Fructose improves titanium dioxide nanoparticles induced alterations in developmental competence of mouse oocytes.

AIMS: We investigated the effects of intraperitoneal injections of titanium dioxide nanoparticles (TiO2 NPs, 100 mg/kg) for 5 consecutive days on the developmental competence of murine oocytes. Furthermore, study the effects of TiO2 NPs on antioxidant and oxidative stress biomarkers, as well as their effects on expression of apoptotic and hypoxia inducing factor-1α (HIF1A) protein translation. Moreover, the possible ameliorating effects of intraperitoneal injections of fructose (2.75 mM/ml) was examined. MATERIALS AND METHODS: Thirty sexually mature (8-12 weeks old; ~ 25 g body weight) female mice were used for the current study. The female mice were assigned randomly to three treatment groups: Group1 (G1) mice were injected intraperitoneal (ip) with deionized water for 5 consecutive days; Group 2 (G2) mice were injected ip with TiO2 NPs (100 mg/kg BW) for 5 consecutive days; Group 3 (G3) mice were injected ip with TiO2 NPs (100 mg/kg BW + fructose (2.75 mM) for 5 consecutive days. RESULTS: Nano-titanium significantly decreased expression of GSH, GPx, and NO, expression of MDA and TAC increased. The rates of MI, MII, GVBD and degenerated oocytes were significantly less for nano-titanium treated mice, but the rate of activated oocytes was significantly greater than those in control oocytes. TiO2 NPs significantly increased expression of apoptotic genes (BAX, Caspase 3 and P53) and HIF1A. Intraperitoneal injection of fructose (2.75 mM/kg) significantly alleviated the detrimental effects of TiO2 NPs. Transmission electron microscopy indicated that fructose mitigated adverse effects of TiO2 NPs to alter the cell surface of murine oocytes. CONCLUSION: Results of this study suggest that the i/p infusion of fructose for consecutive 5 days enhances development of murine oocytes and decreases toxic effects of TiO2 NPs through positive effects on oxidative and antioxidant biomarkers in cumulus-oocyte complexes and effects to inhibit TiO2-induced increases in expression of apoptotic and hypoxia inducing factors.

Periostin's role in uterine leiomyoma development: a mini-review on the potential periostin poses as a pharmacological intervention for uterine leiomyoma.

Uterine leiomyomas, also known as fibroids or myomas, occur in an estimated 70-80% of reproductive aged women. Many experience debilitating symptoms including pelvic pain, abnormal uterine bleeding (AUB), dyspareunia, dysmenorrhea, and infertility. Current treatment options are limited in preserving fertility, with many opting for sterilizing hysterectomy as a form of treatment. Currently, surgical interventions include hysterectomy, myomectomy, and uterine artery embolization in addition to endometrial ablation to control AUB. Non-surgical hormonal interventions, including GnRH agonists, are connotated with negative side effects and are unacceptable for women desiring fertility. Periostin, a regulatory extra cellular matrix (ECM) protein, has been found to be expressed in various gynecological diseases including leiomyomas. We previously determined that periostin over-expression in immortalized myometrial cells led to the development of a leiomyoma-like cellular phenotype. Periostin is induced by TGF-ß, signals through the PI3K/AKT pathway, induces collagen production, and mediates wound repair and fibrosis, all of which are implicated in leiomyoma pathology. Periostin has been linked to other gynecological diseases including ovarian cancer and endometriosis and is being investigated as pharmacological target for treating ovarian cancer, post-surgical scarring, and numerous other fibrotic conditions. In this review, we provide discussion linking pathological inflammation and wound repair, with a TGF-ß-periostin-collagen signaling in the pathogenesis of leiomyomas, and ultimately the potential of periostin as a druggable target to treat leiomyomas.

Cathepsin L regulates oocyte meiosis and preimplantation embryo development.

