关于胚胎植入的关键代谢物研究

Fetus loss in early pregnancy is of major concern to both humans and animals, and this issue is largely influenced by embryo implantation. Chenodeoxycholic acid (CDCA), a primary bile acid, contributes to metabolic improvements and protects against intrahepatic cholestasis of pregnancy. However, the effect of CDCA on embryo implantation during early pregnancy has not been investigated. The present study demonstrated that CDCA administration during early pregnancy improved embryo implantation in sows and rats, thereby improving the pregnancy outcomes of sows. CDCA significantly reduced inflammation, oxidative stress, and insulin resistance. The metabolomics analysis indicated significant differences in the fecal metabolome, especially regarding the level of secondary bile acids, between the control and CDCA-treated sows. CDCA also influenced the serum metabolite profiles in sows, and the serum L-Histidine level was significantly correlated with the abundance of 19 differential fecal metabolites. Importantly, L-Histidine administration improved embryo implantation and metabolic health in rats during early pregnancy. Moreover, CDCA administration during early pregnancy also led to long-term metabolic improvements in sows. Our data indicated that CDCA improved embryo implantation by alleviating inflammation and oxidative stress, improving insulin sensitivity, and modulating the interaction between the gut microbiota and host metabolites. Therefore, CDCA intervention is a potential therapeutic strategy regarding embryo loss during pregnancy.

Background Metabolites in spent embryo culture medium correlate with the embryo’s viability. However, there is no widely accepted method using metabolite dada to predict successful implantation. We sought to combine metabolomic profiling of spent embryo culture medium and clinical variables to create an implantation prediction model as an adjunct to morphological screening of day 3 embryos. Methods This investigation was a prospective, nested case-control study. Forty-two day 3 embryos from 34 patients were transferred, and the spent embryo culture medium was collected. Twenty-two embryos implanted successfully, and the others failed. Metabolites in the medium relevant to implantation were detected and measured by Liquid Chromatography-Mass Spectrometry. Clinical signatures relevant to embryo implantation were subjected to univariate analysis to select candidates for a prediction model. Multivariate logistical regression of the clinical and metabolomic candidates was used to construct a prediction model for embryo implantation potential. Results The levels of 13 metabolites were significantly different between the successful and failed groups, among which five were most relevant and interpretable selected by Least Absolute Shrinkage and Selection Operator regression analysis. None of the clinical variables significantly affected day 3 embryo implantation. The most relevant and interpretable set of metabolites was used to construct a prediction model for day 3 embryo implantation potential with an accuracy of 0.88. Conclusions Day 3 embryos’implantation potential could be noninvasively predicted by the spent embryo culture medium’s metabolites measured by LC-MS. This approach may become a useful adjunct to morphological evaluation of day 3 embryos.

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Evaluating the quality of embryos and implantation potential is a critical determinant of in vitro fertilization and embryo transfer, and it is also one of the main challenges of assisted reproductive technology. A reliable non-invasive method to choose the best candidate with real implantation potential for transfer from two day-3 embryos with equal morphological quality is still lacking clinically. In this article, a sensitive LC-MS method was developed and metabolomic profiling analysis in a 3-day embryo culture medium was performed. Differential metabolites were analysed in two kinds of commercial culture media, and a total of 66 common metabolites were obtained from 106 independent samples in 5 batches. The relationship between changes in key metabolite, benzoic acid, concentration and the embryo implantation result was discovered. This work improved coverage through conditional optimization, enhanced the reliability of omics data through multi-batch validation, and provided a potential biomarker for evaluating the implantation potential of day-3 embryos.

Decidualization of the uterine endometrium is a critical process for embryo implantation in mammals, primarily occurring on gestational day 8 in pregnant mice. However, the interplay between the maternal gut microbiome, metabolism, and the uterus at this specific time point remains poorly understood. This study employed a multi-omics approach to investigate the metabolic, gut microbiome, and transcriptomic changes associated with early pregnancy (gestational day 8 (E8)) in mice. Serum metabolomics revealed a distinct metabolic profile at E8 compared to controls, with the differential metabolites primarily enriched in amino acid metabolism pathways. The gut microbial composition showed that E8 mice exhibited higher alpha-diversity and a significant shift in beta-diversity. Specifically, the E8 group displayed a decrease in pathogenic Proteobacteria and an increase in beneficial Bacteroidetes and S24-7 taxa. Transcriptomics identified myriads of distinct genes between the E8 and control mice. The differentially expressed genes were enriched in pathways involved in alanine, aspartate, and glutamate metabolism, PI3K-Akt signaling, and the PPAR signaling pathway. Integrative analysis of the multi-omics data uncovered potential mechanistic relationships among the differential metabolites, gut microbiota, and uterine gene expression changes. Notably, the gene Asns showed strong correlations with specific gut S24-7 and metabolite L-Aspartatic acid, suggesting its potential role in mediating the crosstalk between the maternal environment and embryo development during early pregnancy. These findings provide valuable insights into the complex interplay between the maternal metabolome, the gut microbiome, and the uterine transcriptome in the context of early pregnancy, which may contribute to our understanding of the underlying mechanisms of embryo implantation and development.

ABSTRACT Intercellular communication is essential for healthy embryo development, yet the role and dynamics of extracellular space in the maternal‐embryonic dialogue remain unclear. Furthermore, little is known of maternal and embryonic metabolic states during early endometrial preparation and after the embryo enters the uterine cavity. Using a human in vitro co‐culture model and extracellular vesicle (EV)‐specific tools, we dynamically tracked EV secretion, uptake and processing between embryonic and endometrial cells at early stages of cell‐to‐cell communication. Hormonal stimulation altered endometrial secretory output, producing distinct EV populations. Stimulated EVs (St‐EVs) differed from non‐stimulated EVs (NSt‐EVs) in size, secretion dynamics, uptake efficiency and metabolic cargo, selectively packaging energy‐related metabolites and aryl hydrocarbon receptor (AhR) ligands. AhR inhibition increased spheroid attachment, suggesting that AhR signalling regulates implantation by modulating the endometrial environment. Additionally, lipid droplets (LDs) affected by endometrial‐ and embryo‐derived EVs were actively secreted and taken up by embryonic cells, highlighting their role in implantation. EVs were not only exchanged between the embryo and endometrium but were also rapidly internalized, influencing mitochondrial activity, lipid metabolism and extracellular matrix remodelling. Translation of EV‐derived mRNA occurred within 1 h of uptake, driving cellular changes and enhancing embryo attachment. These findings suggest EVs, extracellular metabolites and LDs mobilized between the endometrium and embryo coordinate to promote embryo attachment and implantation. This study advances our understanding of embryo‐maternal EV‐mediated communication and provides a valuable model for investigating EV‐mediated simultaneous intercellular bidirectional crosstalk in other biological contexts.

Abstract Objective To investigate whether prior endometrium spectroscopy predicts the success of embryo transfer in patients with recurrent implantation failure (RIF). Materials and methods Twenty women with RIF who were scheduled for IVF-ET and six fertile women were included the study. All women with RIF and fertile controls underwent endometrium spectroscopy before embryo transfer. A multi-voxel spectroscopy sequence was used for the detection of choline (Cho), creatine (Cr), lactate, and lipids. Women with RIF were divided into two subgroups as successful (n = 8) and unsuccessful RIF (n = 12) according to both Cho and Cr metabolite characteristics and whether pregnancy was achieved. Associations between the metabolite intensities of the RIF subjects and the implantation rate (IR) and clinical pregnancy rate (CPR) were assessed. Results While 8 of 20 RIF cases became pregnant, pregnancy could not be achieved in 12 cases. The common spectroscopy finding in all 8 cases who conceived was high Cho and low lactate. The main metabolite change detected in 12 patients who could not conceive was the increase in lactate and lipid signals. The cutoff value of Cho, Cr, lactate and lipid were 1.01 ppm, 1.44 ppm, 0.86 ppm and 1.22 ppm respectively in patients who achieved pregnancy following ET. A positive and significant correlation was found between Cho and Cr intensities and IR or CPR. Conclusions Receptive endometrium represents some specific metabolites in spectroscopy that can be used for prediction of the success or failure of embryo transfer in women suffering implantation failure.

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The anti-implantation effects of high oestradiol (E2) concentrations could be mediated by E2 metabolites. Herein, we examined whether 2-methoxyoestradiol (2ME) impairs embryo implantation via its target protein F-spondin. Mice on Day 3 of pregnancy were treated with E2 concomitantly with the cathecol-O-methyl transferase inhibitor OR486 and the number of implanted embryos was recorded 5 days later. The effect of 2ME or 4-methoxyoestradiol (4ME) on embryo implantation was also investigated. Plasma and uterine levels of 2ME were measured 0.5, 1 or 3h after E2 treatment while the mRNA for spondin 1 (Spon1) and F-spondin were determined in the uterus 3, 6, 12 or 24h after 2ME treatment. Finally, the effect of a neutralising F-spondin antibody on the anti-implantation effect of 2ME was explored. OR486 blocked the anti-implantation effect of E2; 2ME, but not 4ME, affected embryo implantation. The 2ME concentration was increased after 0.5 and 1h in plasma and 3h in uterine fluid following E2 treatment. 2ME increased levels of Spon1 at 12 and 24h although F-spondin was increased at 12h. F-spondin antibody blocked the effect of 2ME on embryo implantation. We conclude that 2ME impairs mouse embryo implantation via activation of F-spondin in the uterus.

