WO2022251582A1 - Densitometry-based sorting for embryo health classification - Google Patents

Densitometry-based sorting for embryo health classification Download PDF

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WO2022251582A1
WO2022251582A1 PCT/US2022/031266 US2022031266W WO2022251582A1 WO 2022251582 A1 WO2022251582 A1 WO 2022251582A1 US 2022031266 W US2022031266 W US 2022031266W WO 2022251582 A1 WO2022251582 A1 WO 2022251582A1
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embryo
embryos
channel
density
hfd
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French (fr)
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Nurlybek MURSALIYEV
Vittorio SEBASTIANO
Utkan Demirci
Naside Gozde DURMUS
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The Board Of Trustees Of The Leland Stanford Junior University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0604—Whole embryos; Culture medium therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2529/00—Culture process characterised by the use of electromagnetic stimulation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/367—Infertility, e.g. sperm disorder, ovulatory dysfunction

Definitions

  • This disclosure relates to the separation of embryos using densitometry-based sorting.
  • one or more embryos are assessed based on a density of an embryo which serves as a proxy for health or viability of the embryo.
  • An embryo in a paramagnetic medium is introduced into a levitation device including a channel that is positioned within in a controlled magnetic field.
  • the embryo in the paramagnetic medium is received in the channel and subjected to the controlled magnetic field in order to levitate the embryo to an equilibrium height against the force of gravity.
  • the equilibrium height of the embryo correlates to the density of the embryo which is dependent at least in part on the lipid content of the embryo.
  • the health or viability of the embryo is assessed based on the density of the embryo.
  • the method may include, either before levitating the embryo and/or periodically between uses to confirm accurate readings, calibrating the levitation device prior to the step of introducing the embryo in the paramagnetic medium into the levitation device.
  • the calibration step may involve establishing a correlation between equilibrium levitation heights of reference objects having known densities in the paramagnetic and their densities such that, when the embryo in a paramagnetic medium is received in the channel, the levitation height of the embryo that is observed can be correlated to the density of the embryo (which serves as a proxy for the health or viability of the embryo in the assessing step).
  • the reference objects may be polyethene beads of known densities.
  • embryos may be sequentially introduced and individually assessed. However, it is also contemplated that embryos may be introduced in sequence to perform a plurality of assessments.
  • the levitation device may include one or more magnets positioned relative to the channel to provide the controlled magnetic field. These may include one magnet or two magnets placed on opposite sides of the channel. It is contemplated that, relative to the direction of gravity, the channel may be generally horizontal, with the magnets positioned above and/or below the channel. It is further contemplated that the channel in question may be a microchannel or microcapillary.
  • the method may further include the step of placing the embryo in the paramagnetic medium before introducing the paramagnetic medium and embryo into the levitation device.
  • the method may further include the step of sorting the embryos based on their equilibrium height.
  • the equilibrium height of a respective embryo may be a function at least in part the intrinsic cellular density based on lipid content.
  • the method may further include the steps of setting a threshold height and sorting a first group of the embryos having respective equilibrium heights above the threshold height from a second group of the embryos having respective equilibrium heights below the threshold height.
  • the channel in which levitation occurs i.e., the levitation channel
  • the channel in which levitation occurs may be in fluid communication with both an upper collection channel and a lower collection channel at an outlet end of the channel.
  • the step of sorting the embryos based on their equilibrium height may involve collecting the first group of the embryos having respective equilibrium heights above the threshold height in an upper collection channel (upper relative to the direction of gravity) and collecting the second group of the embryos having respective equilibrium heights below the threshold height in a lower collection channel (lower relative to the direction of gravity).
  • the upper channel and the lower channel may both be connected to respective pumps.
  • the respective pump for the upper channel can be independent of the respective pump for the lower channel such that just one of the pumps is operated at a particular moment in time.
  • the respective pumps may be part of a dual syringe pump device.