Early embryonic loss, caused by reduced embryo developmental competence, is the major cause of subfertility in humans and animals. This embryo developmental competence is determined during oocyte maturation and the first embryo divisions. Therefore, it is essential to identify the underlying molecules regulating these critical developmental stages. Cathepsin L (CTSL), a lysosomal cysteine protease, is involved in regulating cell cycle progression, proliferation and invasion of different cell types. However, CTSL role in mammalian embryo development is unknown. Using bovine in vitro maturation and culture systems, we show that CTSL is a key regulator for embryo developmental competence. We employed a specific CTSL detection assay in live cells to show that CTSL activity correlates with meiotic progression and early embryo development. Inhibiting CTSL activity during oocyte maturation or early embryo development significantly impaired oocyte and embryo developmental competence as evidenced by lower cleavage, blastocyst and hatched blastocyst rates. Moreover, enhancing CTSL activity, using recombinant CTSL (rCTSL), during oocyte maturation or early embryo development significantly improved oocyte and embryo developmental competence. Importantly, rCTSL supplementation during oocyte maturation and early embryo development significantly improved the developmental competence of heat-shocked oocytes/embryos which are notoriously known for reduced quality. Altogether, these results provide novel evidence that CTSL plays a pivotal role in regulating oocyte meiosis and early embryonic development.

An oocyte meiotic midbody cap is required for developmental competence in mice.

Embryo development depends upon maternally derived materials. Mammalian oocytes undergo extreme asymmetric cytokinesis events, producing one large egg and two small polar bodies. During cytokinesis in somatic cells, the midbody and subsequent assembly of the midbody remnant, a signaling organelle containing RNAs, transcription factors and translation machinery, is thought to influence cellular function or fate. The role of the midbody and midbody remnant in gametes, in particular, oocytes, remains unclear. Here, we examined the formation and function of meiotic midbodies (mMB) and mMB remnants using mouse oocytes and demonstrate that mMBs have a specialized cap structure that is orientated toward polar bodies. We show that that mMBs are translationally active, and that mMB caps are required to retain nascent proteins in eggs. We propose that this specialized mMB cap maintains genetic factors in eggs allowing for full developmental competency.

An oocyte meiotic midbody cap is required for developmental competence in mice.

Embryo development depends upon maternally derived materials. Mammalian oocytes undergo extreme asymmetric cytokinesis events, producing one large egg and two small polar bodies (PB). During cytokinesis in somatic cells, the midbody (MB) and subsequent assembly of the midbody remnant (MBR), a signaling organelle containing RNAs, transcription factors and translation machinery, is thought to influence cellular function or fate. The role of the MB and MBR in gametes, in particular, oocytes, remains unclear. Here, we examined the formation and function of meiotic MBs (mMB) and mMB remnants (mMBRs) using mouse oocytes and demonstrate that mMBs have a specialized meiotic mMB cap structure that is orientated toward PBs. We show that that mMBs are translationally active, and that mMB caps are required to retain nascent proteins in eggs. We propose that this specialized mMB cap maintains genetic factors in eggs allowing for full developmental competency.

IFNT stimulates lysosomal function via type I IFN signaling pathway in pregnant bovine leukocytes.

In brief: Interferon tau (IFNT) stimulates lysosomal activation via the Janus-activated kinase in peripheral blood leukocytes during pregnancy recognition. IFNT-mediated lysosomal activation could serve as a novel marker for early pregnancy in cattle. Abstract: IFNT is important in establishing pregnancy in ruminants. Secreted IFNT in the uterus induces the expression of an interferon-stimulated gene (ISG) in uterine tissues and peripheral blood leukocytes (PBLs). In our previous study, increased lysosome and lysosomal cathepsin (CTS) activity and mRNA expression were observed in PBLs of pregnant cows on day 18 of pregnancy. However, the mechanism of IFNT stimulation in PBLs is unclear. Here, we explored the IFNT-mediated lysosomal activation mechanisms in PBLs during early pregnancy in dairy cows. PBLs collected from the peripheral blood of Holstein cows on day 18 post artificial insemination, after confirmation of their pregnancy status, were used to detect the expression of lysosomal-associated membrane protein (LAMP) 1, 2, CTSB and CTSK. Expression of all genes was significantly higher in PBLs of pregnant cows than in nonpregnant cows. In vitro IFN-mediated stimulation of PBLs collected from cows that did not undergo AI significantly increased lysosomal acidification and expression of LAMP1 and 2, as well as the activities of CTSB and CTSK. Immunodetection analysis showed an increase in LAMP1 and CTSK levels in the PBLs of day 18 pregnant cows. JAK inhibitor significantly decreased lysosomal acidification, CTSK activity, LAMP1, 2, and CTSK expression in the presence of IFNT. These results suggest that IFNT regulates lysosomal function via a type 1IFN-mediated pathway in PBLs during pregnancy recognition.

