stage impacted maternal mRNA degradation during IVM. In addition, the GV-VI group demonstrated significantly lower embryonic developmental competence relative to the MII-VI group. RNA sequencing of 2-cell stage embryos revealed abnormal minor zygotic genome activation in the GV-VI group. Vitrification at the GV stage compromised oocyte quality and reduced developmental competence. Consequently, compared to the GV stage, oocyte vitrification at the MII stage after IVM is more suitable for patients who require immediate FP.

Using of Somatic Embryogenesis for Improving Bolting-resistant New Local Cultivars of Heirloom Carrot Cultivars (Daucus carota) Under Egyptian Conditions

Foreign carrot varieties with desirable morphological traits for the local and global markets must bloom in low temperatures for a long period of time in Egypt, but these climatic conditions are not available for flowering and seed production for these varieties, so it is common to import carrot seeds from abroad. On the other hand, Local cultivars bloom earlier when planted later than the advised date. Due to the heat, it cannot be grown throughout the summer. Pushing the somatic cells to divide and create a new polarity is necessary for the induction of tissues in the lab and the creation of somatic embryos. It was shown that several auxins play a crucial role in the development of these embryos, particularly 2, 4-D (2,4-dichlorophenoxyacetic acid). This experiment was carried out to investigate the effect of five procedures in a tissue culture laboratory using two concentrations of 2, 4-D at a concentration of 2.5 and 5 mg/l for indirect somatic embryos induction from sexual embryos produced from culturing carrot (Daucus carota) seeds of Carrot cv. Kuroda (an heirloom variety). The results confirmed by scanning electron microscopy showed that it is crucial to compare the various processes used to create somatic Embryogenesis to assess their effectiveness. The four stages of somatic embryos, globular, heart, torpedo, and cotyledon form, were observed on the formed callus. The highest amounts of callus fresh weight, the number of globular-shaped stage embryos, the number of heart-shaped stage embryos, the number of torpedo-shaped stage embryos, the number of cotyledon-shaped stage embryos, and the number of seedlings had been recorded in the media supplemented with 2, 4-D at a concentration of 5 mg. This study was able to produce somatic embryos of the foreign carrot cv. Kuroda, Plants obtained from somatic embryos are included in selection programs to produce varieties resistant to early flowering under Egyptian conditions. These results constitute a step in the way in the field of improving bolting-resistant new local cultivars of heirloom carrot cultivars (Daucus carota) under Egyptian conditions.

O-236 Fatty acid supplementation into warming solutions improves pregnancy outcomes after single vitrified-warmed cleavage stage embryo transfer

Abstract Study question Does fatty acid (FA) supplementation into warming solutions affect pre-implantation development, outgrowth competence, and clinical outcomes of human vitrified cleavage stage embryos? Summary answer FA-supplemented solutions improve morphology and outgrowth of blastocysts derived from vitrified cleavage stage embryos and pregnancy outcomes after single vitrified-warmed cleavage stage embryo transfer (SVCT). What is known already Vitrification procedure decreased the intracellular lipid content and subsequent developmental competence. Furthermore, a recent study reported that FA supplementation into warming solutions increased intracellular lipid content and improved developmental competence by stimulating the beta-oxidation pathway in mice and bovines. However, in humans, the effects of FA addition to warming solutions on developmental competence and morphokinetics during the pre-implantation period remain unknown. Additionally, the efficacy of FA supplemented warming solutions in clinical settings has not yet been evaluated. Study design, size, duration A total of 217 discarded human vitrified 4-cell stage embryos donated for research by consenting couples were randomly allocated, to be warmed in solutions either with (FA, n = 111) or without FA (control, n = 106). Embryonic development, morphokinetics, and trophoblast migration were experimentally compared between the groups. Furthermore, a total of 701 clinical records of women who underwent SVCT between April and September 2022 were retrospectively analysed (control, April–June; FA, July–September). Participants/materials, setting, methods Discarded 4-cell stage embryos were warmed and cultured for 72 h in a time-lapse incubator. Furthermore, the blastocysts produced were plated on fibronectin-coated dishes and cultured to assess blastocyst adhesion and outgrowth. In the clinical study, vitrified cleavage stage embryos were warmed in solutions with or without FA and transferred on day 2 after ovulation in the natural cycle. The rates of implantation, clinical pregnancy, and ongoing pregnancy were compared between the groups. Main results and the role of chance The developmental rates were comparable between the groups. However, the rate of morphologically good blastocysts was significantly higher in the FA group than in the control group (P = 0.0089). The developmental timings were comparable between the groups during all stages. Additionally, the incidence of abnormal cleavages, amount of fragmentation at the cleavage and morula stages, and incidence of blastomere exclusion or extrusion during peri-compaction were comparable between the groups. Further, the blastocyst adhesion rate after outgrowth culture was also comparable between the groups. However, the outgrowth area was significantly larger in the FA group than in the control group (P = 0.0438). In the clinical study, the characteristics of the patients and transferred embryos were comparable between the groups. The implantation, clinical pregnancy, and ongoing pregnancy rates were higher in the FA group than in the control group (P = 0.0252, P = 0.0223, and P = 0.0281, respectively). Multivariate logistic regression analysis demonstrated that the probability of ongoing pregnancy was significantly higher in the FA group than in the control group (adjusted odds ratio, 1.46; 95% confidence interval, 1.02–2.06; P = 0.0340). The rate of miscarriage during the first trimester was comparable between the groups. Limitations, reasons for caution This study has some limitations owing to its retrospective design. Further prospective studies are required to validate the clinical efficacy of FA-supplemented solutions. Furthermore, the effects of adding FA to warming solutions on maternal and perinatal outcomes should be assessed in the future. Wider implications of the findings FA supplementation into warming solutions would improve the clinical outcomes of frozen embryo transfers, leading to a shortened treatment period and reduced patient burden in assisted reproductive technologies. Trial registration number not applicable

