Single Embryo Research Articles

Pulsed stimulated Brillouin microscopy enables high-sensitivity mechanical imaging of live and fragile biological specimens

Brillouin microscopy is an emerging optical elastography technique capable of assessing mechanical properties of biological samples in a three-dimensional, all-optical and noncontact fashion. The typically weak Brillouin scattering signal can be substantially enhanced via a stimulated Brillouin scattering (SBS) process; however, current implementations require high pump powers, which prohibit applications to photosensitive or live imaging of biological samples. Here we present a pulsed SBS scheme that takes advantage of the nonlinearity of the pump–probe interaction. In particular, we show that the required pump laser power can be decreased ~20-fold without affecting the signal levels or spectral precision. We demonstrate the low phototoxicity and high specificity of our pulsed SBS approach by imaging, with subcellular detail, sensitive single cells, zebrafish larvae, mouse embryos and adult Caenorhabditis elegans. Furthermore, our method permits observing the mechanics of organoids and C. elegans embryos over time, opening up further possibilities for the field of mechanobiology.

Robust preimplantation genetic testing of the common F8 Inv22 pathogenic variant of severe hemophilia A using a highly polymorphic multi-marker panel encompassing the paracentric inversion

BackgroundHemophilia A (HEMA) is an X-linked bleeding disorder caused by reduced/absent coagulation factor VIII expression, as a result of pathogenic variants in the F8 gene. Preimplantation prevention of HEMA should ideally include direct pathogenic F8 variant detection, complemented by linkage analysis of flanking markers to identify the high-risk F8 allele. Linkage analysis is particularly indispensable when the pathogenic variant cannot be detected directly or identified. This study evaluated the suitability of a panel of F8 intragenic and extragenic short tandem repeat markers for standalone linkage-based preimplantation genetic testing for monogenic disorder (PGT-M) of the Inv22 pathogenic variant, an almost 600 kb paracentric inversion responsible for almost half of all severe HEMA globally, for which direct detection is challenging.MethodsThirteen markers spanning 1 Mb and encompassing both F8 and the Inv22 inversion interval were genotyped in 153 unrelated females of Viet Kinh ethnicity.ResultsAll individuals were heterozygous for ≥ 1 marker, ~ 90% were heterozygous for ≥ 1 of the five F8 intragenic markers, and almost 98% were heterozygous for ≥ 1 upstream (telomeric) and ≥ 1 downstream (centromeric) markers. A prospective PGT-M couple at risk of transmitting F8 Inv22 were fully informative at four marker loci (2 intra-inversion, 1 centromeric, 1 telomeric) and partially informative at another five (2 intra-inversion, 3 centromeric), allowing robust phasing of low- and high-risk haplotypes. In vitro fertilization produced three embryos, all of which clearly inherited the low-risk maternal allele, enabling reliable unaffected diagnoses. A single embryo transfer produced a clinical pregnancy, which was confirmed as unaffected by amniocentesis and long-range PCR, and a healthy baby girl was delivered at term.ConclusionRobust and reliable PGT-M of HEMA, including the common F8 Inv22 pathogenic variant, can be achieved with sufficient informative intragenic and flanking markers.

Melatonin improves the vitrification of sheep morulae by modulating transcriptome.

Embryo vitrification technology is widely used in livestock production, but freezing injury has been a key factor hindering the efficiency of embryo production. There is an urgent need to further analyze the molecular mechanism of embryo damage by the vitrification process. In the study, morulae were collected from Hu sheep uterine horns after superovulation and sperm transfusion. Morulae were Cryotop vitrified and warmed. Nine morulae were in the vitrified control group (frozen), and seven morulae were vitrified and warmed with 10-5 M melatonin (melatonin). Eleven non-frozen morulae were used as controls (fresh). After warming, each embryo was sequenced separately for library construction and gene expression analysis. p < 0.05 was used to differentiate differentially expressed genes (DEG). The results showed that differentiated differentially expressed genes (DEG) in vitrified morulae were mainly enriched in protein kinase activity, adhesion processes, calcium signaling pathways and Wnt, PI3K/AKT, Ras, ErbB, and MAPK signaling pathways compared to controls. Importantly, melatonin treatment upregulated the expression of key pathways that increase the resistance of morulae against vitrification induced damage. These pathways include kinase activity pathway, ErbB, and PI3K/Akt signaling pathway. It is worth mentioning that melatonin upregulates the expression of XPA, which is a key transcription factor for DNA repair. In conclusion, vitrification affected the transcriptome of in vivo-derived Hu sheep morulae, and melatonin had a protective effect on the vitrification process. For the first time, the transcriptome profiles caused by vitrification and melatonin in sheep morulae were analyzed in single embryo level. These data obtained from the single embryo level provide an important molecular mechanism for further optimizing the cryopreservation of embryos or other cells.