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Assisted reproductive technology (ART) is the most effective method to treat infertility and the pathogenesis of implantation failure after in vitro fertilization-embryo transfer (IVF-ET) is a challenging filed in infertility. Microbes in the female reproductive tract are considered to be associated with gynecological and obstetric diseases. However, its effects on embryo implantation failure are unsured. This study aimed to investigate reproductive tract dysbiosis, identify different bacteria in reproductive tract as potential biomarkers of embryo implantation failure and demonstrate the pathogenesis through metabolites analysis. We compared the data from 16S rRNA gene and metagenome in reproductive tracts through QIIME2 and HUMAnN2 by the times of embryo implantation failure on 239 infertile patients and 17 healthy women. Our study revealed a strong positive correlation between Lactobacillus abundance and embryo implantation success (IS) after IVF-ET. The microbial community composition and structure in reproductive tract showed substantially difference between the embryo implantation failure (IF) and healthy control. Moreover, we established a diagnostic model through receiver operating characteristic (ROC) with 0.913 area under curve (AUC) in IS and multiple implantation failures (MIF), verified its effectiveness with an AUC = 0.784 demonstrating microbial community alterations could efficiently discriminate MIF patients. Metagenome functional analyses of vaginal samples from another independent infertile patients after IVF-ET revealed the L-lysine synthesis pathway enriched in IF patients, along with ascended vaginal pH and decreased Lactobacillus abundance. This study clarifies several independent relationships of bacteria in vagina and endometrial fluid on embryo implantation failure and undoubtedly broadens the understanding about female reproductive health.

Background: While economically vital, buffalo exhibits low reproductive efficiency largely due to embryonic losses during implantation. Successful implantation requires precise embryo–maternal communication and metabolic/immune adaptations in the endometrium. We aimed to identify key serum metabolic signatures and associated peripheral immune responses that characterize the endometrial receptivity window during early pregnancy in water buffalo. Methods: Blood samples from pregnant (Preg, n = 12) and non-pregnant (Non-P, n = 10) buffaloes were collected on days 15, 18, and 21 post-artificial insemination (AI). We measured leukocyte counts and hormone levels and performed untargeted serum metabolomic profiling using LC-MS. Results: Pregnant buffaloes showed significantly reduced total white blood cell count, lymphocyte (LYM%), and neutrophil (NEU%) percentages, indicating immune remodeling at the beginning of pregnancy establishment. Metabolomic analysis identified 131 differentially expressed metabolites (DEMs) associated with pregnancy status at different stages. Enriched pathways included steroid hormone synthesis, retinol metabolism, starch/sucrose metabolism, and phenylalanine biosynthesis. Crucially, alterations in unsaturated fatty acids, retinol, and phenylalanine metabolism, along with monocyte (MON%)/LYM% ratios, were strongly linked to receptivity changes and successful implantation. Conclusions: Endometrial receptivity in buffalo during the embryonic implantation window was associated with changes in immune cells and metabolism in the blood, suggesting that immunometabolism may play an essential role in modulating endometrial receptivity during the implantation window. This study provides potential clues and a metabolic framework for understanding the underlying mechanisms of buffalo embryonic implantation.

Assisted reproductive technology (ART) is used widely and efficiently to treat infertility. During the ART procedure, one of the main factors affecting the success rate is abnormal development of preimplantation embryos. The establishment and maintenance of developmental competence are precisely regulated at different levels, while minor errors at early stages may result in adverse outcomes, including developmental arrest and implantation failure. As one of the major inputs, the regulatory mechanisms of metabolites in embryonic development are less known. In this study, we investigated the functional relevance of the metabolic enzyme serine hydroxymethyltransferase 2 (SHMT2) and deoxyribonucleotide (dNTP) metabolites in mouse preimplantation embryonic development. By using a well-characterized SHMT2 inhibitor, SHMT-IN-2, we effectively inhibited the catalytic activity of the SHMT2 enzyme, which led to developmental arrest at the pronuclear stage of the embryo. A low-input liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and applied for detecting dNTP content in embryos. We found that SHMT2 inhibition led to an insufficient dTTP supply and replication stress during the first mitotic cleavage, thereby causing failure of pronuclear fusion and developmental arrest. Our findings demonstrate a specific mechanism where, apart from building blocks of DNA, the availability of dNTPs contributes to the control of mouse preimplantation embryonic development.

Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer ubiquitously found in the environment. Due to its biological activity, it is classified as an endocrine-disrupting chemical and reproductive toxicant. DEHP and its metabolites have been detected in women with various infertility-related pathologies, and their concentrations have been associated with reduced embryo quantity and quality, implantation failure, and miscarriage in humans. The formation of the inner cell mass and trophectoderm in blastocysts is a critical fate decision for continued development and cellular differentiation, accompanied by the expression of GATA6, OCT4, and CDX2. This study tested whether DEHP induces deleterious conformational changes in blastocysts, potentially leading to reduced implantation rates. Adult female CD-1 mice were exposed to vehicle (corn oil) or DEHP (0, 20, 200, or 2000 μg/kg/day) orally for one month. The 2000 μg/kg/day dose induced oocyte and embryo fragmentation. Embryo developmental arrest was evident at DEHP doses of 200 and 2000 μg/kg/day. DEHP affected the levels and expression patterns of GATA6, OCT4, and CDX2 at doses of 200 and 2000 μg/kg/day. These doses also impacted the number and functionality of blastocysts. Furthermore, DEHP doses of 200 and 2000 μg/kg/day impaired endometrial implantation capacity, as evidenced by the failure to implant normal blastocysts from untreated females using transcervical embryo transfer. Collectively, these data suggest that oral exposure to DEHP for one month affects the expression of GATA6, OCT4, and CDX2, consequently reducing implantation capacity.

This study investigated whether metabolomic fingerprints of bovine embryo growth media improve the prediction of successful embryo implantation. In this prospective cohort study, the metabolome from in vitro-produced day 7 blastocysts with successful implantation (n = 11), blastocysts with failed implantation (n = 10), and plain culture media without embryos (n = 5) were included. Samples were analyzed using an AbsoluteIDQ® p180 Targeted Metabolomics Kit with LC-MS/MS, and a total of 189 metabolites were analyzed from each sample. Blastocysts that resulted in successful embryo implantation had significantly higher levels of methionine sulfoxide (p < 0.001), DOPA (p < 0.05), spermidine (p < 0.001), acetylcarnitine-to-free-carnitine ratio (p < 0.05), C2 + C3-to-free-carnitine ratio (p < 0.05), and lower levels of threonine (nep < 0.001) and phosphatidylcholine PC ae C30:0 (p < 0.001) compared to control media. However, when compared to embryos that failed to implant, only DOPA, spermidine, C2/C0, (C2 + C3)/C0, and PC ae C30:0 levels differentiated significantly. In summary, our study identifies a panel of differential metabolites in the culture media of bovine blastocysts that could act as potential biomarkers for the selection of viable blastocysts before embryo transfer.

An effective tool to assess embryo quality in the assisted reproduction clinical practice will enhance successful implantation rates and mitigate high risks of multiple pregnancies. Potential biomarkers secreted into culture medium (CM) during embryo development enable rapid and noninvasive methods of assessing embryo quality. However, small volumes, low biomolecule concentrations, and impurity interference collectively preclude the identification of quality-related biomarkers in single blastocyst CM. Here, we developed a noninvasive trace multiomics approach to screen for potential markers in individual human blastocyst CM. We collected 84 CM samples and divided them into high-quality (HQ) and low-quality (LQ) groups. We evaluated the differentially expressed proteins (DEPs) and metabolites (DEMs) in HQ and LQ CM. A total of 504 proteins and 189 metabolites were detected in individual blastocyst CM. Moreover, 9 DEPs and 32 DEMs were identified in different quality embryo CM. We also categorized HQ embryos into positive implantation (PI) and negative implantation (NI) groups based on ultrasound findings on day 28. We identified 41 DEPs and 4 DEMs associated with clinical implantation outcomes in morphologically HQ embryos using a multiomics analysis approach. This study provides a noninvasive multiomics analysis technique and identifies potential biomarkers for clinical embryo developmental quality assessment.

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Notwithstanding the great improvement of ART, the overall rate of successful pregnancies from implanted human embryos is definitely low. The current routine embryo quality assessment is performed only through morphological criteria, which has poor predictive capacity since only a minor percentage of those in the highest class give rise to successful pregnancy. Previous studies highlighted the potentiality of the analysis of metabolites in human embryo culture media, useful for the selection of embryos for implantation. In the present study, we analyzed in blind 66 human embryo culture media at 5 days after in vitro fertilization with the aim of quantifying compounds released by cell metabolism that were not present as normal constituents of the human embryo growth media, including purines, pyrimidines, nitrite, and nitrate. Only some purines were detectable (hypoxanthine and uric acid) in the majority of samples, while nitrite and nitrate were always detectable. When matching biochemical results with morphological evaluation, it was found that low grade embryos (n = 12) had significantly higher levels of all the compounds of interest. Moreover, when matching biochemical results according to successful (n = 17) or unsuccessful (n = 25) pregnancy, it was found that human embryos from the latter group released higher concentrations of hypoxanthine, uric acid, nitrite, and nitrate in the culture media. Additionally, those embryos that developed into successful pregnancies were all associated with the birth of healthy newborns. These results, although carried out on a relatively low number of samples, indicate that the analysis of the aforementioned compounds in the culture media of human embryos is a potentially useful tool for the selection of embryos for implantation, possibly leading to an increase in the overall rate of ART.