P-256 Large for gestational age in singletons born following frozen embryo transfer: due to freezing technique or to endometrial preparation protocol? Lessons from the French registry

Abstract Study question Is large for gestational age (LGA) observed in singletons born following frozen embryo transfers (FET) either due to freezing technique or to endometrial preparation protocol? Summary answer Artificial cycles were associated with a higher rate of LGA when compared to ovulatory cycles, whereas no difference was observed between the freezing techniques. What is known already Several studies compared neonatal outcomes after fresh embryo transfer (fresh ET) and FET, and showed that FET was associated with improved neonatal outcomes, including reduced risks of preterm birth, low birth weight and small for gestational age (SGA) when compared to fresh ET. However, these studies also revealed an increased risk of LGA after FET. The underlying pathophysiology remains unclear; parental infertility, laboratory procedures including embryo culture conditions and freezing-thawing processes, as well as endometrial preparation treatments might be at play. Study design, size, duration A multicentric epidemiological data study was performed through a retrospective analysis of the standardized individual clinical records of the French national registry of in vitro fertilisation (IVF) from 2014 to 2018, including deliveries resulting from fresh ET and FET that were prospectively collected in fertility centres. Complementary data were collected from the participating fertility centres including the vitrification media and devices, as well as the endometrial preparation protocols. Participants/materials, setting, methods Data were collected from 35 French fertility centres, leading to the inclusion of a total of 72,789 fresh ET, 10,602 slow-freezing FET and 30,062 vitrification FET cycles. Fetal growth disorders were defined in liveborn singletons according to gestational age and sex-specific weight percentile distribution: SGA and LGA if < 10th and >90th percentile, respectively. Analyses were performed using linear mixed models with the centres as random effect. Main results and the role of chance Among a total of 26,585 liveborn singletons, 16,413 babies were born from fresh ET, 1,644 from slow-freezing FET and 8,528 from vitrification FET. Birthweight was significantly higher in the FET groups compared to the fresh ET group, with no difference between the two freezing techniques. Likewise, LGA rates were higher and SGA rates were lower in the FET groups compared to the fresh ET group (12.0% vs 6.4%, p < 0.001, and 7.8% vs 13.4%, p < 0.001 respectively). In a multivariable analysis, the risk of LGA following FET was not different between stimulated and natural cycles (adjusted odds ratio: 1.06, 95% confidence interval: 0.83-1.35, p = 0.64) but was significantly increased in artificial compared to natural cycles (aOR 1.36 [1.11-1.67], p = 0.003). On the contrary, the risk of LGA was not associated with either the freezing mode (slow-freezing vs vitrification) or the embryo stage (cleaved embryo vs blastocyst). When focusing on vitrification, the risk of LGA was not associated with either the freezing medium used or the embryo stage, and no difference was observed according to the vitrification device. Limitations, reasons for caution Most of the vitrification techniques were performed using the same device, and with two major vitrification media, limiting the interpretability of the comparison of the risk for LGA according to the device or the vitrification media. Wider implications of the findings Our results seem reassuring regarding potential fetal growth disorders following embryo vitrification in comparison to slow-freezing. Even if other factors may be involved, the impact of the endometrial preparation treatments seems to prevail for LGA risk following FET. FET during ovulatory cycles could minimize the risk for fetal growth disorders. Trial registration number N/A

P-551 Association between the length of in vitro culture and mode of ART and the initial se-hCG rise in ongoing singleton pregnancy