Embryo ranking agreement between embryologists and artificial intelligence algorithms

To evaluate the degree of agreement of embryo ranking between embryologists and eight AI algorithms. Retrospective study PATIENTS: A total of 100 cycles with at least eight embryos were selected from the Weill Cornell Medicine database. For each embryo, the full-length time-lapse (TL) videos, as well as a single embryo image at 120 h, were given to five embryologists and eight AI algorithms for ranking. None MAIN OUTCOME MEASURE(S): Kendall rank correlation coefficient (Kendall's τ) RESULTS: Embryologists had a high degree of agreement in the overall ranking of 100 cycles with an average Kendall's tau (K-τ) of 0.70, slightly lower than the inter-embryologist agreement when using a single image or video (average K-τ = 0.78). Overall agreement between embryologists and the AI algorithms was significantly lower (average K-τ = 0.53) and similar to the observed low inter-AI algorithm agreement (average K-τ = 0.47). Notably, two of the eight algorithms had a very low agreement with other ranking methodologies (average K-τ = 0.05) and between each other (K-τ = 0.01). The average agreement in selecting the best-quality embryo (1/8 in 100 cycles with an expected agreement by random chance of 12.5%; CI95: 6-19%) was 59.5% among embryologists and 40.3% for six AI algorithms. The incidence of the agreement for the two algorithms with the low overall agreement was 11.7%. Agreement on selecting the same top-two embryos/cycle (expected agreement by random chance corresponds to 25.0%; CI95: 17-32%) was 73.5% among embryologists and 56.0% among AI methods excluding two discordant algorithms, which had an average agreement of 24.4%, the expected range of agreement by random chance. Intra-embryologist ranking agreement (single image vs. video) was 71.7% and 77.8% for single and top-two embryos, respectively. Analysis of average raw scores indicated that cycles with low diversity of embryo quality generally resulted in a lower overall agreement between the methods (embryologists and AI models). To our knowledge, this is the first study that evaluates the level of agreement in ranking embryo quality between different AI algorithms and embryologists. The different concordance methods were consistent and indicated that the highest agreement was intra-embryologist agreement, followed by inter-embryologist agreement. In contrast, the agreement between some of the AI algorithms and embryologists was similar to the inter-AI algorithm agreement, which also showed a wide range of pair-wise concordance. Specifically, two AI models showed intra- and inter-agreement at the level expected from random selection.

Multiple embryo manipulations in PGT-A cycles may result in inferior clinical outcomes

Research questionDo embryos that undergo a thaw, biopsy and re-vitrification (TBR) for pre-implantation genetic testing for aneuploidy (PGT-A) have different ploidy and transfer outcomes compared with fresh biopsied embryos? DesignRetrospective cohort study of all embryos that underwent the following procedures: fresh biopsy for PGT-A (fresh biopsy); embryos that were warmed, biopsied for PGT-A and re-vitrified (single biopsy TBR); embryos with a no signal result after initial biopsy that were subsequently warmed, biopsied and re-vitrified (double biopsy TBR). The patients who underwent transfers of those embryos at a single academic institution between March 2013 and December 2021 were also studied. ResultsAbout 30% of embryos planned for TBR underwent attrition. Euploidy rates were similar after biopsy: fresh biopsy (42.7%); single biopsy TBR (47.5%) (adjusted RR: 0.99, 0.88 to 1.12); and double biopsy TBR 50.3% (adjusted RR: 0.99, 0.80 to 1.21). Ongoing pregnancy over 8 weeks was not statistically significant (double biopsy TBR: 6/19 [31.6%] versus fresh biopsy: 650/1062 [61.2%]) (adjusted RR 0.52, 95% CI 0.26 to 1.03). The miscarriage rate increased (double biopsy TBR: 4/19 [21.1%] versus fresh biopsy: 66/1062 [6.2%])(RR 3.39, 95% CI 1.38 to 8.31). Live birth rate was also lower per transfer for the double biopsy TBR group (double biopsy TBR [18.75%] versus fresh biopsy [53.75%]) (RR 0.35, 95% CI 0.12 to 0.98), though not after adjustment (adjusted RR 0.37, 95% CI 0.13 to 1.09). These differences were not seen when single biopsy TBR embryos were transferred. ConclusionsEmbryos that undergo TBR have an equivalent euploidy rate to fresh biopsied embryos. Despite that, double biopsy TBR embryos may have impaired transfer outcomes.