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Cellular metabolism provides fundamental components for chromatin dynamics and epigenetics. However, metabolic features, and their impact on epigenetic remodeling during mammalian pre-implantation development, remain poorly understood. In this study, we established the metabolic landscape of mouse pre-implantation embryos from zygote to blastocyst, and quantified their absolute carbohydrate metabolites. We found that amino acid/carbohydrate/lipid metabolism increased during pre-implantation development, whereas nucleotide metabolism increased at the 4- and 8-cell stages. We further demonstrated that oxidized nicotinamide adenine dinucleotide (NAD+) is indispensable for mouse pre-implantation development. Mechanistically, NAD+ avoids excessive minor zygotic gene activation (ZGA) by cooperating with deacylase SIRT1 to remove zygotic H3K27ac. In human, NAD+ supplement can promote the removal of zygotic H3K27ac and benefit preimplantation development. Our findings demonstrate that precise and timely regulation of minor ZGA is controlled by metabolic dynamics, and enhance our understanding of the metabolism of mammalian early embryos.

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Graphical abstract Abstract Sex steroids are converted to bioactive metabolites and vice versa by endometrial steroid-metabolising enzymes. Studies indicate that alterations in this metabolism might affect endometrial receptivity. This pilot study determined whether the endometrial formation and inactivation of 17β-oestradiol differed between the supposedly embryo-receptive endometrium and non-receptive endometrium of women undergoing IVF/intracytoplasmic sperm injection (ICSI). Endometrial biopsies were obtained from IVF/ICSI patients 5–8 days after ovulation in a natural cycle, prior to their second IVF/ICSI cycle with fresh embryo transfer (ET). Endometrial biopsies from patients who achieved clinical pregnancy after fresh ET (n = 15) were compared with endometrial biopsies from patients that did not conceive after fresh ET (n = 15). Formation of 17β-oestradiol (oxidative 17β-hydroxysteroid dehydrogenases (HSDs)), oestrone (reductive HSD17Bs) and inhibition of HSD17B1 activity were determined by high-performance liquid chromatography. The endometrial transcriptome was profiled using RNA sequencing followed by principal component analysis and differentially expressed gene analysis. The false discovery rate-adjusted P < 0.05 and log fold change >0.5 were selected as the screening threshold. Formation and inactivation of 17β-oestradiol resulted similar between groups. Inhibition of HSD17B1 activity was significantly higher in the non-pregnant group when only primary infertile women (n = 12) were considered (27.1%, n = 5 vs 16.2%, n = 7, P = 0.04). Gene expression analysis confirmed the presence of HSD17B1 (encoding HSD17B1), HSD17B2 (encoding HSD17B2) and 33 of 46 analysed steroid metabolising enzymes in the endometrium. In the primary infertile subgroup (n = 10) 12 DEGs were found including LINC02349 which has been linked to implantation. However, the exact relationship between steroid-metabolising enzyme activity, expression and implantation outcome requires further investigation in larger, well-defined patient groups. Lay summary Sex hormones are produced and broken down by enzymes that can be found in the endometrium (the inner lining of the womb). This enzyme activity might influence the chances of becoming pregnant. We compared (i) enzyme activity in the endometrium of 15 women who did and 15 women who did not become pregnant in their second in vitro fertilisation attempt, (ii) how enzyme activity can be blocked by an inhibitor, and (iii) differences in gene expression (the process by which instructions in our DNA are converted into a product). Enzyme activity was similar between groups. We found that in women who have never been pregnant in the past, inhibition of enzyme activity was higher and found differences in a gene that has been linked to the implantation of the embryo, but future studies should be performed in larger, well-defined patient groups to confirm these findings.

In vitro fertilization-embryo transfer (IVF-ET) is now widely applied for treating infertility, and unexplained recurrent implantation failure (RIF) has become a substantial challenge. We hypothesize that vaginal microbial dysbiosis is associated with RIF, as it is linked to many female reproductive diseases. In this study, we characterized the vaginal microbiota and metabolomes of patients with unexplained RIF, while patients who achieved clinical pregnancy in the first IVF cycle were set as controls. In general, significant differences were discovered in the vaginal microbiota and metabolomes between the two groups. This study is the first detailed elaboration of the vaginal microbiota and metabolites associated with RIF. We believe that our findings will inspire researchers to consider the dynamics of microbiomes related to the microenvironment as a critical feature for future studies of nosogenesis not only for RIF but also for other reproductive diseases. ABSTRACT Recurrent implantation failure (RIF) refers to repeated failure to become pregnant after transferring embryos with normal morphology. However, the pathogenesis of RIF remains unrevealed, especially for those without any pathological features. In this study, we characterized the vaginal microbiota and metabolomes of patients with unexplained RIF, while patients who achieved clinical pregnancy in the first frozen embryo transfer (FET) cycle were used as controls. Based on 16S rRNA gene sequencing of the vaginal microbiota, the vaginal Lactobacillus showed a significant positive correlation with the pregnancy rate, and the RIF group presented higher microbial α-diversity than the control group (P value = 0.016). The metabolomic profile identified 2,507 metabolites, of which 37 were significantly different between the two groups (P value < 0.05, variable importance for the projection [VIP] > 1). Among them, 2′,3-cyclic UMP and inositol phosphate were the top two metabolites that were higher in the RIF group, while glycerophospholipids and benzopyran were important metabolites that were lower in the RIF group. A lack of lysobisphosphatidic acid and prostaglandin metabolized from glycerophospholipids will lead to deferred implantation and embryo crowding. Benzopyran, as a selective estrogen receptor modulator, may affect the outcome of pregnancy. All of the changes in metabolite profiles may result in or from the differential microbiota compositions in RIF patients. In conclusion, significant differences were presented in the vaginal microbiota and metabolomes between patients with unexplained RIF and women who became pregnant in the first FET cycle. For the first time, this study elaborates the possible pathogenesis of RIF by investigating the vaginal microbiota and metabolites in RIF patients. IMPORTANCE In vitro fertilization-embryo transfer (IVF-ET) is now widely applied for treating infertility, and unexplained recurrent implantation failure (RIF) has become a substantial challenge. We hypothesize that vaginal microbial dysbiosis is associated with RIF, as it is linked to many female reproductive diseases. In this study, we characterized the vaginal microbiota and metabolomes of patients with unexplained RIF, while patients who achieved clinical pregnancy in the first IVF cycle were set as controls. In general, significant differences were discovered in the vaginal microbiota and metabolomes between the two groups. This study is the first detailed elaboration of the vaginal microbiota and metabolites associated with RIF. We believe that our findings will inspire researchers to consider the dynamics of microbiomes related to the microenvironment as a critical feature for future studies of nosogenesis not only for RIF but also for other reproductive diseases.

In cattle, conceptus development after elongation relies on well-characterized, paracrine interactions with the hosting maternal reproductive tract. However, it was unrecognized previously that the pre-hatching, pre-implantation bovine embryo also engages in biochemical signalling with the maternal uterus. Our recent work showed that the embryo modified the endometrial transcriptome in vivo. Here, we hypothesized that the embryo modulates the biochemical composition of the uterine luminal fluid (ULF) in the most cranial portion of the uterine horn ipsilateral to the corpus luteum. Endometrial samples and ULF were collected post-mortem from sham-inseminated cows and from cows inseminated and detected pregnant 7 days after oestrus. We used quantitative mass spectrometry to demonstrate that the pre-hatching embryo changes ULF composition in vivo. Embryo-induced modulation included an increase in concentrations of lipoxygenase-derived metabolites [12(S)-HETE, 15(S)-HETE] and a decrease in the concentrations of amino acids (glycine), biogenic amines (sarcosine), acylcarnitines and phospholipids. The changed composition of the ULF could be due to secretion or depletion of specific molecules, executed by either the embryo or the endometrium, but initiated by signals coming from the embryo. This study provides the basis for further understanding embryo-initiated modulation of the uterine milieu. Early embryonic signalling may be necessary to guarantee optimal development and successful establishment of pregnancy in cattle.

Exploring strategies to prevent miscarriage in women or early pregnancy loss in mammals is of great importance. Manipulating maternal lipid metabolism to maintain sufficient progesterone level is an effective way. To investigated the embryo loss and progesterone synthesis impacts of short and medium chain fatty acids on the lipid metabolism, pregnancy outcome and embryo implantation were investigated in rats fed the pregnancy diets supplemented without or with 0.1% sodium butyrate (SB), 0.1% sodium hexanoate (SH), or 0.1% sodium caprylate (SC) during the entire pregnancy and early pregnancy, respectively, followed with evaluation of potential mechanisms. Maternal SB, SH, or SC supply significantly improved live litter size and embryo implantation in rats. Serum progesterone, arachidonic acid, and phospholipid metabolites levels were significantly increased in response to maternal SB, SH, and SC supply. The expression of key genes involved in ovarian steroidogenesis and granulosa cell luteinization were elevated in ovaries and primary cultured granulosa cells, including cluster of differentiation 36 (CD36), steroidogenic acute regulatory protein (StAR), and cholesterol side-chain cleavage enzyme (CYP11A1). Additionally, the expression of lysophosphatidic acid receptor 3 (LPA3) and cyclooxygenase-2 (COX2) related with phospholipid metabolism were enhanced in uterus in vivo and in in vitro cultured uterine tissue. In conclusion, maternal SB, SH and SC supply reduced early pregnancy loss through modulating maternal phospholipid metabolism and ovarian progesterone synthesis in rats. Our results have important implications that short or medium chain fatty acids have the potential to prevent miscarriage in women or early pregnancy loss in mammals.