Sire modulates developmental kinetics and transcriptome of the bovine embryo.

In brief: The first week of gestation is a period of major pregnancy loss in cattle, this study reveals that the male plays a key role in regulating embryonic development during this time. Abstract: The impact of sire on preimplantation embryonic development in cattle remains poorly understood. This study evaluated differences in embryos produced in vitro from sires with varying capacities to produce blastocysts. Sires classified as high (HP) and low performing (LP) based on their ability to produce embryos were used to better understand how sire regulates embryonic development. By monitoring development, it was determined that the most common arrest stage was the five- to six-cell stage. Embryos (four to six cells) from HP and LP sires were then analyzed for autophagic activity, where embryos for LP sires exhibited increased autophagy than HP-derived embryos. Transcriptome analysis of four-cell embryos found that embryos from LP sires might have issues in sperm mitochondrial clearance, histone retention, and DNA damage, while HP sires had increased expression of genes involved in transcription, chromosome segregation, and cell division. In conclusion, LP sires had an increased proportion of embryos arresting at the five- to six-cell stage, and these embryos had higher rates of cellular stress due to paternal contributions from the spermatozoon.

Initial spindle positioning at the oocyte center protects against incorrect kinetochore-microtubule attachment and aneuploidy in mice.

Spindle positioning within the oocyte must be tightly regulated. In mice, the spindle is predominantly assembled at the oocyte center before its migration toward the cortex to achieve the highly asymmetric division, a characteristic of female meiosis. The significance of the initial central positioning of the spindle is largely unknown. We show that initial spindle positioning at the oocyte center is an insurance mechanism to avoid the premature exposure of the spindle to cortical CDC42 signaling, which perturbs proper kinetochore-microtubule attachments, leading to the formation of aneuploid gametes. These findings contribute to understanding why female gametes are notoriously associated with high rates of aneuploidy, the leading genetic cause of miscarriage and congenital abnormalities.

A re-appraisal of mesenchymal-epithelial transition (MET) in endometrial epithelial remodeling.

Mesenchymal-epithelial transition (MET) is a mechanism of endometrial epithelial regeneration. It is also implicated in adenocarcinoma and endometriosis. Little is known about this process in normal uterine physiology. Previously, using pregnancy and menses-like mouse models, MET occurred only as an epithelial damage/repair mechanism. Here, we hypothesized that MET also occurs in other physiological endometrial remodeling events, outside of damage/repair, such as during the estrous cycle and adenogenesis (gland development). To investigate this, Amhr2-Cre-YFP/GFP mesenchyme-specific reporter mice were used to track the fate of mesenchymal-derived (MD) cells. Using EpCAM (epithelial marker), EpCAM+YFP+ MD-epithelial cells were identified in all stages of the estrous cycle except diestrus, in both postpartum and virgin mice. EpCAM+YFP+ MD-epithelial cells comprised up to 80% of the epithelia during estrogen-dominant proestrus and significantly declined to indistinguishable from control uteri in diestrus, suggesting MET is hormonally regulated. MD-epithelial cells were also identified during postnatal epithelial remodeling. MET occurred immediately after birth at postnatal day (P) 0.5 with EpCAM+GFP+ cells ranging from negligible (0.21%) to 82% of the epithelia. EpCAM+GFP+ MD-epithelial cells declined during initiation of adenogenesis (P8, avg. 1.75%) and then increased during gland morphogenesis (P14, avg. 10%). MD-epithelial cells expressed markers in common with non-MD-epithelial cells (e.g., EpCAM, FOXA2, ESR1, PGR). However, MD-epithelial cells were differentially regulated postnatally and in adults, suggesting a functional distinction in the two populations. We conclude that MET occurs not only as an epithelial damage/repair mechanism but also during other epithelial remodeling events, which to our knowledge has not been demonstrated in other tissues.