The neglected emotional drawbacks of the prioritization of embryos to transfer

In recent years, increasing efforts have been made to develop advanced techniques that could predict the potential of implantation of each single embryo and prioritize the transfer of those at higher chance. The most promising include non-invasive preimplantation genetic testing for aneuploidy and artificial intelligence-based algorithms using time lapse images. The psychological effect of these add-ons is neglected. One could speculate that embarking on another transfer after one or more failures with the prospect of receiving an embryo of lower potential may be distressing for the couple. In addition, the symbolic and mental representation of an embryo with ‘lower capacity to implant’ is currently unknown but could affect couples’ choices and wellbeing. These emotional responses may also undermine adherence to the programme and, ultimately, its real effectiveness. Future trials aimed at evaluating the validity of prioritization procedures must also consider the emotional burden on the couples.

Antibiotic cured chronic endometritis remains a risk factor for early pregnancy loss in the subsequent frozen euploid embryo transfer

Research questionDo patients with antibiotic-cured chronic endometritis (CCE) have a comparable pregnancy outcome to those with non-chronic endometritis (NCE) in the subsequent frozen embryo transfer (FET) cycle? DesignA retrospective cohort analysis included 833 patients in their first FET cycles with single euploid embryo transfer. Chronic endometritis (≥5 CD138+ plasma cells per high-power field [CD138+/HPF]) was treated with standard antibiotic therapy. Patients were classified into two groups: the NCE group (n = 611, <5 CD138+/HPF) and the CCE group (n = 222, ≥5 CD138+/HPF and cured after antibiotic treatment). Pregnancy outcomes were compared. NCE group was divided into subgroup 1 (CD138+/HPF = 0) and subgroup 2 (CD138+/HPF = 1–4) for further analysis. ResultsThe rate of early pregnancy loss (EPL), incorporating all losses before 10 weeks’ gestation, was significantly higher in the CCE group than the NCE group (21.2% versus 14.2%, P = 0.016), and the difference was statistically significant (adjusted odds ratio [AOR] 1.68, 95% confidence interval [CI] 1.11–2.55). No significant differences were observed between the two groups with regard to other pregnancy outcomes. In the subgroup analysis, the EPL rate and biochemical pregnancy rate were significantly higher in subgroup 2 than subgroup 1 (17.2% versus 9.4%, AOR 2.21, 95% CI 1.30–3.74; 12.2% versus 6.9%, AOR 2.01, 95% CI 1.09–3.68). ConclusionsChronic endometritis cured by standard antibiotic therapy remains a risk factor for EPL in FET cycles, although no differences were found in live birth rates between patients with CCE or with NCE.

A cyclin-dependent kinase-mediated phosphorylation switch of disordered protein condensation

Cell cycle transitions result from global changes in protein phosphorylation states triggered by cyclin-dependent kinases (CDKs). To understand how this complexity produces an ordered and rapid cellular reorganisation, we generated a high-resolution map of changing phosphosites throughout unperturbed early cell cycles in single Xenopus embryos, derived the emergent principles through systems biology analysis, and tested them by biophysical modelling and biochemical experiments. We found that most dynamic phosphosites share two key characteristics: they occur on highly disordered proteins that localise to membraneless organelles, and are CDK targets. Furthermore, CDK-mediated multisite phosphorylation can switch homotypic interactions of such proteins between favourable and inhibitory modes for biomolecular condensate formation. These results provide insight into the molecular mechanisms and kinetics of mitotic cellular reorganisation.