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An increasing number of studies have related the mitochondrial DNA (mtDNA) content to embryo viability and transfer outcomes. However, previous studies have focused more on the relationship between mtDNA and embryo implantation, few studies have studied the effect of the mtDNA content on live birth. In the study, we investigated whether mtDNA content is a reliable screening biomarker for live birth after single blastocyst transfer. A total of 233 couples with 316 blastocyst stage embryos undergoing in vitro fertilization treatment and pre-implantation genetic testing analysis were included in the study. All embryos were chromosomally normal and had undergone single-embryo transfers. There was no significant difference observed in the blastocyst mtDNA content among the live birth, miscarriage and non-implanted groups (p=0.999), and the mtDNA content in blastocysts from the miscarriage and live birth groups was similar [median (interquartile range), 1.00*108(7.59*107- 1.39*108) vs 1.01*108 (7.37*107- 1.32*108)]. Similarly, no significant association was observed between mtDNA content and embryo implantation potential (p=0.965). After adjusting for multiple confounders in a logistic regression analysis with generalized estimating equations, no associations between mtDNA content and live birth were observed in all blastocysts, Day-5 and Day-6 blastocysts (p=0.567, p=0.673, p=0.165, respectively). The live birth rate was not significantly different between blastocysts with an elevated mtDNA content and blastocysts with a normal mtDNA content (26.7% vs 33.6% p=0.780). Additionally, there was no linear correlation between the mtDNA content and maternal age (p=0.570). In conclusion, the mtDNA content does not seem to be a potential biomarker for embryo transfer outcomes (i.e., implantation and live birth) based on the existing testing tools. Embryos with an elevated mtDNA content also have development potential for successful live birth.

The relationship between mitochondrial DNA (mtDNA) copy number and the outcome of embryo transfer is under debate. The aim of this study was to explore the relationship between mtDNA copy number in human blastocysts and embryonic development to determine whether mtDNA represents a novel biomarker for the prediction of implantation potential. A total of 246 blastocysts were analyzed by next-generation sequencing. There was no correlation between mtDNA copy number and maternal age in all blastocysts groups and euploid blastocysts groups. Additionally, the mtDNA copy number was not significantly higher in aneuploid blastocysts. Subsequently, no relationship was observed between mtDNA copy number and blastocyst quality. The assessment of clinical pregnancy outcome after the transfer of euploid blastocysts to the uterus indicated that the mtDNA copy number was significantly lower in the clinical pregnancy group than in those who failed implantation. The cut-off value of mtDNA copy number was 320.5, which was a highly predictive value. Blastocysts with an elevated mtDNA copy number had lower implantation potential and mtDNA copy number was largely equal in terms of maternal age, chromosome ploidy, and quality of blastocysts.

Background It is highly desirable to develop new strategies based on secretomics to more accurately selection of embryos with the highest developmental potential for transfer. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been reported to promote embryo development and pregnancy establishment. However, the predictive value of GM-CSF in single blastocyst selection remains unclear. This study is to determine the concentration of GM-CSF in human single-blastocyst conditioned medium (SBCM) and to evaluate its association with embryo quality and pregnancy outcome. Methods The patients with ≤38 years of age receiving the first cycle of assisted reproductive therapy were included in this study. The patients who had <4 top-quality embryos formed by the fertilized two pronuclear zygotes on day 3 were excluded. A total of 126 SBCM samples (SBCMs) were included, of which blastocysts from 77 SBCMs were later transferred in subsequent frozen-thawed embryo transfer. The concentrations of GM-CSF were detected by single-molecule array (SIMOA) and analyzed for their possible association with embryo quality and pregnancy outcomes. The top-quality embryo (TQ), positive HCG (HP), clinical pregnancy (CP), and ongoing pregnancy (OP) rates were determined and compared between groups divided based on GM-CSF concentrations. Results The detection rate of GM-CSF was found to be 50% in all SBCMs. There were significant differences in TQ rate, HP rate, CP rate and OP rate among high concentration group, medium concentration group and low concentration group. Both GM-CSF alone or GM-CSF combined with the morphological score (MS) had a greater AUC of ROC curve than that of MS alone to predict the pregnancy outcome, and GM-CSF combined with MS had the highest AUC. Conclusions The concentration of GM-CSF in SBCM was detected at fg/ml levels, which was associated with embryo quality and pregnancy outcome. Collectively, GM-CSF may be used as a biomarker for prediction of pregnancy outcome and selection of embryos with high developmental potential for transfer in assisted reproductive technology (ART).

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MicroRNAs, secreted by the embryo in blastocoel fluid (BF) and embryo spent culture medium (SCM), regulate important cellular pathways controlling the stemness of inner cell mass, trophectoderm differentiation, and the dialogue between blastocyst and maternal tissues. In recent years, their role as non-invasive biomarkers of embryo quality has been deeply investigated. We compared the expression profiles of 96 microRNAs between BF and SCM from the same embryos, highlighting the differences between these two compartments. We found 10 and 6 microRNAs specifically expressed in BF and in SCM, respectively; 22 microRNAs significantly up-regulated in BF; and 2 significantly up-regulated in SCM. To investigate the role of SCM microRNAs in implantation, we focused on the microRNAs specifically expressed/up-regulated in SCM and absent in blank medium. We deepened our understanding of SCM microRNA’s biological role by building a network of miRNA–mRNA interaction within the signalling pathways crucial in embryo implantation success. We demonstrated that BF and SCM contain different sets of microRNAs playing different and unique roles in embryo implantation and development. Finally, we suggest that there is not a single “ideal” technique to identify the most competent embryo, but an integrated approach is needed to obtain informative results on the health of the embryo.

Objective This study aims to find whether microRNAs (miRNAs) detected in the culture medium of embryos produced in vitro could be potential biomarkers of embryo implantation. Methods Culture media samples from 36 embryos, derived from patients undergoing intracytoplasmic sperm injection (ICSI) in a private university-affiliated IVF center, were collected between January/2015 and November/2015. Samples were collected on day three and embryo transfers were performed on day five and all embryos reached the blastocyst stage. Samples were split into groups according to the embryo implantation result: Positive-Implantation-Group (n=18) or Negative-Implantation-Group (n=18). For the first analysis, samples were pooled in three sets for each group (6-7 spent media per pool). MicroRNAs were extracted from spent media and cDNA was synthesized. C. elegans miR-39 was used as RNA spike-in to normalize the gene expression analysis. The expression of microRNAs into the spent media from the Positive-Implantation-Group was compared with those from the Negative-Implantation-Group. A set of seven miRNAs (miR-21, miR-142-3p, miR-19b, miR-92a, miR-20b, miR-125a and miR148a) selected according with the literature, was tested. To check whether miRNAs could be detected in individual samples of culture media, in a second analysis, ten more samples were tested for miR-21 and miR-142-3p. Results From the sevens tested miRNAs, a significant increased expression of miR-142-3p could be noted in the Negative-Implantation-Group (P<0.001). For other three miRNAs (miR-21, miR-19b and miR-92a) a difference in expression was observed, however it did not reach a statistical significance. In addition, when ten non-redundant samples were tested to check if miRNAs could be detected in individual samples of culture media, the highly specific amplification of mature miRNAs, including miR-142-3p, could be noted. Conclusion Our findings suggest that miR-142-3p, previously described as a tumor suppressor and cell cycle inhibitor, may be a potential biomarker of blastocyst implantation failure. The identification of miRNAs on individual culture medium samples offers unique opportunities for non-invasive early diagnosis of blastocyst implantation.

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The rate of pregnancy can be affected by many factors in assisted reproductive technology (ART), and one of which is the quality of embryos. Therefore, selecting the embryos with high potential is crucial for the outcome. 15 spent blastocyst medium (SBM) samples were collected from 14 patients who received in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), 7 from high-grade embryos and 8 from low-grade embryos. Cell-free RNA (cf-RNA) profile of SBM samples were analyzed by RNA sequencing in this study. It was found that a large amount of cf-RNA were released into SBM, including protein coding genes (68.9 %) and long non-coding RNAs (lncRNAs) (17.26 %). Furthermore, a high correlation was observed between blastocyst genes and SBM genes. And the cf-mRNAs of SBM were highly fragmented, and CDS and UTR regions were released equally. 232 differentially expressed genes were identified in high-grade SBM (hSBM) and low-grade SBM (lSBM), which could be potential biomarker in distinguishing the embryos with different quality as an alternative or supplementary approach for subjective morphology criteria. Hence, cf-RNAs sequencing revealed the characterization of circulating transcriptomes of embryos with different quality. Based on the results, the genes related to blastocyst quality were screened, including the genes closely related to translation, immune signaling pathway and amino acid metabolism. Overall, this study showed the types of SBM cf-RNAs, and the integrated analysis of cf-RNAs profiling with morphology grading displayed its potential in predicting blastocyst quality. This study provided valuable scientific basis for non-invasive embryo selection in ART.