Abstract Study question Does the length in vitro and mode of ART (fresh and frozen cycles) impact the se-hCG rise in cycles with ongoing single implantation pregnancy? Summary answer Length in vitro and mode of ART alter the hCG. Blastocysts in FET induced higher hCG than FET clevage stage embryos and fresh blastocyst transfers. What is known already Human chorionic gonadotropin (hCG) is produced by placental trophoblasts providing the first measurable sign of pregnancy. Factors, such as multiple pregnancies, advanced maternal age and gender of the fetus are known to influence hCG levels. Other factors such as mode of ART and length in vitro have been considered to alter the hCG kinetics but with conflicting results. Some studies have shown that hCG levels are higher after fresh blastocyst transfer compared with fresh cleavage stage embryo transfer (ET). Other studies found frozen-thawed embryo transfer (FET) results in higher hCG compared with fresh ET, other studies concluded the opposite. Study design, size, duration Multicenter historical cohort study based on clinical data. The study included 5271 women undergoing in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), insemination (IUI) or FET with single ET followed by hCG measurement day 15-19 after ovulation induction (IVF/ICSI/IUI/natural cycle FET) or progesterone administration (estrogen/progesterone substituted FET cycles). All included cycles resulting in single implantation and ongoing pregnancy verified by ultrasound in week 7-8. Data was collected between January 2014 and December 2021. Participants/materials, setting, methods Data was prospectively collected from the Danish Medical Data Centre used by three public fertility clinics, Copenhagen University hospitals. The association between length in vitro (no in vitro period (IUI), day2/3, day5, day6) and mode of ART (IVF/ICSI, FET) and the initial hCG rise was tested both with linear regression and multivariable regression. The results were based on mean serum hCG and presented in percentage with the corresponding 95% confidence interval (CI). Main results and the role of chance After accounting for the pre-defined exclusion criteria (i.e. oocyte donation, pre-implantation genetic testing, more than one gestational sac), 5251 cycles were included in the study. 2122 were FET, 2521 were fresh ET and 608 were IUI. The initial hCG rise was overall lower for fresh ET compared to IUI (no in vitro period). For cleavage stage ET, hCG was 18% (95% CI: 13% - 23%, p < 0.001) lower, and for blastocyst transfer 23% (95% CI: 18% - 28%, p < 0.001) lower. In FET, hCG was 26% (95% CI: 13% - 40%, p < 0.001) higher for blastocyst transfer compared to cleavage stage ET. When comparing blastocyst transfer in FET vs. fresh ET cycles, hCG was 33% (95% CI: 27% - 45%, p < 0.001) higher in FET cycles. Stratifying FET cycles in natural and substituted cycle did not alter the result. All results remained significant after adjusting for referral diagnosis, women age and treatment center. Limitations, reasons for caution It cannot be excluded that the higher level of hCG in IUI pregnancies is due to an additional vanished implantation. Wider implications of the findings The mechanisms in the embryo and endometrial interplay are far from understood. The present data add to the knowledge regarding this, pointing towards alteration in the implantation window in fresh stimulated cycles. Studies following the more detailed hCG rise are needed for further elucidating how ART affect the early implantation. Trial registration number Journal-nr.: 21019857

P-518 The effect of inactivated SARS-CoV-2 vaccination on the outcome of the first frozen-thawed embryo transfer cycle

Abstract Study question Is there an effect of inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination on the outcome of the first frozen-thawed embryo transfer(FET) cycle. Summary answer Inactivated SARS-CoV-2 vaccination did not affect the outcome of the first FET cycle, including clinical pregnancy rate, early miscarriage rate and newborns’ birth weight. What is known already SARS-CoV-2 vaccination can significantly reduce the risk of severe disease and death after SARS-CoV-2 infection. However, due to the lack of vaccine safety data, the infertile population who plan to undergo ART treatment have high concerns about the safety of vaccine vaccination. To date, there are merely few researches aimed to determine the impact of inactivated SARS-CoV-2 vaccination on reproductive and neonatal outcomes of women attempting FET. Study design, size, duration This was a retrospective cohort study of patients who underwent the first FET cycle in the Reproductive Center of Ningbo Women and Children Hospital from November 1st 2021 to December 19th 2022. All patients were followed up until January 19th 2023. Patients whose age was >40 years old were excluded. The patients were organized into groups based on age, anti-Müllerian hormone (AMH) levels, antral follicle count (AFC), body mass index (BMI), and vaccination or not. Participants/materials, setting, methods Patients fully vaccinated with two doses of Sinopharm or Sinovac inactivated vaccines(Group A, n = 452) were compared with unvaccinated patients(Group B, n = 452) who were matched by age, AMH, AFC and BMI. The baseline characteristics and clinical outcomes of each group were compared.SPSS version 26.0 (SPSS Inc., USA) was used for all data analyses. Main results and the role of chance There were no statistically significant differences in age, AMH, AFC, BMI, infertility duration, infertility type, infertility causes, fertilization type, ovarian stimulation protocol, number of oocytes retrieved(9.24 vs 9.92, p = 0.102), endometrial preparation protocol, endometrial thickness(9.40 vs 9.29mm, p = 0.344), embryo stage, number of embryos for transfer(1.4 vs 1.42, p = 0.589) between two groups. No difference was found between vaccinated and unvaccinated patients in clinical pregnancy rate(55.31% vs 57.52%, p = 0.502), early miscarriage rate(10.4% vs 11.54%, p = 0.681). The newborns’ birth weight were comparable between two groups(2971.44 vs 3156.83g, p = 0.66). Limitations, reasons for caution This is a single-center retrospective study and further accumulated data is warranted to validate the findings. Because of the short follow-up time, some patients are still pregnant, hence we await live birth outcomes. Wider implications of the findings These findings contribute to the evidence regarding the reproductive safety of inactivated SARS-CoV-2 vaccination for fertility-seeking women, and it can provide further theoretical reassurance for doctors and patients to vaccinate before pregnancy. Trial registration number not applicable