Correlations between a deep learning-based algorithm for embryo evaluation with cleavage-stage cell numbers and fragmentation

Research questionDo cell numbers and degree of fragmentation in cleavage-stage embryos, assessed manually, correlate with evaluations made by deep learning algorithm model iDAScore v2.0? DesignRetrospective observational study (n = 5040 embryos; 1786 treatments) conducted at two Swedish assisted reproductive technology centres between 2016 and 2021. Fresh single embryo transfer was carried out on days 2 or 3 after fertilization. Embryo evaluation using iDAScore v2.0 was compared with manual assessment of numbers of cells and grade of fragmentation, analysed by video sequences. ResultsData from embryos transferred on days 2 and 3 showed that having three or fewer cells compared with four or fewer cells on day 2, and six or fewer cells versus seven to eight cells on day 3, correlated significantly with a difference in iDAScore (medians 2.4 versus 4.0 and 2.6 versus 4.6 respectively; both P < 0.001). The iDAScore for 0–10% fragmentation was significantly higher compared with the groups with higher fragmentation (P < 0.001). When combining cell numbers and fragmentation, iDAScore values decreased as fragmentation increased, regardless of cell number. iDAScore discriminated between embryos that resulted in live birth or no live birth (AUC of 0.627 and 0.607), compared with the morphological model (AUC of 0.618 and 0.585) for day 2 and day 3, respectively. ConclusionsThe iDAScore v2.0 values correlated significantly with cell numbers and fragmentation scored manually for cleavage-stage embryos on days 2 and 3. iDAScore had some predictive value for live birth, conditional that embryo selection was based on morphology.

In vivo direct cell-penetrating peptide mediated protein transduction system in Acyrthosiphon pisum

ObjectiveThe principal delivery method for CRISPR-based genome editing in insects is now based on microinjection into single cells or embryos. The direct protein transduction systems cannot be employed in aphids because oogenesis occurs without apparent vitellogenesis. Given the limited timing of injection into the embryonic stage in oviparous aphids, a protein delivery system from the hemolymph to the germline and embryos would be a useful tool for genome editing. This study reports a newly developed direct protein delivery system for aphids using cell-penetrating peptides (CPPs). CPPs are short peptides that translocate across the plasma membrane when bound to cargo proteins.ResultsPenetratin (PEN), a widely conserved CPP among insects, was identified in this study. We used mVenus, a recombinant fluorescent protein, as a visual marker for CPP availability assessments, and fused it with PEN by bacterial protein expression. The mVenus-PEN recombinant proteins were introduced into the hemolymph of adult unwinged Acyrthosiphon pisum females using a nanoinjector. Fluorescence emitted by mVenus-PEN was observed in various tissues, such as the gut, trachea, bacteriocytes, and their progeny. This study shows that PEN can deliver exogenously expressed proteins into tissues in vivo, indicating that CPPs are powerful tools for protein transduction.

Live birth after single euploid frozen embryo transfer in a 39-year-old woman with high-grade mosaic Turner syndrome

Objective To describe the reproductive and obstetric outcomes of an intracytoplasmic sperm injection cycle with preimplantation genetic testing for aneuploidy in an advanced reproductive-age woman with high-grade mosaic Turner syndrome. Methods Case report of a 39-year-old woman diagnosed with mosaic Turner Syndrome 45,X[90]/46,XX[10] karyotype who underwent in vitro fertilization treatment with blastocyst trophectoderm biopsy for preimplantation genetic testing using next-generation sequencing. Result(s) Two of the four blastocysts biopsied were euploid. The patient achieved ongoing pregnancy after the first single euploid frozen embryo transfer, followed by the birth of a healthy child. Conclusion Autologous intracytoplasmic sperm injection cycles can be considered in a select group of advanced reproductive-age women diagnosed with high-grade mosaic Turner syndrome.