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The hyperglycemia in pregnancy (HIP) is classified as the gestational diabetes mellitus (GDM) and pre-gestational diabetes mellitus (PGDM). Metabolomic changes during pregnancy have been suggested to underlie the etiology of HIP, which influence fetal organogenesis and placental development. On one hand, the PGDM women suffer from hyperglycemic exposure in earlier gestation than GDM. On the other, type 2 diabetes mellitus (T2DM) is the most common type of PGDM. Therefore, it is of clinical implication to detect the metabolic alterations of T2DM pregnant women, especially for those with refractory and uncontrolled hyperglycemia. We aimed to figure out the metabolic profile of umbilical cord blood among pregnant women with T2DM and its influences on the fetal growth. We included 48 uncontrolled T2DM singleton pregnancies and 52 matched healthy singleton pregnancies. Metabolites in umbilical cord blood were measured by liquid chromatography‒mass spectrometry analysis. We observed significant increases in the levels of several metabolites in umbilical cord blood samples from patients with uncontrolled T2DM, including alisol A, glycodeoxycholic acid, 3-quinolinecarboxylic acid- 7,8-dichloro-1,4-dihydro-4-oxo-, fraxetin, sphingosine, alpha-D-glucose, L-glutamine, isoalantolactone, L-leucine, and voriconazole. The abundance of alisol A was negatively correlated with the level of glucose in umbilical cord blood, and the abundance of L-leucine was positively correlated with the level of insulin in umbilical cord blood. The pathways of biosynthesis of amino acids and butanoate metabolism were enriched in the uncontrolled T2DM group. Further, the abundance of alisol A and L-leucine were positively correlated with the fetal weight in the uncontrolled T2DM group. The metabolic profile of umbilical cord blood among pregnant women with uncontrolled T2DM was different from that of the healthy pregnant women. The metabolic profile of umbilical cord blood from the uncontrolled T2DM women was characterized by the enrichment of the pathways of biosynthesis of amino acids and butanoate metabolism and the metabolites of alisol A and L-leucine.

Blood vessel formation is a critical process for successful pregnancy establishment and placenta formation. Angiogenic factors such as vascular endothelial growth factor (VEGF), angiopoietins (ANGPTs) or fibroblast growth factor 2 (FGF2) are known to be involved in angiogenesis. However, the mechanism regulating their expression in the porcine endometrium and trophoblast has not been described during early pregnancy establishment. Recently, we reported an important role for prostaglandin F2α (PGF2α) in supporting processes accompanying the peri-implantation period in the pig. The aim of the present study was to determine the effect of PGF2α on angiogenic factor gene and protein expression at the embryo-maternal interface and on capillary-like structure formation by endometrial endothelial cells. In the present study, we used various in vitro models involving endometrial tissue explants, primary porcine trophoblast and endometrial endothelial cells, as well as a swine umbilical vein endothelial cell line (G1410). ANGPT1, ANGPT2 and FGF2 gene expression was analyzed in porcine endometrial explants and in primary trophoblast cells incubated with PGF2α (100 nM, 1 μM). VEGFA gene expression and protein secretion by porcine primary trophoblast cells were studied in vitro using primary trophoblast cells. A network formation assay using the G1410 cell line and primary endothelial cells of endometrial origin was performed to assess the effect of PGF2α on capillary-like structure formation. We found that PGF2α stimulated VEGFA gene expression (1 μM) and secretion of this protein (100 nM) by porcine trophoblast cells (P < 0.05). In endometrial explants, PGF2α increased the expression of the ANGPT1, ANGPT2 and FGF2 genes (P < 0.05). PGF2α stimulated the formation of capillary-like structures acting on porcine endometrial endothelial cells on days 15 and 20 of pregnancy and in the G1410 cell line (P < 0.05). PGF2α-stimulated endothelial cell network formation was diminished by using a MEK kinase inhibitor in G1410 cells. Our results indicate an important role for PGF2α in the regulation of angiogenesis at the embryo-maternal interface. PGF2α promotes angiogenesis in the porcine endometrium by activating the MAPK signaling pathway. The stimulating effect of PGF2α on the formation of capillary-like structures by endothelial cells, together with our previous findings, supports the hypothesis that PGF2α is an important factor promoting the development of the placenta during early pregnancy in the pig.

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The initiation of human pregnancy is marked by the implantation of an embryo into the uterine environment; however, the underlying mechanisms remain largely elusive. To address this knowledge gap, we developed hormone-responsive endometrial organoids (EMO), termed apical-out (AO)–EMO, which emulate the in vivo architecture of endometrial tissue. The AO-EMO comprise an exposed apical epithelium surface, dense stromal cells, and a self-formed endothelial network. When cocultured with human embryonic stem cell–derived blastoids, the three-dimensional feto-maternal assembloid system recapitulates critical implantation stages, including apposition, adhesion, and invasion. Endometrial epithelial cells were subsequently disrupted by syncytial cells, which invade and fuse with endometrial stromal cells. We validated this fusion of syncytiotrophoblasts and stromal cells using human blastocysts. Our model provides a foundation for investigating embryo implantation and feto-maternal interactions, offering valuable insights for advancing reproductive medicine.

Summary Pregnancy disorders in patients with autoimmune diseases or viral infections are often associated with an excessive response of type I interferons. We identify radical S-adenosyl methionine domain containing 2 (RSAD2) as a pathogenic interferon-stimulated gene (ISG) associated with pregnancy complications in systemic lupus erythematosus (SLE). The increased expression of RSAD2 mainly occurs in macrophages and structural cell populations at the maternal-fetal interface of pregnant patients with SLE. The elevation of RSAD2 leads to the accumulation of diacylglycerol lipids in the placenta, impairing the necessary vascular development for the fetus. Depletion of Rsad2 in pregnant mice models exposed to type I interferon inducers significantly reduces lipid accumulation, vascular injury, and embryo development disorders. An RSAD2 inhibitor, L-chicoric acid (LCA), alleviates lipid accumulation and vascular damage, improving pregnancy outcomes in SLE-induced and spontaneous mouse models. This study proposes the potential of targeting RSAD2 to improve pregnancy outcomes in individuals with heightened type I interferon response.

ETHNOPHARMACOLOGICAL RELEVANCE The classic TCM prescription, Shoutai Wan (STW), is extensively used in clinical settings to manage threatened miscarriage and Recurrent spontaneous abortion (RSA). The complexity of pregnancy physiology, coupled with diverse etiologies, and the specificity of energy metabolism for normal embryo attachment and development,pose challenges to clinical diagnosis and treatment. The specific molecular mechanisms of how STW regulates these biological processes and contributes to the treatment of RSA remain to be elucidated. AIM OF THE STUDY This study aims to investigate the causes of early pregnancy loss in RSA mice and explore how STW mitigates this loss. MATERIALS AND METHODS An RSA mouse model will be established and treated with STW and Dydrogesterone (DYD). Embryo loss will be quantified on the 14th day of pregnancy, and embryos will be collected on the 6th and 10th days to observe the embryonic condition and assess pathological changes. The study will analyze aerobic glycolysis and angiogenesis at the maternal-fetal interface (MFI). Additionally, STW on a knockdown LDHA mouse model and Human Endometrial Microvascular Endothelial Cells (HEMECs) in vitro will also be examined to verify the mechanism. RESULTS Compared with the control group, the RSA group exhibited significant embryo loss, and reduced levels of aerobic glycolysis at the MFI, the precarious acidic microenvironment (AME), and the PI3K/AKT/mTOR signaling axis downregulated, leading to impaired angiogenesis, which ameliorated following STW treatment. STW treatment enhanced key aerobic glycolysis enzymes-HK2, PKM2, LDHA-and lactate levels, thereby maintaining the AME and upregulating the PI3K/AKT/mTOR axis. This, in turn, promoted the expression of angiogenesis-related factors (VEGFA and VEGFR2) at the MFI, thereby improving angiogenesis, and the same was seen in sh-LDHA mice. In vitro studies confirmed that STW could counteract the glycolysis decline caused by increased oxygen levels, a recovery that was impaired after LDHA knockdown or PI3K inhibition. CONCLUSIONS In RSA mice, disturbances in aerobic glycolysis at the MFI prevent the maintenance of a stable AME, thus impairing angiogenesis and leading to embryo loss, and STW effectively improve early pregnancy outcomes, and laying the foundation for uterine spiral artery remodeling.

Embryo implantation is a decisive process in pregnancy that highly relies on effective cell communication at the maternal-fetal interface. Embryo implantation failure is frequently caused by gram-negative bacterial infection, therefore, this study aimed to investigate the effect of Lipopolysaccharides (LPS)-induced inflammation on cellular composition, cell-cell interaction and key signaling pathways at the maternal-fetal interface using Single-cell RNA-Sequence (scRNA-Seq). LPS exposure significantly up-regulated the expression of pro-inflammatory cytokines, CCL-2, TNF-α, and IL-1β in maternal-fetal interface tissues as well as triggered the recruitment of neutrophils, monocytes and eosinophils into peripheral blood. scRNA-Seq revealed endometrial epithelial cells (EpCs), stromal cells (ESCs), Fibroblasts (FiCs) and 15 other cell types. LPS administration significantly shifted the cellular proportions, increased populations of immune cells and fibroblasts while decreased ESCs and EpCs. Cellular differentiation indicated that all ESCs originated from ESC8 while ESC2 and 7 were the most differentiated ESC subtypes. Likewise, cellular communication demonstrated notable differences, reversed interactions were observed exclusively on the LPS exposure between luminal epithelial (LE) and glandular epithelial (GE) cells. ESC8 was inactive in the control group but exhibited robust interactions in the LPS group. Furthermore, the communication analysis predicted significant disruptions in the signaling pathways: Embryo-maternal communications (DHEA, BMP, LIFR, EDN, and NEGR pathways). Endometrial stromal-epithelial crosswalks (5αP, CAECAM, DHEAS and HH pathways) and Endometrial stromal-immune cell interactions (EGF and NCAM pathways). Our findings suggest that signaling pathways are essential for maternal-fetal communication. The disruption of the pathways in response to LPS may provide new molecular targets for diagnosing and treating implantation failure and recurrent pregnancy loss.