The Effects of Periostin Expression on Fibroid-Like Transition of Myometrial Cells.

Fibroids, benign tumors of the myometrium, are the most common tumors in women and are associated with spontaneous abortion, preterm birth, placenta abruption, and infertility, among others. The incidence of fibroids in reproductive aged women is 20-89%. Fibroids are characterized by high production of extracellular matrix (ECM), particularly collagens, which play a role in their growth. However, their pathogenesis is poorly understood. Recently, we and others have found periostin (POSTN), a regulatory ECM protein, to be overexpressed in the majority of fibroids analyzed. Periostin is an ECM protein that is a critical regulator and well-established biomarker for fibrosis in tissues such as the lung, skin, and kidney. Our hypothesis was that periostin plays a role in the fibrotic transition of myometrial cells to fibroid cells. To test this, we evaluated the effects of POSTN overexpression in myometrial cells. Telomerase-immortalized myometrial cells were transduced with control or POSTN-overexpression lentivirus particles, generating one control (dCas9-Mock) and two overexpression (dCas9-POSTN-01, dCas9-POSTN-02) cell lines. Overexpression of POSTN in immortalized myometrial cells resulted in a change in phenotype consistent with fibroid cells. They upregulated expression of key fibroid genes and had increased proliferation, adhesion, and migration in vitro. Here, we show a potential role for periostin in the transition of myometrial cells to fibroid cells, giving rationale for future investigation into the role of periostin in fibroid pathogenesis and its potential as a therapeutic target.

Synergistic effect of standardized extract of Asparagus officinalis stem and heat shock on progesterone synthesis with lipid droplets and mitochondrial function in bovine granulosa cells.

Progesterone (P4) is a well-known steroid hormone that plays a key role in oocyte growth and the maintenance of pregnancy in mammals, including cattle. Heat stress (HS) has an adverse effect on P4 synthesis through an imbalance in the cellular redox status. We have recently revealed that a standardized extract of Asparagus officinalis stem (EAS) increases P4 through non-HS induction of heat shock protein 70 (HSP70) and a synergistic increase of HSP70 by enhancing the intracellular redox balance, which was adversely affected by HS in bovine granulosa cells (GCs). Bovine GCs collected from bovine ovarian follicles were cultured at 38.5 °C and 41 °C for 12 h with or without 5 mg/mL EAS. After treatment, cells and culture suppernatant were collected for the analysis. Enzyme-linked immunosorbent assay (ELISA) was performed to detect in P4 levels. Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) was used to detect expression of steroidogenesis related genes. Fluorescence staining was used to detect mitochondrial activity and lipid droplet. P4 level was increased by EAS treatment in association with increase in steroidogenic acute regulatory protein (STAR), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), mitochondrial membrane activity and lipid droplet both under non-HS and HS conditions. Notably, synergistic effect of EAS with HS co-treatment was observed to show a greater increase in P4 synthesis when comparison with EAS treatment under non-HS condition. Furthermore, inhibition of HSP70 significantly reduced EAS-induced P4 synthesis, mitochondrial activity and synthesis of lipid droplets. These results suggest that P4 synthesis by EAS is mediated by the steroidogenesis pathway via HSP70-regulated activation of STAR and 3ß-HSD, together with improved mitochondrial activity and lipid metabolism in bovine GCs. Moreover, effect of EAS has a synergistic effect of with HSP70-regulated steroidogenesis pathway.

Adverse PFAS effects on mouse oocyte in vitro maturation are associated with carbon-chain length and inclusion of a sulfonate group.