Risk factors associated with monozygotic twinning in offspring conceived by assisted reproductive technology.

What are the factors influencing the occurrence of monozygotic (MZ) twins in offspring conceived by assisted reproductive technology (ART)? Parental ages, the transfer of fresh versus frozen embryos, and the grade of blastocysts are all related to MZ twinning in ART offspring. Offspring conceived by ART have significantly increased risk of MZ twins, which may be due to the characteristics of the infertile population. The objective of this study was to explore the incidence of monozygotic (MZ) twins after ART and to clarify the risk factors for MZ twinning. A total of 255 monozygotic twins were enrolled in this cohort study, and then matched with singletons at a ratio of 1:4 randomly (with 1020 in the control group). All offspring were conceived by single embryo transfer. The collected data were divided into the following three aspects for analysis: characteristics of the infertile population, gamete or embryo manipulations, and factors related to embryo development. The incidence of MZ twins was 1.638% (255 out of 15 567 pregnancies after single embryo transfers). Compared to singleton births, a significantly lower rate of frozen embryo transfers (FET; 78.0% vs 86.1% P = 0.002) was seen amongst the MZ twins. Amongst fresh ETs, the rate of blastocyst transfers in the MZ twins group was higher compared to that in the control group (92.9% vs 75.4%, P = 0.005). We also found that certain grades of blastocysts in terms of trophectoderm (TE) development, inner cell mass + TE development and the classification of 'top-quality' embryos were associated with the incidence of MZ twinning (P = 0.025, P = 0.012, P = 0.020, respectively). Logistic regression analysis revealed that higher paternal age (odds ratio (OR) = 0.94, 95% CI = 0.89-1.00, P = 0.029) and FET (OR = 0.48, 95% CI = 0.33-0.68, P = 0.001) may be protective factors against MZ twinning. However, higher maternal age (OR = 1.07, 95% CI = 1.01-1.13, P = 0.027) and the transfer of blastocysts (OR = 4.31, 95% CI = 1.46-12.73, P = 0.008) appeared to be associated with an increased risk of MZ twinning. Amongst blastocyst transfers, a C grade TE may be protective factor against MZ twinning (B: OR = 1.90, 95% CI = 1.18-3.07, P = 0.009; A: OR = 1.58, 95% CI = 0.93-2.67, P = 0.089). First, our definition of MZ twins was based on twins' birth after single embryo transfers (SET), rather than ultrasound examination during early pregnancy. Second, the parental characteristics of the two groups were homogenous, so it was difficult to find any associations between infertility factors and the incidence of MZ twins. This multifaceted analysis of the risk factors for MZ twinning provides some information for clinical interventions in high-risk populations. This study was supported by the National Key Technology Research and Developmental Program of China (2022YFC2704404), CAMS Innovation Fund for Medical Sciences (2021-I2M-5-001), Taishan Scholars Program for Young Experts of Shandong Province (tsqn201909195), the Basic Science Center Program (31988101), and the Shandong Provincial Key Research and Development Program (2020ZLYS02). All authors have no conflicts of interest to declare. N/A.

Methods to improve frozen-thawed blastocyst transfer outcomes- the IVF laboratory perspective

During the last few years, the number of frozen-thawed embryo transfer cycles (FET) significantly increased due to the universal application of more efficient cryopreservation techniques in the IVF laboratory and the improved survival rates of blastocyst stage embryos and the wide implementation of “freeze all” IVF cycles to prevent OHSS, or for preimplantation genetic testing for aneuploidy (PGT-A). Blastocyst cryopreservation allows single embryo transfers to reduce the rate of multiple pregnancies and improve perinatal outcomes. There is no consensus regarding the optimal laboratory protocol for blastocyst cryopreservation, and research is ongoing for its amelioration. This review summarizes different laboratory methods that may improve frozen-thawed blastocyst embryo transfer outcomes, alone or in combination. Some of the techniques relate to embryo survival; some of them work on endometrial receptivity.