  • the method may further involve, immediately after levitating of a respective embryo in the channel, determining whether the respective embryo in the channel is above or below the threshold height by visually imaging the respective embryo in the channel and then operating only one of the respective pumps to collect the respective embryo in the upper collection chamber or in the lower collection chamber based on the equilibrium height of the respective embryo relative to the threshold height.
  • the visually imaging may be live imaging (for example, real time imaging) of the respective embryo in the channel.
  • the channel may be a capillary.
  • the method may further involve the step of transplanting an embryo after assessment into a mother.
  • FIG. 1A-1C provides a characterization of High Fat Diet ("HFD") female mice and their oocyte qualities.
  • FIG. IB shows oocyte numbers extracted per female.
  • FIG. ID shows High Fat Diet content as % kcal.
  • FIG. IE shows body fat percentage as measured by total abdominal fat ratio to total weight.
  • FIG. 2C presents the oocyte numbers extracted per female of control and ob/ob mice.
  • N normal
  • D deformed
  • FIGS. 3A-3G provides a characterization of oocytes and embryonic development of control and HFD females.
  • FIG. 3A is a schematic of the in vitro generation of embryos from female mice.
  • FIG. 3A is a schematic of the in vitro generation of embryos from female mice.
  • pIVF 72h post IVF
  • FIG. 3E compares E18.5 heights and
  • FIG. 3F compares weights from control and HFD females.
  • FIGS. 4A-4J provides a characterization of oocytes and preimplantation embryos from control and ob/ob females.
  • FIG. 4A compares fertilization efficiency of control and HFD eggs, as measured by 2C stage 24h pIVF.
  • FIG. 4B compares percent of dead blastocysts at E3.5 from control and HFD mice.
  • FIG. 4E compares the number of fetuses per litter.
  • FIG. 4A compares fertilization efficiency of control and HFD eggs, as measured by 2C stage 24h pIVF.
  • FIG. 4B compares percent of dead blastocysts at E3.5 from control and HFD mice.
  • FIG. 4F is a schematic of the in vitro generation of embryos from female control and ob/ob mice.
  • FIG. 41 compares percent of dead blastocysts at E3.5 from control and ob/ob mice.
  • FIGS. 5A-5G provides transcriptomic characterization of 4-cell stage embryos from control and obese embryos.
  • FIG. 5A is a schematic of events and stages of preimplantation embryos (ZGA- zygotic genome activation).
  • FIG. 5B provides principal component analysis of mRNA expression showing HFD embryos clustered away from Control embryos.
  • FIG. 5C illustrates hierarchical clustering using Euclidean distance with average linkage of all expressed genes from Control and HFD embryos.
  • FIG. 5D provides volcano plots showing upregulated differentially expressed genes (DEG) in green [rightmost dashed box], and downregulated DEGs is in red [leftmost dashed box]. Insignificant genes are in black and primarily outside of the dashed boxes.
  • FIG. 5E provides the gene ontology analysis of Control and HFD DEGs.
  • FIG. 5F provides functional annotation analysis with UniProt Keywords with DEGs.
  • FIG. 5G is a heatmap of expression of genes involved in AMPK pathway.
  • FIG. 6A provides an analysis of differentially expressed genes (DEG).
  • the leftmost column of FIG. 6A provides a heatmap of Log2 of 92 DEGs with hierarchical clustering of genes and different treatments.
  • the rightmost four columns of FIG. 6A provide a heatmap of Log2 of genes from gene ontology groups.
  • the bottommost column of FIG. 6A is a heatmap of Log2 expression of Gpx7 and Entpd6.
  • FIG. 6B illustrates principal component analysis showing ob/ob embryos clustered away from HFD and Control embryos.
  • FIG. 6C shows volcano plots showing upregulated differentially expressed genes (DEG) in green [right box], and downregulated DEGs is in red [left box]. Insignificant genes are in black [all others]
  • FIG. 6D provides a Venn diagram of shared DEGs between HFD and ob/ob embryos and a Pearson correlation analysis and heat map of 40 genes. Expression patterns of 42 shared genes. Red are upregulated and blue are downregulated genes compared to control embryos.