OBJECTIVES: Per- and polyfluoroalkyl substances (PFAS) are man-made chemicals that are widely used in various products. PFAS are characterized by their fluorinated carbon chains that make them hard to degrade and bioaccumulate in human and animals. Toxicological studies have shown PFAS toxic effects: cytotoxicity, immunotoxicity, neurotoxicity, and reproductive toxicity. However, it is still unclear how the structures of PFAS, such as carbon-chain length and functional groups, determine their reproductive toxicity. METHODS AND RESULTS: By using a mouse-oocyte-in-vitro-maturation (IVM) system, we found the toxicity of two major categories of PFAS, perfluoroalkyl carboxylic acid (PFCA) and perfluoroalkyl sulfonic acid (PFSA), is elevated with increasing carbon-chain length and the inclusion of the sulfonate group. Specifically, at 600 µM, perfluorohexanesulfonic acid (PFHxS) and perfluorooctanesulfonic acid (PFOS) reduced the rates of both germinal-vesicle breakdown (GVBD) and polar-body extrusion (PBE) as well as enlarged polar bodies. However, the shorter PFSA, perfluorobutanesulfonic acid (PFBS), and all PFCA did not show similar adverse cytotoxicity. Further, we found that 600 µM PFHxS and PFOS exposure induced excess reactive oxygen species (ROS) and decreased mitochondrial membrane potential (MMP). Cytoskeleton analysis revealed that PFHxS and PFOS exposure induced chromosome misalignment, abnormal F-actin organization, elongated spindle formation, and symmetric division in the treated oocytes. These meiotic defects compromised oocyte developmental competence after parthenogenetic activation. CONCLUSIONS: Our study provides new information on the structure-toxicity relationship of PFAS.

Multi-Photon Laser Ablation of Cytoplasmic Microtubule Organizing Centers in Mouse Oocytes.

The fidelity of oocyte meiosis is critical for generating developmentally competent euploid eggs. In mammals, the oocyte undergoes a lengthy arrest at prophase I of the first meiotic division. After puberty and upon meiotic resumption, the nuclear membrane disassembles (nuclear envelope breakdown), and the spindle is assembled mainly at the oocyte center. Initial central spindle positioning is essential to protect against abnormal kinetochore-microtubule (MT) attachments and aneuploidy. The centrally positioned spindle migrates in a time-sensitive manner toward the cortex, and this is a necessary process to extrude a tiny polar body. In mitotic cells, spindle positioning relies on the interaction between centrosome-mediated astral MTs and the cell cortex. On the contrary, mouse oocytes lack classic centrosomes and, instead, contain numerous acentriolar MT organizing centers (MTOCs). At the metaphase I stage, mouse oocytes have two different sets of MTOCs: (1) MTOCs that are clustered and sorted to assemble spindle poles (polar MTOCs), and (2) metaphase cytoplasmic MTOCs (mcMTOCs) that remain in the cytoplasm and do not contribute directly to spindle formation but play a crucial role in regulating spindle positioning and timely spindle migration. Here, a multi-photon laser ablation method is described to selectively deplete endogenously labeled mcMTOCs in oocytes collected from Cep192-eGfp reporter mice. This method contributes to the understanding of the molecular mechanisms underlying spindle positioning and migration in mammalian oocytes.

Importance of Antioxidant Supplementation during In Vitro Maturation of Mammalian Oocytes.

The in vitro embryo production (IVEP) technique is widely used in the field of reproductive biology. In vitro maturation (IVM) is the first and most critical step of IVEP, during which, the oocyte is matured in an artificial maturation medium under strict laboratory conditions. Despite all of the progress in the field of IVEP, the quality of in vitro matured oocytes remains inferior to that of those matured in vivo. The accumulation of substantial amounts of reactive oxygen species (ROS) within oocytes during IVM has been regarded as one of the main factors altering oocyte quality. One of the most promising approaches to overcome ROS accumulation within oocytes is the supplementation of oocyte IVM medium with antioxidants. In this article, we discuss recent advancements depicting the adverse effects of ROS on mammalian oocytes. We also discuss the potential use of antioxidants and their effect on both oocyte quality and IVM rate.