Abstract The purinergic system is composed of purine nucleotides, enzymes, transporters, and receptors and is involved in a variety of physiological processes in the body, including development, metabolism, immunity, tumorigenesis, and reproduction. The importance of the purinergic system for embryo implantation in the endometrium and in pathophysiological conditions has been shown in some species. However, the expression, regulation, and function of purinergic system molecules at the maternal–conceptus interface in pigs is not fully understood. Therefore, we determined the expression of purinergic system molecules in the endometrium during the estrous cycle and pregnancy and in the conceptus and chorioallantoic tissues during pregnancy in pigs. The expression of many purinergic system molecules in the endometrium changed dynamically during pregnancy, with the expression of several components in the endometrium greater on day 15 of pregnancy than day 15 of the estrous cycle, and it was regulated by conceptus-derived interleukin-1beta and interferon-gamma. Purinergic system molecules were also expressed in conceptuses during early pregnancy and in chorioallantoic tissues during mid- to term pregnancy. Furthermore, adenosine triphosphate, a major purinergic signaling molecule, increased migration of endometrial epithelial and conceptus trophectoderm cells, the expression of prostaglandin synthetic enzymes, and the secretion of prostaglandin F2alpha in endometrial epithelial cells in vitro. These data suggest that the purinergic system molecules expressed in the endometrium, conceptus, and chorioallantoic tissues might play an important role in the establishment and maintenance of pregnancy by regulating various cellular functions at the maternal–conceptus interface in pigs. Summary Sentence The purinergic system molecules are dynamically expressed and regulated at the maternal–conceptus interface during pregnancy in pigs. Graphical Abstract

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BACKGROUND Miscarriage has the characteristics of recurrent attacks and complex etiology, so it is gradually attracted the wide attention of scholars in the fields of reproduction. Potential association between gut microbiome (GM) and pregnancy disorders has been investigated. Jianwei Shoutai pills (JWP), as a representative formula, have been proven to have protective effect in both clinical and experimental research in miscarriage. However, the specific mechanism of JWP in miscarriage through GM remains unclear. PURPOSE To investigate the underlying mechanism of JWP against miscarriage through the gut-uterus axis. METHODS The effects of JWP on an RU486-induced rat model of miscarriage were evaluated by embryo resorption rate, vaginal bleeding rate, and appearance of the uterus and embryo. We used 16S rRNA sequencing to measure the extent of the effect of JWP on GM of rats with miscarriage. Bile acid (BA) content of the feces of rats treated with JWP was evaluated by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS). The activation of bile acid-associated receptor, Farnesoid X receptor (FXR), was evaluated by immunofluorescence. The expression level of NLRP3 inflammasome-associated protein was detected by Western blot or Elisa. Fecal microbiota transplantation (FMT) was used to confirm that GM was essential for the therapeutic effect of JWP in miscarriage. RESULTS JWP significantly ameliorated miscarriage symptoms and embryo resorption rate caused by RU486-induced miscarriage as well as restored the abnormal activation of NLRP3-inflammasome at the maternal-fetal interface. Furthermore, JWP can significantly regulated GM dysbiosis and closely associated with BA metabolism by KEGG pathway prediction analysis. Several BA content were significantly restored by HPLC-MS. The expression of NLRP3 inflammasome-associated protein at maternal-fetal interface was reversed by JWP. Combined with FMT, JWP could regulate activation of NLRP3 at the maternal-fetal interface by BAs produced by GM. CONCLUSION JWP restored abnormal activation of the NLRP3-inflammasome in an RU486-induced miscarriage rat model, and corrected the BA disorder by regulating imbalance of the GM.

There have been over 8 million babies born through in vitro fertilization (IVF) and this number continues to grow. There is a global trend to perform elective single embryo transfers, avoiding risks associated with multiple pregnancies. It is therefore important to understand where current research of noninvasive testing for embryos stands, and what are the most promising techniques currently used. Furthermore, it is important to identify the potential to translate research and development into clinically applicable methods that ultimately improve live birth and reduce time to pregnancy. The current focus in the field of human reproductive medicine is to develop a more rapid, quantitative, and noninvasive test. Some of the most promising fields of research for noninvasive assays comprise cell-free DNA analysis, microscopy techniques coupled with artificial intelligence (AI) and omics analysis of the spent blastocyst media. High-throughput proteomics and metabolomics technologies are valuable tools for noninvasive embryo analysis. The biggest advantages of such technology are that it can differentiate between the embryos that appear morphologically identical and has the potential to identify the ploidy status noninvasively prior to transfer in a fresh cycle or before vitrification for a later frozen embryo transfer.

In vitro fertilization occurs in a controlled laboratory setting, where oocytes are fertilized by sperm, and the resulting embryos are cultured to the blastocyst stage before transfer to the uterus. The secreted/consumed substances by the embryo in the extracellular environment (secretome) contain a variety of molecules that may provide insights into embryo quality. This study presents new perspectives on the non-invasive and cost-effective assessment and evaluation of embryos during the IVF process, utilizing a spent embryo culture medium (SECM). The SECM was used from blastocysts prepared for a single blastocyst transfer and was analyzed in two groups—the SECM with successful (F) (n = 30) and unsuccessful (N) (n = 36) embryo implantation in the woman's uterus. Building on our previous next-generation sequencing results, we decided to validate the expression levels of specific miRNAs, particularly hsa-miR-16-5p and hsa-miR-92a-3p, to assess their potential to predict embryo implantation success. Our results demonstrate different expression levels of miRNA molecules in the monitored groups, which could lead to their use in non-invasive analysis of the implantation potential of embryos in the IVF process. In this study, we employed a metabolomics approach using 3D fluorescence analysis of SECM to identify differences between the studied groups, F and N. Our preliminary results indicate a slightly increased metabolic activity in the group with unsuccessful embryo implantation group. This is our pilot study where we demonstrated the use of two approaches in analyzing the SECM to predict the implantation potential of embryos in the IVF process which promises further development.

The lipidomic secretions of embryos provide a unique opportunity to examine the cellular processes of the early conceptus. In this study we profiled lipids released by the early equine conceptus, using high-resolution mass spectrometry to detect individual lipid species. This study examined the lipidomic profile in embryo-conditioned media from in vivo-produced, 8–9 day-old equine embryos (n = 3) cultured in vitro for 36 h, analyzed over 3 timepoints. A total of 1,077 lipid IDs were recorded across all samples, containing predominantly glycerolipids. Seventy-nine of these were significantly altered in embryo conditioned-media versus media only control (p < 0.05, fold-change >2 or < 0.5). Fifty-five lipids were found to be released into the embryo-conditioned media, of which 54.5% were triacylglycerols and 23.6% were ceramides. The sterol lipid, cholesterol, was also identified and secreted in significant amounts as embryos developed. Further, 24 lipids were found to be depleted from the media during culture, of which 70.8% were diacylglycerols, 16.7% were triacylglycerols and 12.5% were ceramides. As lipid-free media contained consistently detectable lipid peaks, a further profile analysis of the various components of non-embryo-conditioned media consistently showed the presence of 137 lipids. Lipid peaks in non-embryo-conditioned media increased in response to incubation under mineral oil, and contained ceramides, diacylglycerols and triacylglycerols. These results emphasize the importance of a defined embryo culture medium and a need to identify the lipid requirements of the embryo precisely. This study sheds light on early embryo lipid metabolism and the transfer of lipids during in vitro culture.

Abstract The aim of this study was to develop an advanced in vitro model of human endometrium using single-cell-derived endometrial epithelial organoids, enabling the study of embryo secretome–endometrial crosstalk at the maternal–fetal interface. Single-cell-derived organoids generated from endometrial tissue of a parous 39-year-old woman recapitulated hormone-responsive decidualization, as demonstrated by expression of SPP1 and acetyl-α-tubulin. When cultured in embryo culture media, organoid viability was maintained with no cytotoxicity, but proliferation was suppressed, likely due to the lower concentrations of the required factors in organoid growth media. Organoids were stimulated with culture supernatants from morphologically good-quality embryos with known pregnancy outcomes (live birth (n = 4) vs no pregnancy (n = 4)). Transcriptomic profiling (RNA-sequencing) revealed that 32 genes were differentially expressed (DEGs) in organoids exposed to the culture supernatants from live-birth embryos versus non-pregnant outcomes: 24 upregulated and 8 downregulated. These DEGs were enriched for biological processes related to cell motility and cytoskeletal dynamics. In conclusion, soluble factors secreted by human blastocysts achieving live birth selectively modulate the endometrial epithelial transcriptome, enhancing pathways involved in cytoskeletal remodeling and immune modulation. This embryo-directed remodeling likely facilitates endometrial receptivity for successful implantation to occur. Our organoid model provides a robust platform for further investigating implantation failure.