  • FIG. 6E shows gene ontology analysis of ob/ob DEGs.
  • FIG. 6F provides a heatmap of genes involved in AMPK signaling pathway that are downregulated in ob/ob embryos.
  • FIGS. 7A-7D illustrate downregulation of AMPK activity and metabolic disruption in HFD embryos.
  • FIG. 7B provides [left panel]
  • FIGS. 8A-8D illustrate downregulation of AMPK activity and metabolic disruption in HFD embryos.
  • FIGS. 9A-9C illustrate downregulation of AMPK activity and metabolic disruption in HFD embryos.
  • 9C provides [left panels] immunofluorescent images of NucView488 and Gadd45a staining of control and HFD blastocysts and [right panels] further quantification of NucView488 and Gadd45a positive cells per blastocyst.
  • FIGS. 10A-10C illustrate downregulation of AMPK activity and metabolic disruption in ob/ob embryos.
  • FIGS. 11A-11C illustrate density measurements of 4Cell stage embryos from HFD mothers.
  • FIG. 11A shows [left panel] magnetic levitation representative images of oocyte and preimplantation embryo stages and [right panel] quantifications of density measurements.
  • FIG. 11B is an illustration of magnetic density device and experimental procedure.
  • FIG. 12A shows density measurement of polyethylene beads (1.031 g-mL-1, 1.064 g-mL-1, and 1.089 g-mL-1) in the magnetic levitation platform with [left panel] representative images of levitated beads and [right panel] the relationship between the beads and levitation heights in 30mM Gd+ concentration.
  • FIG. 12B includes representative images of magnetic levitations of blastocysts with large and small diameters.
  • FIG. 12C shows the relationship between the blastocyst sizes and density measurements.
  • FIG. 12D shows [left panel] representative images of control and density measured embryo and [right panel] percentage of blastocyst formation between control and density measured embryos.
  • FIG. 12A shows density measurement of polyethylene beads (1.031 g-mL-1, 1.064 g-mL-1, and 1.089 g-mL-1) in the magnetic levitation platform with [left panel] representative images of levitated beads and [right panel] the relationship between the beads and levit
  • FIGS. 13A-13F illustrate transplantation of density sorted embryos into foster mothers and post-implantation embryo assessment.
  • FIG. 13A provides an illustration of workflow of density sorting by density and transplanting into foster mothers.
  • FIG. 13E shows E18.5 placental weights of highl and low- density embryos.
  • FIG. 14A-14C provides an evaluation of low- and high-density embryos after sorting.
  • FIG. 14A provides lipid staining with Bodipy493/503 of 4Cell embryos from HFD mice sorted by densitometry device.
  • FIG. 14B provides single embryo fluorescent intensity quantification of high density and low-density sorted embryos.
  • FIG. 14C provides blastocysts and morula ratio at 72h post IVF of low-density and high-density cohorts.
  • FIG. 14D shows efficiency of transplantation of high density and low density embryos.
  • FIG. 14E shows litter sizes of high and low density cohorts.
  • FIG. 14F shows average placenta/fetus ratio per litter in high- and low- density embryos.
  • FIGS. 15A-15E provide an automated density-based embryo sorting device.
  • FIG. 15A provides an illustration of workflow.
  • FIG. 15B shows the real-time observation of single embryos sorted by density in which an embryo with lower density was sorted to Top Syringe (Sorted Up) and an embryo with higher density was sorted to Bottom Syringe (Sorted Down).
  • FIG. 15C shows [top panel] representative images of levitated 4 Cell embryos in which the dotted line denotes separation height and [bottom panel] density quantification of 4 Cell embryos sorted down and sorted up.
  • FIG. 15A provides an illustration of workflow.
  • FIG. 15B shows the real-time observation of single embryos sorted by density in which an embryo with lower density was sorted to Top Syringe (Sorted Up) and an embryo with higher density was sorted to Bottom Syringe (Sorted Down).