P-276 Telomeres cooperate in zygotic genome activation by affecting DUX4/Dux transcription

Abstract Study question Whether the activation of DUX4, a key inducer in the process of zygotic genome activation (ZGA), is associated with telomere length. Summary answer Telomeres regulate the expression of DUX4/Dux through chromatin remodeling and are thereby involved in ZGA. What is known already In human early embryos, the expression of DUX4 is activated as a key inducer in the initial stage of ZGA, and it, in turn, activates hundreds of genes in the cleavage-stage embryo. Human DUX4 is localized to the subtelomeric region 4q35.2 with a D4Z4 repeat of approximately 10 to 100 units that encodes a homeodomain transcription factor. DUX4 expression is inversely proportional to the telomere length in myoblasts/myotubes derived from FSHD patients Study design, size, duration We characterize the dynamics of telomeres during preimplantation development, and assessed the relationship between the expression of DUX4/Dux and telomere length in preimplantation embryos and human embryonic stem cells. Participants/materials, setting, methods All sperm and immature oocytes were collected after obtaining written informed consent from the donor couples. Telomere length in gametes and early embryos by telomere-specific quantitative fluorescence in situ hybridization (Q-FISH). Main results and the role of chance Zygotic genome activation (ZGA) is initiated once the genome chromatin state is organized in the newly formed zygote. While telomeres are specialized chromatin structures at the ends of chromosomes and are reset during early embryogenesis, the details and significance of telomere changes in preimplantation embryos remain unclear. We demonstrated that the telomere length was shortened in the minor ZGA stage and significantly elongated in the major ZGA stage of human and mouse embryos. Expression of the ZGA pioneer factor DUX4/Dux was negatively correlated with the telomere length. ATAC-sequencing suggested that the chromatin accessibility peaks on the DUX4 promoter region (i.e., the subtelomere of chromosome 4q) were transiently augmented in human minor ZGA. Reduction of telomeric heterochromatin H3K9me3 in the telomeric region also synergistically activated DUX4 expression with p53 in hESCs. We propose herein that telomeres regulate the expression of DUX4/Dux through chromatin remodeling and are thereby involved in ZGA. Limitations, reasons for caution Since the mouse Dux gene is not located at the end of the chromosome, the classical telomere position effect (TPE) pathway may not involve in its activation in early embryos. 3D analysis as presented by Hi-C may be able to make a greater breakthrough in confirming the relationship. Wider implications of the findings We herein provided detailed data on changes in the telomere length during ZGA in human and mouse preimplantation embryos, explored the possibility that the TPE affects regulation of DUX4/Dux gene expression in embryos, and suggest that telomere chromatin remodeling is involved in the ZGA process. Trial registration number not applicable

P-667 The attenuated response to elevated serum progesterone levels on pregnancy outcomes of fresh IVF/ICSI cycles in older patients