To elucidate the role of IL-6 in folliculogenesis and embryo development during human in-vitro culture High IL-6 levels in follicular-fluid positively correlate with oocyte maturity status whereas higher IL-6 secretome in day5/day6 spent culture media inversely correlates with blastocyst/ICM grades Although some studies have attempted to explore the correlation of FF IL-6 with fertilization and embryo development rates; the results have been inconclusive. Similarly, studies comparing FF IL-6 levels with endometrial receptivity and pregnancy outcomes have been ambiguous and contradictory. Recent research has reported improvement in bovine blastocyst ICM grades in culture media supplemented with IL-6. No study has yet evaluated the role of IL-6 as a biomarker of oocyte maturity nor investigated IL-6 as an embryokine that could regulate embryo developmental competence in humans. We have employed the FF metabolomic and Secretomic approach for IL-6 in human IVF cycles. Prospective study in women (25-35 years) undergoing IVF cycles (n = 256) for unexplained/tubal-factor infertility from 2018-2020. Only those women whose FF and day5/6 spent culture media sample could be obtained, were included. Women with endometriosis, PID, Genital Kochs were excluded. IL-6 levels were measured by diagnostic kit in spent micro-drops and FF (pooled per patient per cycle). Cycles were divided into Low and High IL-6 groups (FF: ≤ 47.5 and >47.5pg/ml; Spent Medium: <0.5 and ≥0.5pg/ml). Antagonist cycles involving COH with r-FSH and ICSI for all. Noted oocyte Nuclear maturity (GV/MI/MII). Oocyte Cytoplasmic maturity graded as Top and non-Top, based on presence of granulation, refractile bodies, sER, visco-elasticity. Post ICSI, oocytes cultured individually in 50µl single-step media micro-drops till day3 and shifted to fresh 50µl micro-drops for extended culture till day5/6. Blastocyst gradation was done using Gardner’s classification for expansion, ICM and trophectodermal cells. Statistical analysis done using graph-pad prism VI High FF/IL-6 group (n = 103) had significantly higher proportion of MII (92.08 vs. 76.33%; p < 0.001) and top-quality (88.10 vs. 70.0%; p < 0.001) oocytes and fertilization rates (89.45 vs. 77.30%; p = 0.009) compared to Low FF/IL-6 group (n = 153) although total number of days of stimulation/gonadotropin dose, mean oocytes retrieved did not differ significantly between the two groups. Cleavage (84.64 vs. 75.92%; p = 0.061) and blastocyst formation (53.86 vs. 42.37%; p = 0.053) rate, although higher, did not differ significantly between the two FF/IL-6 groups. FF/IL-6 level correlated strongly with overall oocyte maturity status (Pearson r = 0.61, 95% CI 0.52-0.8). Threshold FF/IL-6 level for obtaining top-quality MII oocytes was >39.2pg/ml (ROCAUC = 86%, Sensitivity = 78%, Specificity = 83%). In D5/6 spent culture drops, likelihood of top-quality blastocyst formation (expansion grade ≥3 and ICM/TE grades AA/AB/BA) increased by 41% (odds ratio: 0.44, 95%CI: 0.4-0.5; χ2 p < 0.0001) if IL-6 concentration was <0.5 pg/ml. Lower culture-drop IL-6 levels correlated strongly with top ICM grades (Pearson r = −0.66, 95% CI 0.6-0.7). Highest IL-6 levels were found in spent culture-drops containing grade-C blastocysts whether on day5 or day6. IL-6 levels were comparatively higher in day6 than in day5 micro-drops, correlating with higher percentage of top-quality blastocysts on day5 than on day6. Threshold micro-drop IL-6 level for top-grade blastocyst <0.42 pg/ml. We estimated IL-6 levels in pooled FF (per patient, per cycle). Although cumbersome & time-consuming, it would be worthwhile to evaluate individual FF (per follicle, per patient, per cycle). Also, measuring IL-6 in spent media involving group embryo culture, may facilitate study of the probable paracrine effects of this ‘embryokine’. IL-6 is a pleiotropic cytokine and its role/s in folliculogenesis, embryo development and endometrial receptivity is not yet clearly etched out. This study attempts to elucidate the functions of IL-6 in a systematic manner by deriving threshold values for obtaining top quality oocytes and blastocysts in human in-vitro culture conditions. NOT APPLICABLE

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Morphological assessment at defined developmental stages is the most important method to select viable embryos for transfer and cryopreservation. Timing of different developmental stages in embryo development has been shown to correlate with its potential to develop into a blastocyst. However, improvements in pregnancy rates by using time-lapse techniques have been difficult to validate scientifically. Therefore, there is a need for new methods, preferably non-invasive methods based on metabolomics, genomics and proteomics, to improve the evaluation of embryo quality even further. The aim of this study was to investigate if different levels of caspase-3 and histidine-rich glycoprotein (HRG), secreted by the embryo into the culture media, can be used as biomarkers of embryo quality. In this study, a total of 334 samples of culture media were collected from in vitro fertilization (IVF) treatments at three different clinics. Protein analysis of the culture media was performed using multiplex proximity extension protein analysis to detect levels of caspase-3 and HRG in the embryo secretome. Protein levels were compared in secretome samples from high- and low-quality blastocysts and embryos that became arrested during development. Correlation between protein levels and time to morula formation was also analyzed. Furthermore, protein levels in secretomes from day-2 cultured embryos were compared on the basis of whether or not pregnancy was achieved. The results showed that caspase-3 levels were lower in secretomes from high-quality vs. low-quality blastocysts and those that became arrested (p ≤ 0.05 for both). In addition, higher HRG levels correlated with a shorter time to morula formation (p ≤ 0.001). Caspase-3 levels were also lower in secretomes from day-2 cultured embryos resulting in a pregnancy vs. those that did not (p ≤ 0.05). Furthermore, it was shown that caspase-3 might be used as a marker for predicting potential success rate after transfer of day-2 cultured embryos, where a caspase-3 cutoff level of 0.02 gave a prediction probability of 68% (p = 0.038). In conclusion, in future prediction models, levels of caspase-3 and HRG might be used as potential markers of embryo quality, and secreted caspase-3 levels could to some extent predict the outcome after transfer of day-2 cultured embryos.

BACKGROUND: The developing embryo secretes a wide range of cytokines that are essential for establishing a “dialogue” between the embryo and the endometrium, as well as for autocrine effects on the embryo. Non-invasive assessment of the embryo using the study of its secretome is of great interest in reproductive medicine, as it can be used for early selection of the most viable embryos in assisted reproductive technology programs. AIM: The aim of this study was to determine the prognostic significance of cytokine levels in media after embryo culture and their impact on endothelial function in achieving clinical pregnancy in assisted reproductive technology protocols, as well as the effect of these media on endothelial function. METHODS: The presence of cytokines [interleukin-1b, -4, -5, -6, -8, -10, growth factors (granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor)] in embryo-conditioned culture media was determined in patients undergoing infertility treatment using assisted reproductive technology. To detect cytokine levels below the sensitivity threshold, we studied the effect of conditioned culture media on the proliferation and migration of endothelial cells. RESULTS: The patients were divided into 2 groups: in group 1 (n = 34), no cytokines were found, whereas in group 2 (n = 14), at least one of the studied interleukins or growth factors was detected. Patients in group 2 had more oocyte-cumulus complexes, 2PN2PB zygotes, embryos on day 3 of culture, embryos of quality ≥ 6B on day 3 of culture, blastocysts, and blastocysts of quality ≥ 3BB. Additionally, the effective doses of gonadotropins per oocyte and per embryo were lower in group 2. The clinical pregnancy rate was also higher in group 2. We found no differences in endothelial cell proliferation and migration parameters. CONCLUSION: A higher concentration of cytokines in media after embryo culture may indicate greater implantation potential; therefore, cytokine assessment in culture media could be used as an additional non-invasive method for embryo evaluation.

Objective Morphologic assessment of an embryo is a valuable indicator for determining embryo health; however, it does not provide information on the chromosomal status of an embryo. Therefore, this study aimed to investigate the levels of Apolipoprotein A1 secreted by day-5 embryos in the spent media of euploid and aneuploid human embryos. Methods This study utilized 131 spent culture media samples from 22 infertile couples who were referred to the fertility clinic of Royan Institute. Following ovulation induction, retrieved oocytes were fertilized by intracytoplasmic sperm injection. For pre-implantation genetic diagnosis, embryos were frozen and thawed on days 2 to 3 and a single blastomere was isolated from each embryo for the assessment of chromosomal abnormalities by fluorescence in situ hybridization. Five days after fertilization, the levels of Apolipoprotein A1 were determined in the spent media of normal embryos, aneuploid embryos (with chromosome abnormalities), and the control group (medium without any embryos) using enzyme-linked immunosorbent assay. Results The Apolipoprotein A1 levels in the secretome of euploid cleavage-arrested embryos were significantly lower than those in the control group (p<0.04). However, Apolipoprotein A1 levels increased significantly in groups of euploid blastocysts, aneuploid cleavage-arrested embryos, aneuploid morulae, and aneuploid blastocysts compared to the control group (p<0.04). Furthermore, the Apolipoprotein A1 levels in the spent media of euploid early blastocysts were significantly higher compared to euploid hatching blastocysts and aneuploid blastocysts in the early, mid, and late stages (p<0.03). Conclusions This study highlights the significant potential of Apolipoprotein A1 as a developmental bi-omarker to distinguish between euploid and aneuploid embryos.