  • FIG. 15C shows [top panel] representative images of levitated 4 Cell embryos in which the dotted line denotes separation height and [bottom panel]
  • FIG. 15D shows lipid staining with Bodipy493/503 of 4Cell sorted down and sorted up embryos.
  • mice To study obesity in mice, two categories of mouse models are investigated: first, mouse models with genetic mutations (ob/ob, Tallyho etc.) and second, genetically intact animals exposed to obesogenic environments (Diet induced model). Genetic mutation in leptin mouse ob/ob is one of the most studied models of obesity and it has been recognized since 1950s. ob/ob mice are morbidly obese and experience hyperphagia, glucose intolerance, and are infertile. However, ob/ob female infertility was concluded only by absence of pregnancy after natural mating, and the underpinning cause (maternal versus embryonic) is not known.
  • DIO Diet induced obesity
  • HFD fat calorie diet
  • Oocytes were collected from HFD and control females and they were in vitro fertilized (IVF) with sperm from males fed with a normal diet.
  • IVF vitro fertilized
  • the preimplantation development and metabolic state from obese mothers were first characterized.
  • transcriptional profiles at late 4 cell stage embryos were analyzed and disruption in metabolic genes was shown.
  • Abnormal metabolism with downregulated AMPK, and elevated lipid content in embryos from HFD mothers was confirmed.
  • MagneticDense single-cell magnetic levitation densitometry device
  • Gadd45a is associated with early lineage commitments in preimplantation embryos, and knockout of Gadd45a/b genes in preimplantation embryos caused lethality at 4-cell stage. Overall, this indicates that Gadd45a plays important role in preimplantation development, and this necessitates deeper examination of its role in embryos under maternal obesity. As Gadd45a is regulated by AMPK via FOX03 signaling pathway, AMPK and Gadd45a reduction in obese embryos can potentially be activated by small molecule drugs, such as resveratrol and metformin. An accumulation of lipids in embryos from mice was also observed, which was also seen in embryos from ob/ob mice.
  • lipid accumulation has been observed in human embryos as well, suggesting its clinical relevance. Since lipid accumulation is associated with low embryonic quality, lipid accumulation in obese embryos was used as a proxy for low embryo quality.
  • MagDense a novel magnetic densitometry device
  • FIG. 15A Low density was shown to be correlated with higher lipid content in embryos. For the first time, single embryo densities was able to be measured using the MagDense device and density measurements used as a proxy for embryonic quality in embryos from fetal growth retardation obese mothers.
  • the densitometry approach can be used to non-invasively assess the lipid content of human embryos before implantation, as the lipid content was correlated with preimplantation embryonic arrest.
  • Example I Characterization of oocytes from obese females
  • B6.Cg-Lepob/J (ob/ob) females were also investigated, which have a spontaneous mutation of leptin gene ob/ob mice are characterized by obesity, hyperphagia, and infertility ob/ob females gained considerable weight (FIG. 2A), and their blood metabolic panel was elevated (FIG. 2B).
  • ob/ob females were able to generate Mil oocytes upon superovulation, but the oocyte number was lower (FIG. 2C) and the percentage of deformed oocytes was increased (FIG.
  • FIG. 3D the fetuses at E18.5 were evaluated and smaller fetal sizes (FIG. 3D) were measured in heights (FIG. 3E) and weights (FIG. 3F). Moreover, the average weight of fetuses in a litter was smaller as well (FIG. 4C). Even though there was no significant difference in placental weights (FIG. 4D), the ratio of placenta to embryo was elevated in HFD embryos compared to controls (FIG. 3G). Moreover, the litter sizes were smaller in HFD mice compared to control mice (FIG.4E).
  • Example III Transcriptomic analysis of embryos from HFD mothers revealed disruption in metabolic genes and genes involved in energy regulation
  • Embryonic transcriptome is established by a sequence of well-regulated events, including maternal mRNA degradation and chromatin remodeling, that are initiated with fertilization and almost completed by the 4-cell stage. Therefore, to evaluate the transcriptomes of embryos from HFD mothers, 4 cell stage embryos were analyzed where embryonic state has been established (FIG. 5A). Principal component analysis showed that 4 cell embryos from HFD (HFD 4C embryos) and control mothers seem to be separated more on Principal Component 1 (PCI) (FIG. 5B), which is defined by 66% of genes rather than Principle Component 2 (PC2) defined by 24% of genes.