Abstract Study question Whether there are different impacts of serum progesterone (P) levels on the day of trigger on pregnancy outcomes in patients of different age groups. Summary answer Our results suggest that the pregnancy outcome was less sensitive to progesterone elevation in older patients. What is known already It is now generally believed that the P elevation at the late follicular phase reduces the pregnancy rate by opening the implantation window prematurely and disrupt the embryo-endometrial synchrony. Previous studies further investigated the effect of developmental stages of embryo, number of oocytes retrieved, cause of infertility and different stimulation regimens on the association of P level and the likelihood of pregnancy. However, the study of maternal age on such association is scarce. A previous study found that LH receptor (LHCGR) and progesterone receptor (PGR) were up regulated in advanced age group. Study design, size, duration This was a non-interventional, retrospective cohort study of patients undergoing routine practice in a university-based fertility center between 2015 and 2021. One thousand five hundred and seventeen IVF/ICSI cycles among 1305 patients were included in the analysis. Participants/materials, setting, methods Fresh ET using autologous oocytes with serum P level measured on the day of hCG administration were included. Exclusion criteria include cycles without P measurement, using frozen oocytes, PGT cycles, freeze-all cycles, oocyte donation and no embryo transfer cycles. The patient age was divided into 3 groups: group 1 included patients at age 35 or below, group 2 included patients at age 36 to 39, group 3 included those at 40 years old or above. Main results and the role of chance We performed logistic regression analysis for each group to determine the contributing factors to the live birth outcome. In group 1, age, serum P level, number of usable embryos, embryo grading and endometrial thickness were found to be significantly associated with live birth. In group 2, age and number of usable embryos were significantly associated with live birth. However, total dose of gonadotropin was found to be the sole contributing factor to live birth in group 3. We also divided the P levels into low (≤0.50 ng/mL), median (0.51-1.00 ng/mL) and high (>1.00 ng/mL) and compared the live birth rate in each age groups. The live birth rate was found to be significantly reduced with increasing P levels (40.3% vs. 35.5% vs. 23.6%, p = 0.005). No significant difference in live birth rate between different P levels was found in age group 2 and 3. Limitations, reasons for caution The limitation of the study is its retrospective nature which may have led to bias in the interpretation of the data. There were also significantly higher proportion of cleavage-stage embryo to blastocyst transfer in the older group. Wider implications of the findings The negative association of premature progesterone rise at pre-ovulatory phase to the IVF fresh ET outcome was more prominent for younger patients. This may be one of the contributing factors that accounts for the great heterogeneity in the cut-off of P and its predicting power in different studies. Trial registration number Not applicable

P-469 Body mass index impacts ectopic pregnancy during in vitro fertilization: are we jumping the gun? An analysis of 42,362 clinical pregnancy cycles

Abstract Study question Does female body mass index (BMI) have impact on ectopic pregnancy (EP) in women undergoing in vitro fertilization (IVF) and embryo transfer (ET) treatment? Summary answer EP rate is higher in underweight patients in fresh cycles. However, BMI is not a risk factor for EP after controlling confounders. What is known already It has been shown that tubal factor infertility, stage of embryos transferred (cleavage stage or blastocyst stage), high estrogen levels during ovarian stimulation, and endometrial thickness, have impact on EP rate in IVF cycles. In addition, other potential factors including ovarian stimulation protocols, number of embryos transferred, and type of embryos (fresh or frozen thawed) are also seemed to be associated with EP rate. However, BMI has interactions with these confounding factors mentioned above. Study design, size, duration This was a retrospective cohort study. From January 2010 to June 2022, patients between the ages of 20 and 45 years who had their first fresh/frozen thawed ET cycles, and reported as clinical pregnant were included. BMI was categorized into three groups: underweight (< 18.5 Kg/m2), normal weight (18.5–23.9 Kg/m2), overweight and obesity (≥ 24 Kg/m2). Participants/materials, setting, methods A total of 42,362 cycles were included in the final analysis. The overall ectopic pregnancy rate was 2.57% (1,089/42,362). Firstly, possible factors affecting ectopic pregnancy were screened in both fresh and frozen thawed ET cycles. Then, multivariate logistic regression analysis was used to adjust confounding risk factors. Main results and the role of chance EP rates in fresh, and frozen thawed transfer cycles were 2.43% (672/27,600) and 2.82% (417/14,762), respectively. In fresh ET cycles, the ectopic pregnancy rate was significantly higher in underweight patients when compared with that in normal and overweight/obesity patients (3.29% vs. 2.29% vs. 2.54%; P = 0.029). However, the ectopic pregnancy rate was not differ among patients with different BMI (2.72% vs 2.76% vs 2.96%; P = 0.782) in frozen thawed cycles. In fresh ET cycles, secondary infertility, tubal infertility, elevated estrogen level, thin endometrial thickness, and cleavage stage embryo transfer were risk factors with ectopic pregnancy. However, female BMI was not associated with ectopic pregnancy (adjusted OR: 0.98, P = 0.894, for BMI 18.5-23.9 Kg/m2; adjusted OR: 0.89, P = 0.205, for BMI ≥ 24 Kg/m2. Reference = BMI < 18.5 Kg/m2). In frozen thawed embryo transfer cycles, only thin endometrial thickness, and cleavage stage embryo transfer were risk factors with ectopic pregnancy. Female BMI was not predictable for ectopic pregnancy, either (adjusted OR: 1.15, P = 0.513, for BMI 18.5-23.9 Kg/m2; adjusted OR: 1.24, P = 0.367, for BMI ≥ 24 Kg/m2. Reference = BMI < 18.5 Kg/m2). Limitations, reasons for caution This is a retrospective study with 12 years period. New embryo culture methods and embryo transfer strategy emerged. It is possible that not all confounders have been controlled. Meanwhile, the definition of tubal infertility and pelvic disease need more caution as the lack of laparoscopic examination in some cases. Wider implications of the findings Understanding the impact of BMI on EP during IVF treatment may be useful for predicting results. At the first glance, underweight patients are more likely to encounter with EP during fresh ET. However, it is the high estrogen level, but not low BMI, that contributes to this phenomenon. Trial registration number N/A