Impact of Lactobacillus in the uterine microbiota on in vitro fertilization outcomes

Abundant intrauterine Lactobacillus is associated with good in vitro fertilization (IVF) outcomes; however, whether specific species of Lactobacillus have any benefit remains unclear. So we examine the effect of Lactobacillus on the clinical outcomes of IVF at the species level. Uterine microbiota were classified as either Lactobacillus-dominant (LD) or non-Lactobacillus-dominant. In the LD group, we further investigated the clinical results for each Lactobacillus species and evaluated them in relation to IVF outcomes. In Uterine microbiome analysis, Lactobacillus was the most abundant, with the four species of L. crispatus, L. iners, L. gasseri, and L. jensenii accounting for the great majority. We compared the clinical outcomes of single frozen-thawed embryo transfer conducted by Lactobacillus species and found that the implantation rate was lowest in those in whom L. iners was dominant. This study is the first to conduct a species-level analysis of the uterine microbiota and report on a detailed investigation of Lactobacillus, which was believed to be particularly helpful for pregnancy.

CRIOTOLERANCIA DE EMBRIONES BOVINOS CULTIVADOS INDIVIDUALMENTE EN UN MEDIO SUPLEMENTADO CON L-CARNITINA

&lt;p&gt;&lt;strong&gt;Background:&lt;/strong&gt; L-carnitine is a lipid metabolism enhancer and a potent antioxidant that prevents oxidative damage and improves cryotolerance of bovine embryos. &lt;strong&gt;Objective:&lt;/strong&gt; To determine the effect of L-carnitine during oocyte maturation on developmental competence and cryotolerance of single bovine embryo cultured. &lt;strong&gt;Methodology:&lt;/strong&gt; Embryos were produced in vitro using abattoir-derived cumulus-oocyte complexes (COCs). In experiment 1, two individual maturation, fertilization and culture systems were used in 24-well plates with 20 µL drops of medium covered with mineral oil and 96-well plates with 30 µL drops of medium. In experiment 2, oocytes were randomly distributed into two groups and single matured in 96-well plates in medium supplemented or not with 0.6mg/mL L-carnitine. On day 7 post fertilization, blastocysts were vitrified on solid surface in Fiberplug. Non-vitrified blastocysts were used as control. Embryonic survival after devitrification was determined by blastocysts re-expansion and hatching rate at 24 and 48 hours of post-devitrification culture. Total cell number and apoptotic rate by TUNEL-DAPI staining were used as quality and cryotolerance indicator. In both cases, cleavage and blastocyst rates were evaluated at 48 hours and 7 days post fertilization, respectively. &lt;strong&gt;Results:&lt;/strong&gt; No significant differences were found for embryonic development between single culture systems. There was no effect of L-carnitine supplementation during maturation on embryo development, but embryo survival had increased (P &amp;lt; 0.05) at 24- and 48-hours post devitrification. &lt;strong&gt;Implications:&lt;/strong&gt; Treatment with L-carnitine had increased (P &amp;lt; 0.05) post-thaw re-expansion rates (86.8 ± 3.0 vs 70.0 ± 4.4) and it was similar to non-vitrified control (89.7 ± 2.6). Mean cell number and apoptotic cell index, were similar for all treatment groups. &lt;strong&gt;Conclusion:&lt;/strong&gt; L-carnitine supplementation during maturation, does not improve division rate and subsequent development of single cultured embryos, however increases cryotolerance post devitrification.&lt;/p&gt;

Cooperative contributions of the klf1 and klf17 genes in zebrafish primitive erythropoiesis

Krüppel-like transcription factors (Klfs), which are characterized by the three conserved C-terminal zinc fingers, are involved in various biological processes, such as haematopoiesis and angiogenesis. However, how the Klf family of transcription factors cooperate in organogenesis remains elusive. During zebrafish embryogenesis, both klf1 and klf17 are expressed in the intermediate cell mass (ICM), where primitive erythroid cells are produced. Using CRISPR–Cas9 genome editing technology, we established klf1-klf17 double mutant zebrafish to investigate the functionally interactive roles of the klf1 and klf17 genes. The klf1-klf17 mutant exhibited a diminished number of circulating primitive erythroid cells at 2 days postfertilization (dpf), while klf1 or klf17 single mutants and wild-type embryos produced comparable numbers of primitive erythroid cells. Circulating erythroid cells from the klf1-klf17 mutant possessed larger nuclei at 2 dpf than wild-type cells, suggesting the impairment of primitive erythroid cell maturation. The expression of the erythroid cell maturation markers band3 and mitoferrin, but not the haematopoietic progenitor markers c-myb and scl, was decreased in the klf1-klf17 mutant at 1 dpf. Thus, these results illustrate the cooperative function of klf1 and klf17 in the maturation processes of zebrafish primitive erythroid cells.