  • PCI Principal Component 1
  • Gpx7 and Entpd6 were downregulated in HFD embryos (FIG. 6, center bottom).
  • Gpx7 Glutathione Peroxidase 7
  • ROS reactive oxygen species
  • Entpd6 ectonucleoside triphosphate diphosphohydrolase 6
  • Purine and pyrimidine metabolism is part of purine and pyrimidine metabolism and it is associated with obesity in humans.
  • AMP-activated protein kinase AMP-activated protein kinase
  • Idua alpha-L-iduronidase
  • AMPK AMPK pathway
  • Ppp2r2c protein phosphatase 2, regulatory subunit B, gamma
  • PP2A is a heterotrimeric serine/threonine phosphatase that has been shown to inactivate AMPK.
  • the expression of Ppp2r2c is also upregulated in HFD 4C embryos (FIG.
  • Eef2k eukaryotic elongation factor 2 kinase
  • AMPK eukaryotic elongation factor 2 kinase
  • FIG. 5G Eef2k downregulated in HFD 4C embryos was observed (FIG. 5G), which correlates with AMPK downregulation in HFD 4C embryos.
  • Gadd45a Growth Arrest And DNA Damage Inducible Alpha
  • Example IV Embryos from ob/ob mothers have similar transcriptomic disruptions to embryos from HFD females
  • Example V Immunofluorescent imaging revealed potential role o/Gadd45a in apoptotic cells at blastocysts from obese females
  • Gadd45a have been shown to be important in normal preimplantation embryo development and it is associated with early lineage commitments in preimplantation embryos, and knockout of GADD45a/ genes in preimplantation embryos caused lethality at 4 cell stage. Gadd45a has not been investigated in the context of obesity in preimplantation embryos, therefore it was decided to explore its role in preimplantation embryos from obese mice. To confirm downregulation of Gadd45a observed in RNA-seq analysis, protein levels of Gadd45a was investigated by immunofluorescence and confirmed Gadd45a downregulation in 4 cell embryos from HFD mothers (FIG. 9A) and ob/ob mothers (FIG. 10A).
  • Gadd45a (Growth Arrest and DNA Damage Inducible Alpha) plays an important role in DNA damage response and regulates apoptosis, and DNA damage and apoptosis is mostly observed in blastocysts stage of preimplantation embryos, thus, we sought to investigate the role of Gadd45a in apoptosis in blastocysts.
  • FIG. 9C Increased apoptosis in blastocysts from obese mothers was observed (see FIG. 9C, FIG. 4J), therefore the DNA damage in obese blastocysts was assessed and increased levels of DNA damage observed as measured by phospho-yH2A.X signal (FIG. 9B). Similar increase in embryos from ob/ob mice was confirmed as well (FIG. 10B). Then we sought to investigate the relationship of Gadd45a in apoptotic cells in blastocysts with co staining NucVew488 and Gadd45a antibody. In control blastocysts, we saw Gadd45a is co expressed in apoptotic cells (FIG. 9C).
  • Example VI Embryos from obese mothers have abnormal metabolism and disruption in cellular energy homeostasis
  • Example VII Non-invasive cellular density measurements can predict embryo quality at 4 cell stage
  • Magnetic levitation densitometry allows the measurement of cellular density at the single cell level, without compromising cellular integrity and without the use of biomarkers, antibodies or tags.
  • the device consists of a glass microcapillary or channel held between two permanent magnets with same poles facing each other (although a magnetic field may be produced in other ways as well using fewer or more magnets and in various possible configuration).
  • Cells are spiked or introduced into the capillary or channel with paramagnetic medium. Under the magnetic field, cells are levitated at specific heights when gravitational, buoyancy, and magnetic forces reach an equilibrium. This equilibrium mainly depends on intrinsic cellular density.