P-669 The effect of ovarian follicle size on oocyte fate and embryology outcome

Abstract Study question Does ovarian follicle size influence ultimate oocyte fate and embryology outcome? Summary answer Per punctured follicle, higher oocyte yield, MII oocytes, 2PN fertilisation, and utilisable blastocyst is observed as follicle increases in size up to threshold of 15.5mm What is known already It is well known that oocyte maturity and probability of successful fertilisation is correlated to the size of follicles where oocyte originated from. However the true cutoff and ideal size at oocyte retrieval remains elusive. Furthermore, there is no conclusive evidence that embryological outcome in terms of blastocyst morphology and utilisation differs in the mid to large ovarian follicular sizes. Study design, size, duration Prospective observational study undertaken in a tertiary fertility referral centre over 10 months. 50 participants recruited and underwent conventional controlled ovarian stimulation as per unit protocol. A total of 288 follicular punctures performed in the study period. Participants/materials, setting, methods 50 oocyte retrieval (OR) procedures were performed. The size of ovarian follicles was individually measured using transvaginal ultrasound. 288 follicular punctures were performed and up to 3 oocytes per patient were collected for the study to be individually cultured to allow for tracking of oocyte and embryo outcomes. Follicular fluid and spent culture media were collected separately for subsequent metabolomics analysis. Follicular sizes were divided into 6 groups ( < =9.5, 10-12.5, 13-15.5, 16-19.5, 20-23.5, > =24). Main results and the role of chance Oocyte fate included 113 mature oocytes, 83 bipronuclear (2PN) oocytes, 7 cleavage stage embryos, and 39 good quality blastocysts suitable for utilisation in fresh or frozen transfer cycles. When compared to overall average per punctured follicle, there is an association of higher oocyte yield, proportion of MII oocytes, 2PN fertilisation, and utilisable blastocyst as follicle size increases. The threshold for a change in fate appears to be when follicle size increases beyond 15.5mm where optimal follicle size may reach plateau. Proportion of good-quality blastocysts appears to be associated with size of follicles at rates as follow- 0% (less than 10mm), 11.1% (10-12.5mm), 13.2% (13-15.5mm), 15.9% (16-19.5mm), 14.5% (20-23.5mm), and 20% (above 24mm). 23 embryos have been utilised to date, yielding a 50% positive pregnancy rate. Of these utilised embryos resulting in a positive pregnancy test, 81.8% of embryos originated from follicles above 15.5mm. Metabolomics analysis for follicular fluid and spent culture media is ongoing. Limitations, reasons for caution The observational nature of the study along with a small sample size are limitations that did not allow clinical significance to be observed. Wider implications of the findings Oocyte retrieved from follicles less than 10mm is futile. There appears to be a plateau in optimal follicle size where no real detriment is demonstrated. This is an important findings to guide stimulation, especially in cases of asynchronous growth where sacrifice in larger follicles is commonly assumed. Trial registration number Not applicable

O-145 From mathematical modeling to real-life clinical data: live birth rates up until the tenth blastocyst transfer without PGT-A