Frozen embryo transfer outcomes decline with increasing female body mass index in female but not male factor infertility: analysis of 56,564 euploid blastocyst transfers

ObjectiveTo evaluate the association of body mass index (BMI) with cycle outcomes after euploid frozen blastocyst transfer. DesignRetrospective cohort study. SettingNot applicable. Patient(s)A total of 56,564 first single autologous euploid frozen embryo transfers (FET) from the 2016-2019 Society for Assisted Reproductive Technology database were analyzed by BMI and by World Health Organization BMI cohorts. Sub-analyses were performed on cycles among patients with a sole diagnosis of polycystic ovary syndrome (PCOS) (n = 4,626) and among patients with only male factor (n = 10,854). Intervention(s)None. Main outcome measure(s)Clinical pregnancy, pregnancy loss, and live birth. Results(s): Success rates and adjusted odds ratios (aORs) with 95% confidence intervals (CIs) for all outcomes were most favorable among those with normal BMI and progressively worsened with increasing BMI. These trends persisted among patients with PCOS for clinical pregnancy (aOR 0.99, 95% CI 0.98-0.997, p 0.003), pregnancy loss (aOR 1.02, 95% CI 1.01-1.04, p 0.01), and live birth (aOR 0.98, 95% CI 0.97-0.99, p <0.001), but not among patients with male factor only for clinical pregnancy (aOR 1.00, 95% CI 0.99-1.01, p 0.425), pregnancy loss (aOR 1.01, 95% CI 0.99-1.03, p 0.07), or live birth (aOR 0.99, 95% CI 0.98-1.00, p 0.08). Conclusion(s): In the largest cohort to date, increasing BMI was associated with decreased pregnancy and live birth and increased pregnancy loss following euploid FET among the entire cohort and among patients with a sole diagnosis of PCOS; however, these results were attenuated among patients with a sole diagnosis of male factor infertility, suggesting that associated female infertility diagnoses and not BMI alone may underly this trend.

Increased incidence of live births in implanted day 5 versus day 6 blastocysts following single embryo transfers with PGT-A

Elective single-embryo transfers of euploid or low-level mosaic blastocysts were analyzed in this retrospective study to determine the correlations of live birth (LB) probability with embryonic developmental features of implanted day 5 (D5, n = 245) or day 6 (D6, n = 73) blastocysts using time-lapse (TL) monitoring. According to the logistic regression analyses (adjusted odds ratio [OR] = 0.341, 95% confidence interval [CI] = 0.169–0.685, P < 0.05), the LB probability was negatively associated with the D6 group. The LB rate of the D5 group was higher than the D6 group (88.2% vs. 75.3%; P < 0.05). Compared with the D5 blastocysts, the D6 blastocysts exhibited comparable dysmorphisms except for the multinucleation at the 4-cell stage (10.9% vs. 2.9%, P < 0.05). Moreover, D6 blastocysts had considerably slower developmental kinetics and poorer blastocyst morphologies. Further analysis confirmed that the LB rate was not associated with developmental kinetics or dysmorphisms but rather with blastocyst morphology (inner cell mass [ICM] grade ≤ C vs. ICM grade A, adjusted OR = 0.155, 95% CI = 0.04–0.596, P < 0.05; trophectoderm [TE] grade ≤ C vs. TE grade A, adjusted OR = 0.157, 95% CI = 0.032–0.760, P < 0.05). In conclusion, D6 implanted blastocysts have a considerably lower LB rate than D5 implanted blastocysts. As determined by TL monitoring, the diminished blastocyst morphology can be one of the primary reasons underlying the decreased likelihood of LB.