  • ZGA pre-zygotic genome activation
  • Lipids are buoyant macromolecules that affect cellular density. Recently, we showed that iPSC-derived cardiomyocytes with high lipid content have lower densities and these minute density differences were leveraged to separate diseased cell populations based on their intracellular lipid content by using MagDense. We also sought to utilize MagDense device to sort embryos based on their inherent magnetic levitation profiles driven by their lipid content (FIG. 13B). Since we observed elevated lipid content in embryos from HFD mothers, we assessed whether embryos are also characterized by lower density due to higher lipid content. Interestingly, we detected a significant decrease in density measurements (FIG. 11C) which is inversely correlated with the lipid content observed in HFD embryos.
  • Average densities for control and HFD 4 Cell embryos were measured as 1.0862 ⁇ 0.002 g/ml and 1.084 ⁇ 0.002 g/ml, respectively. Since we saw increased lipid content in 4 cell embryos from ob/ob females, we tested their densities as well and saw similar low-density measurements (FIG. 12E). Average densities for control and ob/ob 4 Cell embryos were measured as 1.087 ⁇ 0.004 g/ml and 1.082 ⁇ 0.0006 g/ml, respectively. This indicates that maternal obesity (HFD and ob/ob mothers) were able to influence embryonic density characteristics as well.
  • FIG. 15A To test if the embryonic densities at early preimplantation stage (4 Cell stage) play significant role in further embryonic development, we sought to sort embryos based on their densities (FIG. 15A).
  • FIG. 15B At 4 cell stage, we measured the densities at single embryo level and sorted them onto high- and low-density cohorts based on the average density measurement (FIG. 15B). Average density of 4 Cell embryos were 1.085 ⁇ 0.002 g/ml, and the average measurements of high- and low-density embryos were 1.083 ⁇ 0.001 g/ml and 1.087 ⁇ 0.001 g/ml, respectively.
  • Example VIII Automated Density Based Embryo Sorting Device
  • CD1 mice 3 weeks old CD1 mice (Crl:CDl(ICR), 022) were purchased from Charles River Laboratories. CD1 mice were randomly divided into two diet groups; one group received a HFD (TD.180431, Teklad Custom Diet, Envigo) and the other group received a Control Diet (TD.08806, Envigo) for 16 weeks. CD1 male sperm were used for in vitro fertilization procedure.
  • mice Female mice were superovulated by injecting lOOul of CARD HyperOva ® followed by
  • COCs human chorionic gonadotropin
  • zygotes were transferred into preincubated KSOM medium (K0101, CytoSpring) and cultured up to the blastocyst stage at 37 °C in a humidified atmosphere of 5% C0 2 , 5% 0 2 and 90% N 2 . Every day the developmental rate of embryos was assessed, by counting number of embryonic stages. After density measurements, the embryos were transferred into foster mothers. The embryo transfer procedure is followed from previous work. Foster mother selected were 4-5-week-old CD1 females under normal diet. To generate pseudopregnant females, proestrus stage females were selected based on the appearance of their external genital tract and sterile mated with vasectomized males. E3.5 embryos were transferred into
  • mice 2.5 dpc pseudo-pregnant CD1 females. Mice were anesthetized and oviducts from both uterine horns were surgically pulled out for a transfer. 15 embryos were transferred per mouse (7-8 embryos per horn). Recipients were killed at E18.5, and fetuses were collected for evaluation.
  • mice bedding was added into female mice cage to induce estrus cycle. After 2 days, a male was placed with 2 females. Next day, males were removed from the cage and presence of vaginal plug was checked in females. For gestational age, midnight of the day of mating was designated as day 0. On E18.5 females were euthanized, and embryos were collected, and evaluated for size. RNA sequencing of 4 cell embryos
  • NucView ® 488 Caspase-3 Enzyme Substrate (10402-T, Biotium) according to manufacturer's protocols. 5 uM NucView ® 488 substrate was added to 50ul KSOM medium and preincubated for at least 30min. When embryos reached blastocyst stage (E3.5) embryos were transferred into this preincubated NucView ® 488 -KSOM medium for 30 minutes. Afterwards, the zona pellucida was removed by Acidic Tyrode's solution (CytoSpring) and embryos were fixed in 4% PFA in PBS for 20 min at 4° C. Embryos were proceeded to immunostaining and imaging.