Abstract Study question What is the cumulative live birth rate (CLBR) from the first until the tenth consecutive blastocyst transfer, across all age-groups, if no PGT-A is applied? Summary answer Our clinical data confirm existing mathematical models: without PGT-A, a CLBR of 70.0% after three, 88.9% after seven and 95.9% after ten transfers was observed. What is known already The rate of ‘true’ repeated implantation failure (RIF) from endometrial origin is an important topic of debate in our scientific community. Its existence was challenged with the reporting of CLBRs >90% after three consecutive euploid blastocyst transfers. These findings are not directly applicable when PGT-A is not routinely performed. Mathematical models have been developed to tackle this issue by taking into account (1) the anticipated euploidy rate based on the oocyte’s age and (2) the probability of an euploid embryo to implant. So far, no actual patient data have confirmed the validity of this model. Study design, size, duration This is a single-center, retrospective, observational cohort study including 4320 unique women who underwent up to ten consecutive blastocyst transfers between 2010 and 2020. All age categories were included, as well as fresh/frozen and single/double embryo transfers. Patients using donor oocytes, PGT or cleavage stage embryo transfers were excluded or led to drop-out in the study. Participants/materials, setting, methods Patient characteristics were retrieved and cumulative outcomes were analyzed. A Kaplan-Meier curve was plotted for CLBR. For each transfer cycle the live birth rate (LBR), double embryo transfer rate (DET) and multiple pregnancy rate were calculated. Main results and the role of chance The mean age of the patients included in the study was 31.8 (±4.5) years at the time of the first ovarian stimulation. The mean body mass index was 23.7 (± 4.3) kg/m2 and mean basal FSH 6.8 (±2.3) IU/L. The mean number of stimulations per patient was 1.21 (±0.5) and a total mean number of 4.6 (±3.2) blastocysts were obtained per patient. The mean time to the pregnancy leading to live birth was less than one year (0.3±0.7). We observed the highest LBR (46.3%, 2002/4320) after the first blastocyst transfer followed by 35.4% (593/1672), 32.3% (275/851), 32.2% (147/456), 32.8% (78/238), 21.3% (26/122), 23.9% (17/71), 25.6% (10/39), 31.8% (7/22), 18.2% (2/11) for the second until the 10th transfer, respectively. The Kaplan-Meier curve showed CLBRs to mount from 70.0% after the third, up to 88.9% after the seventh and 95.9% after the tenth blastocyst transfer. We noticed a steady increase in DET rate from only 6.4% in the first up to > 27% as of the fourth and >44% as of the seventh transfer with acccompanied multiple pregnancy rates of 1.3% over 10.9% and 11.8%, respectively. Limitations, reasons for caution The main limitation is the study’s retrospective nature. As our center performs cleavage stage and blastocyst transfer, the included population is a good prognosis one as embryology allowed to perform extended culture. Nevertheless, to investigate RIF due to endometrial causes, this could also be seen as an asset. Wider implications of the findings These patient data confirm mathematical models and further question the prevalence of insurmountable, endometrial origin RIF. Preserved LBRs (even in higher cycle ranks) and a CLBR >95% provide hope and reassurance to couples with failed embryo transfers and encourage them to continue treatment if blastocysts are available. Trial registration number NA

O-050 The impact of embryo biopsy on obstetric and neonatal outcomes

Abstract Background Preimplantation genetic testing of embryos (PGT) was performed first in 1990 to prevent the transmission of X-linked disease. Since then, the technique has improved to include couples who are both carriers of the same autosomal recessive disorder, to those with balanced translocations having recurrent pregnancy loss, and to women with advanced maternal age who are at higher risk for having aneuploid embryos. Originally, PGT involved biopsy of the polar body or of one blastomere of a cleavage stage embryo for diagnosis. The contemporary practice of PGT involves the culture of embryos to the blastocyst stage (5-7 days after fertilization), with biopsy of several trophectoderm cells that would become the placenta, transport of biopsy tissue off-site for genetic analysis, and cryopreservation of blastocysts until biopsy results are available. A single euploid embryo is then transferred into the uterus as a frozen embryo transfer. Objective We sought to determine if removal of these cells for preimplantation genetic testing was associated with adverse obstetrical or neonatal outcomes after frozen-thawed single embryo transfer compared to frozen-thawed single embryo transfer without biopsy. Further, we review other studies of contemporary practice of PGT to draw conclusions about the safety of the practice. Study design We linked assisted reproductive technology surveillance data from the Society for Assisted Reproductive Technology Clinic Outcome Reporting System to birth certificates and maternal and neonatal hospitalization discharge diagnoses in Massachusetts, USA, from 2014-2017, considering singleton births after frozen-thawed single embryo transfer. We compared outcomes of cycles having embryo biopsy (n = 585) to those having no biopsy (n = 2,191), adjusting for mother’s age, race, education, parity, body mass index, birth year, insurance, and all infertility diagnoses (Sites CK et al, Am J Obstet Gynecol 2021;225:285.e1-7). Results With no biopsy used as the reference, we found no differences between no biopsy and biopsy groups with respect to preeclampsia, pregnancy-induced hypertension, placental disorders (placental abruption, placenta previa, placenta accreta, placenta increta, and placenta percreta), preterm birth, low birthweight, cesarean delivery, gestational diabetes mellitus, or maternal or infant length of stay after delivery. Comparing our outcomes to 4 other studies of contemporary PGT practice of frozen-thawed embryo transfers (Zhang WY Fertil Steril 2019, Li M Am J Obstet Gynecol 2020, Makhijani R Hum Reprod 2021, He H Fertil Steril 2019), our data are consistent with no effect of biopsy on low birth weight, pregnancy-induced hypertension, gestational diabetes, placenta previa, and placenta accreta. A slight decrease in preterm delivery (RR 1.10, CI 1.02-1.18), slight reduction in cesarean section (RR 0.90, CI 0.82-0.99), and increase in intrauterine growth restriction (RR 1.21, CI 1.06-1.38) are reported with biopsy in a systematic review (Hou W, Fertil Steril 2021). The increase in risk for intrauterine growth restriction with biopsy was influenced by one study (Li M Am J Obstet Gynecol 2020). Conclusion Embryo biopsy of trophectoderm cells for PGT in contemporary practice appears to be safe generally with respect to maternal and neonatal outcomes. There is no clear increase in diagnoses related to placentation (preeclampsia, pregnancy-induced hypertension, placental disorders, preterm birth, low birthweight), cesarean delivery, gestational diabetes mellitus, or maternal or infant length of stay after delivery. Further study of a possible increase in intrauterine growth restriction with biopsy is indicated.