Lycopene Reduces the In Vitro Aging Phenotypes of Mouse Oocytes by Improving Their Oxidative Status.

Postovulatory aging is a major problem that limits the success of many assisted reproductive technologies (ARTs). Oxidative stress is a leading cause of oocyte aging. This study investigated the effects of lycopene supplementation of in vitro maturation (IVM) medium during the aging of mouse oocytes on the oocytes' morphology and oxidative stress status. Mouse cumulus-oocyte complexes (COCs) were collected and cultured in the IVM medium either for 17 h, (freshly matured oocytes), or for 48 h, (in vitro-aged oocytes), with or without lycopene. The rate of fragmented and degenerated oocytes and the oocyte levels of hydrogen peroxide (H2O2), malondialdehyde (MDA), total antioxidant capacity (TAC), reduced glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD) were estimated and compared. Oocytes aged with 200 nM lycopene revealed significantly less fragmentation and degeneration, lower H2O2 and MDA levels, and higher TAC, GSH and SOD levels than those aged without lycopene. CAT levels were unchanged by lycopene treatment. Taken together, our data showed beneficial effects of lycopene during in vitro aging of mouse oocytes by reducing the oxidative stress damages that lead to their apoptosis. The present study introduces lycopene as a natural supplement to reduce the postovulatory aging-dependent abnormalities of mammalian oocytes.

Effect of E-64 Supplementation during In Vitro Maturation on the Developmental Competence of Bovine OPU-Derived Oocytes.

Recovery of bovine oocytes using the ovum pick-up (OPU) technique offers the advantage of rapid genetic improvement through propagation of desired genes from animals with high genetic qualities. However, the developmental competence of OPU-derived immature oocytes remains relatively poor. We previously found that cathepsin B gene expression and activity are increased in poor quality oocytes and embryos compared to good quality ones. In this study, we investigated the effect of E-64 (cathepsin B inhibitor) supplementation during in vitro maturation (IVM) on the developmental competence of OPU-derived immature oocytes and the quality of the produced blastocysts. Our results showed that supplementation of IVM medium with E-64 significantly improved the developmental competence of OPU-derived immature oocytes as evidenced by the significant increase of the blastocyst rate. Importantly, the presence of E-64 during IVM also significantly improved blastocyst quality by increasing the total cell number and decreasing the percentage of TUNEL positive cells. These results indicate that E-64 supplementation during IVM is a promising tool to improve the efficiency of OPU-IVF program by improving the developmental competence of OPU-derived immature oocytes.

Microtubule organizing centers regulate spindle positioning in mouse oocytes.

During female meiosis I (MI), spindle positioning must be tightly regulated to ensure the fidelity of the first asymmetric division and faithful chromosome segregation. Although the role of F-actin in regulating these critical processes has been studied extensively, little is known about whether microtubules (MTs) participate in regulating these processes. Using mouse oocytes as a model system, we characterize a subset of MT organizing centers that do not contribute directly to spindle assembly, termed mcMTOCs. Using laser ablation, STED super-resolution microscopy, and chemical manipulation, we show that mcMTOCs are required to regulate spindle positioning and faithful chromosome segregation during MI. We discuss how forces exerted by F-actin on the spindle are balanced by mcMTOC-nucleated MTs to anchor the spindle centrally and to regulate its timely migration. Our findings provide a model for asymmetric cell division, complementing the current F-actin-based models, and implicate mcMTOCs as a major player in regulating spindle positioning.

Serum supplementation during bovine embryo culture affects their development and proliferation through macroautophagy and endoplasmic reticulum stress regulation.