Association between oocyte donors’ or recipients’ body mass index and clinical outcomes after first single blastocyst transfers—the uterus is the most affected

ObjectiveTo assess if high BMI in either oocyte donors or recipients is associated with poorer outcomes after first single blastocyst transfers. DesignRetrospective study including 1394 first blastocyst single embryo transfers (SETs) conducted by 1394 recipients during oocyte donation cycles with the gametes retrieved from 1394 women (Jan2019-July2021). Four BMI clusters were defined for both donors and recipients (underweight: <18.5; normal weight: 18.5-24.9; over-weight: 25-29.9; obese: ≥30). Subjects1394 recipients, 42.4±4.0 years old and with a BMI 23.2±3.8, and 1394 donors, 26.1±4.2 years old and with a BMI 21.9±2.5. InterventionAll oocytes were vitrified at two egg banks and warmed at eight IVF clinics part of the same network. ICSI, blastocyst culture and either fresh or vitrified-warmed SETs were conducted. Putative confounders were investigated, and the data adjusted through regression analyses. Main outcome measuresThe primary outcome was the live birth rate (LBR) per SET according to donors’ and/or recipients’ BMI. The main secondary outcome was the miscarriage rate (<22nd gestational weeks) per clinical pregnancy. ResultsThe LBR per blastocyst SET showed no significant association with donors’ BMI. Regarding recipients’ BMI, instead, the multivariate-OR was significant in obese versus normal weight recipients (0.58, 95%CI 0.37-0.91, p=0.019). The miscarriage rate per clinical pregnancy was also significantly associated with recipients’ obesity with a multivariate-OR of 2.31 (95%CI 1.18-4.51, p=0.014) versus normal weight patients. A Generalized Additive Model (GAM) method was used to represent the relationship between predicted LBR or miscarriage rates with donors’ or recipients’ BMI; it pictured a scenario where the former outcome moderately but continuously decreases with increasing recipients’ BMI to then sharply decline in the BMI range 25-35. The miscarriage rate, instead, increases almost linearly with respect to both donors’ and recipients’ increasing BMI. ConclusionObesity mostly effects the uterus, especially due to higher miscarriage rates. Yet, poorer outcomes can be appreciated already with a BMI of 25 in both oocyte donors and recipients. Finer markers of nutritional homeostasis are therefore desirable, recipients should be counselled about poorer expected outcomes in case of overweight/obesity, and oocyte banks should avoid assigning oocytes from overweight donors to overweight/obese recipients.

Endometrial microbiota profile in in-vitro fertilization (IVF) patients by culturomics-based analysis.

It is well recognized that the human uterus and adjoining tissues of the female reproductive tract exist in a non-sterile state where dysbiosis can impact reproductive outcomes. The endometrial microbiota is a part of this greater milieu. To date, it has largely been studied using 16S rRNA or metagenomics-based methodologies. Despite the known advantages of sequencing analysis, several difficulties have been noted including sample contamination and standardization of DNA extraction or sequencing. The aim of this study was to use a culturomics-based method to analyze the endometrial microbiota and correlate the results with ongoing pregnancy rates. A prospective cohort study was performed at the University of Naples from June 2022 to December 2022. Ninety-three patients undergoing an IVF cycle with single embryo transfer (ET) (fresh or frozen) were enrolled in the study. Following ET, the catheter tip was inserted into brain heart infusion (BHI) medium under sterile conditions for culture. After 24h and 48h of incubation the microorganisms in the colonies were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Overall, 68 (73,92%) patients resulted positive for one or more microbes and 25 patients (26,08%) had no microbial growth. Across all participants, the four most important phyla were Firmicutes (87,76%), Proteobacteria (27,94%), Actinobacteria (10,29%) and Ascomycota (8,82%). Lactobacillus species, in particular, was significantly correlated with ongoing pregnancy rate (p=0,05). On the other hand, Staphylococcus subspecies (spp.) (p<0,05) and Enterobacteriaceae (p<0,001) were found to have a negative impact on the implantation rate. Detection of bacteria by culturomics from catheter tips used for embryo transfer has been shown to be a reliable method to detect pathogen growth. Endometrial microbiota testing in clinical practice could certainly offer a means to further improve diagnosis and treatment strategies in IVF patients.