P-228 Reliability of blastomere versus trophectoderm biopsy in preimplantation genetic testing for mitochondrial DNA disorders

Abstract Study question Can a single blastomere or trophectoderm (TE) biopsy accurately reflect the heteroplasmy levels of the whole embryo for mitochondrial DNA (mtDNA) disorders? Summary answer Heteroplasmy levels of single blastomere and TE are comparable to those of the rest of the embryo, including the inner cell mass (ICM). What is known already Oocytes of women carrying mtDNA mutations can display varying mutation loads – percentage of mutated mtDNA copies. Preimplantation genetic testing (PGT) aims at reducing the risk of having affected children by screening for embryos with minimal mtDNA heteroplasmy. The technique involves either a blastomere biopsy on cleavage stage embryos or TE biopsy from blastocysts, followed by genetic analysis to select embryos suitable for transfer. However, it remains unclear whether there is a uniform segregation of heteroplasmic mtDNA during the early stages of embryogenesis, and therefore whether the mutation load from blastomere or TE biopsies are representative of the whole embryo. Study design, size, duration We investigated the suitability of PGT for mtDNA disorders by comparing the mutation load between a single blastomere recovered at the eight-cell stage, TE cells and rest of the same blastocyst (n = 15) in a mouse model heteroplasmic for the m.5024C>T mutation. We also compared the levels of heteroplasmy between TE and ICM of heteroplasmic human blastocysts (n = 5) and between blastomeres of arrested cleavage stage embryos (n = 4) donated by two mtDNA disease patients. Participants/materials, setting, methods To obtain heteroplasmic mouse embryos, we mated female mice carrying a mitochondrial tRNAAla mutation (m.5024C>T) with wild-type males. Whereas in human experiments, we analysed preimplantation embryos from two different carriers of the m.3243A>G mutation, associated with MELAS syndrome, who showed 16% and 32% mutation load in their peripheral blood. After embryo biopsy, NGS was performed in three independent technical replicates and profiles were compared between samples from the same embryo to assess heteroplasmy levels. Main results and the role of chance Analysis of heteroplasmic mouse embryos showed a strong correlation in mtDNA heteroplasmy between blastomere and the corresponding embryo (r2 = 0.76), and between TE and the rest of the blastocyst (r2 = 0.94) (n = 15). Moreover, a strong correlation was observed between the biopsied blastomere and TE from the same embryo (r2 = 0.90). Heteroplasmy levels in the embryos analysed ranged from 55.3% to 88.3%, and the mean overall mutation load was 73.7 ± 7.9%. Development of biopsied embryos was comparable to those of non-manipulated mutant (n = 10) and wild-type embryos (n = 10) (blastocyst rate = 85.7%). For human blastocysts (n = 5), statistical analysis showed a strong correlation in heteroplasmy levels between ICM and TE portions (r2 = 0.98). Moreover, a modest correlation was observed among blastomeres of cleavage stage human embryos (r2 = 0.60) (n = 24 from 4 embryos). The mutation load in the embryos analysed ranged from 10.3% to 57.4%, and the mean overall heteroplasmy was 42.8 ± 12.2% in cleavage stage embryos and 38.6 ± 17.3% in blastocysts. Overall, concordance in heteroplasmy levels between single blastomeres, TE portions and corresponding blastocyst was established in human and mouse embryos. Limitations, reasons for caution These results should be further validated by increasing the sample size and by the inclusion of different mtDNA mutations. Furthermore, current data are suggestive, but not definitive, to guarantee that the mtDNA mutation load in the embryo will remain constant throughout life. Wider implications of the findings Taken together, results suggest that TE biopsy may be better than blastomere biopsy for mtDNA disorders. Moreover, biopsy at the blastocyst stage is likely less harmful for embryo development. Following, we will also investigate how heteroplasmic mtDNA segregates at the peri-implantation stages after the resumption of mtDNA replication. Trial registration number Not applicable