Serum supplementation during bovine embryo culture has been demonstrated to promote cell proliferation and preimplantation embryo development. However, these desirable outcomes, have been associated with gene expression alterations of pathways involved in macroautophagy, growth, and development at the blastocyst stage, as well as with developmental anomalies such as fetal overgrowth and placental malformations. In order to start dissecting the molecular pathways by which serum supplementation of the culture medium during the preimplantation stage promotes developmental abnormalities, we examined blastocyst morphometry, inner cell mass and trophectoderm cell allocations, macroautophagy, and endoplasmic reticulum stress. On day 5 post-insemination, > 16 cells embryos were selected and cultured in medium containing 10% serum or left as controls. Embryo diameter, inner cell mass and trophectoderm cell number, and macroautophagy were measured on day 8 blastocysts (BL) and expanded blastocysts (XBL). On day 5 and day 8, we assessed transcript level of the ER stress markers HSPA5, ATF4, MTHFD2, and SHMT2 as well as XBP1 splicing (a marker of the unfolded protein response). Serum increased diameter and proliferation of embryos when compared to the no-serum group. In addition, serum increased macroautophagy of BL when compared to controls, while the opposite was true for XBL. None of the genes analyzed was differentially expressed at any stage, except that serum decreased HSPA5 in day 5 > 16 cells stage embryos. XBP1 splicing was decreased in BL when compared to XBL, but only in the serum group. Our data suggest that serum rescues delayed embryos by alleviating endoplasmic reticulum stress and promotes development of advanced embryos by decreasing macroautophagy.

Loop-mediated isothermal amplification (LAMP) and machine learning application for early pregnancy detection using bovine vaginal mucosal membrane.

An early and accurate pregnancy diagnosis method is required to improve the reproductive performance of cows. Here we developed an easy pregnancy detection method using vaginal mucosal membrane (VMM) with application of Reverse Transcription-Loop-mediated Isothermal Amplification (RT-LAMP) and machine learning. Cows underwent artificial insemination (AI) on day 0, followed by VMM-collection on day 17-18, and pregnancy diagnosis by ultrasonography on day 30. By RNA sequencing of VMM samples, three candidate genes for pregnancy markers (ISG15 and IFIT1: up-regulated, MUC16: down-regulated) were selected. Using these genes, we performed RT-LAMP and calculated the rise-up time (RUT), the first-time absorbance exceeded 0.05 in the reaction. We next determined the cutoff value and calculated accuracy, sensitivity, specificity, positive prediction value (PPV), and negative prediction value (NPV) for each marker evaluation. The IFIT1 scored the best performance at 92.5% sensitivity, but specificity was 77.5%, suggesting that it is difficult to eliminate false positives. We then developed a machine learning model trained with RUT of each marker combination to predict pregnancy. The model created with the RUT of IFIT1 and MUC16 combination showed high specificity (86.7%) and sensitivity (93.3%), which were higher compared to IFIT1 alone. In conclusion, using VMM with RT-LAMP and machine learning algorithm can be used for early pregnancy detection before the return of first estrus.

Capture and metabolomic analysis of the human endometrial epithelial organoid secretome.

Suboptimal uterine fluid (UF) composition can lead to pregnancy loss and likely contributes to offspring susceptibility to chronic adult-onset disorders. However, our understanding of the biochemical composition and mechanisms underpinning UF formation and regulation remain elusive, particularly in humans. To address this challenge, we developed a high-throughput method for intraorganoid fluid (IOF) isolation from human endometrial epithelial organoids. The IOF is biochemically distinct to the extraorganoid fluid (EOF) and cell culture medium as evidenced by the exclusive presence of 17 metabolites in IOF. Similarly, 69 metabolites were unique to EOF, showing asymmetrical apical and basolateral secretion by the in vitro endometrial epithelium, in a manner resembling that observed in vivo. Contrasting the quantitative metabolomic profiles of IOF and EOF revealed donor-specific biochemical signatures of organoids. Subsequent RNA sequencing of these organoids from which IOF and EOF were derived established the capacity to readily perform organoid multiomics in tandem, and suggests that transcriptomic regulation underpins the observed secretory asymmetry. In summary, these data provided by modeling uterine luminal and basolateral fluid formation in vitro offer scope to better understand UF composition and regulation with potential impacts on female fertility and offspring well-being.