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In vitro fertilization (IVF) is the most common assisted reproductive technology used to treat infertility. Embryo selection for transfer in IVF cycles relies on the morphological evaluation by embryologists, either by conventional microscopic assessment or more recently by time-lapse imaging systems. Despite the introduction of time-lapse imaging improvements in IVF success rates have failed to materialize, therefore alternative approaches are needed. Recent studies have shown that embryos resulting in successful pregnancy differ in their secretome and metabolism compared to embryos that fail to implant, suggesting that molecular analysis of embryo culture medium could assist in non-invasive single embryo selection. However, this approach has yet to be adopted clinically due to the lack of appropriate highly sensitive screening technologies needed to assess volume-limited samples. Here we report the detection of hCGβ, IL-8 and TNFα from conditioned culture media of single human embryos using electrochemical impedance spectroscopy. The impedimetric immunosensors revealed that morphologically non-viable embryos produce higher levels of IL-8 and TNFα, associated with abnormal cell division and cell death, respectively. More importantly, hCGβ detection was able to discriminate apparently morphologically identical viable embryos. This work brings an objective dimension to embryo selection, which could overcome the major limitations of morphology-based embryo selection for implantation. Future work should include the validation of these biomarkers in a large patient cohort.

PurposeSelecting an embryo at the transfer stage with the best chance of a successful pregnancy is still largely dependent on preceding subjective evaluation of morphokinetics. Expensive prenatal genomic profiling has been so far proved ineffective. Proteomics and metabolomics are promising new approaches to assess embryo viability, but methodologies are often complex and do not lend themselves to rapid analysis in the critical time between blastocyst formation and embryo transfer. Here, we used matrix-assisted laser desorption ionization time-of-flight (MALDI ToF) mass spectrometry to assess the secretome of blastocysts in the minutes prior to embryo transfer and correlated spectral features with pregnancy outcome.MethodsFour hundred one samples of spent blastocyst culture media were collected from embryo cultures at the time of embryo transfer, of which 136 were used to construct the predictive model. The media samples were frozen at − 20 °C and stored for analysis. Sample analysis was conducted in batches using 1 μl of spent embryo in direct MALDI ToF mass spectral analysis. Quantitative characteristics within this mass range (2000–17,000 m/z) were used to generate a score for selected mass regions (bins) in order to predict pregnancy outcome for each sample.ResultsWith a simple algorithm based on nine mass bins within the 2000–10,000 m/z region, it was possible to identify samples with the best chance of becoming an ongoing pregnancy (positive predictive value of 82.9%, p = 0.0018).ConclusionA simple, direct and rapid analysis of spent culture fluid from blastocysts at the point of embryo transfer can quickly identify optimal embryos with the best chance of achieving ongoing pregnancy. Methods like this, which take less than 20 min to perform, could dramatically improve the approach to embryo selection and live births.

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Abstract STUDY QUESTION What is the composition of currently available commercial human embryo culture media provided by seven suppliers, for each stage of human preimplantation embryo development? SUMMARY ANSWER While common trends existed across brands, distinct differences in composition underlined the absence of a clear standard for human embryo culture medium formulation. WHAT IS KNOWN ALREADY The reluctance of manufacturers to fully disclose the composition of their human embryo culture media generates uncertainty regarding the culture conditions that are used for human preimplantation embryo culture. The critical role of the embryo culture environment is well-recognized, with proven effects on IVF success rates and child outcomes, such as birth weight. The lack of comprehensive composition details restricts research efforts crucial for enhancing our understanding of its impacts on these outcomes. The ongoing demand for greater transparency remains unmet, highlighting a significant barrier in embryo culture medium optimization. STUDY DESIGN, SIZE, DURATION For this study, 47 different human embryo culture media and protein supplements were purchased between December 2019 and June 2020; they comprise complete media (n = 23), unsupplemented media (n = 14), and supplements (n = 10). Unsupplemented media were supplemented with each available supplement from the same brand (n = 33 combinations). All samples were directly frozen in liquid nitrogen and stored at −80°C until composition analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS We determined the concentrations of 40 components in all samples collected (n = 80). Seven electrolytes (calcium, chloride, iron, magnesium, phosphate, potassium, sodium), glucose, immunoglobulins A, G, and M (IgA, IgG, IgM), uric acid, alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), and albumin, as well as the total protein concentration, were determined in each sample using a Cobas 8000 Analyser (Roche Diagnostics). Analysis of pyruvate, lactate, carnitine, and 21 amino acids was achieved with Ultra-High Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS/MS). MAIN RESULTS AND THE ROLE OF CHANCE Our analysis showed that generally, the concentrations of components of ready-to-use human embryo culture media align with established assumptions about the changing needs of an embryo during early development. For instance, glucose concentrations displayed a high-low-high pattern in sequential media systems from all brands: 2.5–3 mM in most fertilization media, 0.5 mM or below in all cleavage stage media, and 2.5–3.3 mM in most blastocyst stage media. Continuous media generally resembled glucose concentrations of cleavage stage media. However, for other components, such as lactate, glycine, and potassium, we observed clear differences in medium composition across different brands. No two embryo culture media compositions were the same. Remarkably, even embryo culture media from brands that belong to the same parent company differed in composition. Additionally, the scientific backing for the specific concentrations used and the differences in the composition of sequential media is quite limited and often based on minimal in vivo studies of limited sample size or studies using animal models. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION We used a targeted approach and performed a selection of tests which limit the composition analysis to this set of analytes. WIDER IMPLICATIONS OF THE FINDINGS Comprehensive disclosure and complete transparency concerning the composition of human embryo culture media, including the exact concentration of each component, are crucial for evidence-based improvements of culture media for human preimplantation embryos. STUDY FUNDING/COMPETING INTEREST(S) This research was supported by ZonMw (https://www.zonmw.nl/en), Programme Translational Research 2 (project number 446002003). M.G. declares an unrestricted research grant from Ferring not related to the presented work, paid to the institution VU Medical Center. The remaining authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER N/A.

Culture media supplemented with reproductive fluids (RF) have been used in livestock species, improving the efficiency and quality of in vitro produced embryos. However, usefulness in humans is still unknown. In this study, we collected human reproductive fluids (HRFs) ex vivo (from 25 patients undergoing abdominal hysterectomy plus bilateral salpingectomy) and in vivo (from 31 oocyte donors). Afterward, protocols to evaluate their osmolality, pH, total protein concentration, endotoxin level, and sterility were optimized, establishing security ranges for their use as natural additives. In addition, a functional assay was developed with bovine embryos grown in vitro in a medium supplemented with 1% of collected HRFs. Finally, a proof of concept was performed with six patients on post ovulation day 2 to evaluate the full-term viability of embryos grown in media supplemented with autologous uterine fluid, collected under in vivo conditions. Two of the embryos resulted in successful pregnancy and delivery of healthy babies. In conclusion, this study establishes a complete quality control sheet of HRFs as additives for embryo culture media and shows first preliminary data on obtaining healthy offspring derived from embryos grown in media supplemented with HRFs.

INTRODUCTION Benchtop incubators with small individual chambers have been developed in order to improve the stability of embryo culture conditions reducing the environmental stress during the embryo development. These new dry incubators were designed without any air humidification system in order to prevent bacterial proliferation and to enable the use of timelapse system. However, an elevated evaporation of the culture media could occur in these conditions. The main objective of the study is to analyse the impact of the used incubator type on the embryo culture media osmolality. MATERIALS AND METHODS Micro-drops of 50 µL of culture media were placed in 60 mm diameter culture dishes, and quickly covered with either 7 or 8 mL of mineral oil in an IVF workstation with laminar airflow. Two series of culture dishes have been randomly placed either in a humidified incubator or in a dry benchtop incubator. The micro-drops of each culture dishes were sampled at D0, D1, D2, D3, and D5 respectively to measure the osmolality in triplicate using a cryoscopic osmometer. The mean values of osmolality in each incubator have been compared respectively on D0, D1, D2, D3 and D5 with appropriate statistical tests, and considered statistically significant when p < 0.05. RESULTS The osmolality of the micro-drops placed in the dry benchtop incubator differed significantly after the third day of culture, regardless of the level of mineral oil in the culture dishes. Indeed, using Petri dishes covered respectively with 7 or 8 mL of mineral oil, osmolality values of samples from the dry incubator were significantly higher than those from the humidified one, at D3 and D5 (D3/7 mL : 273 ± 2.1 vs. 268 ± 1.0 mOsm/kg ; p = 0.02; D3/8 mL : 282 ± 8.0 vs. 270 ± 0.7 mOsm/kg ; p = 0.04) and D5 (D5/7 mL : 283 ± 1.5 vs. 270 ± 3.6 mOsm/kg ; p = 0.004; D5/8 mL: 287 ± 5.6 vs. 268 ± 2.3 mOsm/kg ; p = 0.005). Furthermore, the analysis on paired samples showed that the osmolality in the dry benchtop incubator at D5 using 7 mL of oil (283 ± 1,5 mOsm/kg ; p = 0.003) and at D3 ( 273 ± 2.1 mOsm/kg ; p = 0.016) and D5 (287 ± 5.6 mOsm/kg ; p = 0.009) using 8 mL of oil was significantly higher than that measured at D0 (265 ± 1.9 mOsm/kg). CONCLUSION A significant increase of the embryo culture media osmolality was observed in the dry benchtop incubator with ambient hygrometry in our standard conditions. Adding 1 mL of oil was not sufficient to reduce the evaporation of the media. Although maintained at a physiological level, the impact of the osmolality changes on the in vitro embryo development has to be further determined.

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