  • the zona pellucida was removed by Acidic Tyrode's solution (CytoSpring) and embryos were fixed in 4% PFA in PBS for 20 min at 4° C. After permeabilization in 0.2% Triton-X, 0.1% BSA in PBS for 10 min at RT blastocysts were blocked overnight in 0.1% BSA in PBS at 4° C.
  • Embryos were then incubated with primary antibodies in blocking solution for 3-4 h at RT at following conditions: 1:200 Gadd45a (mouse, Santa Cruz), 1:200 pAMPK (rabbit, 50081S), 1:3000 Phospho-Histone H2A.X (rabbit, 9718T), 1:500 Anti- -actin (mouse, A5441).
  • After several washes in blocking solution at RT blastocysts were incubated with secondary antibodies using 488, 594 or 680 Alexa Fluor conjugates (Invitrogen) at 1:1000 dilution, and/or with BODIPYTM 493/503 (D3922) 1:1000 dilution for 1-2 h at room temperature.
  • Fatty acid assay kit was purchased from Biovision (# K408-100), and fatty acid incorporation was measured according to manufacturer's protocols with some modifications. Instead of plate reader we used fluorescent microscope to measure fatty acid uptake per embryo. After growth in KSOM media without Phenol, the Morula stage embryos were transferred into glass bottom plate with prewarmed Uptake Assay buffer at 37°C, 20-30 embryos per 50ul droplet and were incubated for lh. Afterwards, 2x solution of quenched Uptake Reaction Mix was added to the droplets with embryos. The fluorescence was immediately measured in EVOS FL Digital Microscope. Fluorescent intensity per embryo was measured in ImageJ. Empty regions without embryos were used as a background control signal.
  • Mitochondrial potential measurement was performed by MitoView ® 633 (70055-T, Biotium) according to manufacturer's protocols.
  • lOOnM MitoView ® 633 solution was made in KSOM medium and preincubated at 37°C for at least 30min. 4 cell stage embryos were transferred into this preincubated lOOnM MitoView ® 633 - KSOM medium for 30 minutes. Afterwards, embryos were washed one time, and imaged using EVOS FL Digital Microscope. Fluorescent intensity per embryo was measured in ImageJ.
  • Embryos were levitated in a paramagnetic medium: 30 mM Gd (gadolinium-based paramagnetic medium) in KSOM media. Before loading embryos into levitation capillary, they were preincubated in 30 mM Gd KSOM medium for 3-5 minutes. Afterwards, embryos were transferred into capillary with 30 mM Gd KSOM and levitated for 10 minutes. In this unique device configuration, embryos can be levitated and separated based on their inherent density without the use of labels, antibodies or tags.
  • Gd gadolinium-based paramagnetic medium
  • the magnetic susceptibility difference between an embryo and its surrounding paramagnetic medium causes it to move away from a higher (i.e., close vicinity at the magnets) to a lower magnetic field strength site (i.e., away from the magnets) until gravitational, buoyancy and magnetic forces acting on the embryos reach an equilibrium. Embryos are levitated at a final position between the two magnets, where the magnetic force (Fmag) equals the buoyancy force (Fb).
  • Polyethylene beads with known densities 1.031, 1.064, and 1.089 g rnL -1 ) were used to generate a standard curve to calculate embryo densities based on their levitation heights. Levitation heights were quantified by ImageJ software.
  • the embryos were separated into two cohorts: i) embryos levitating above (low density); and ii) below (high density) the average levitating height. High- and low- density embryos were then cultured separately in KSOM until blastocyst stage (E3.5).
  • Automated Density Based Embryo Sorting Device is a densitometry and imaging platform which uses the principles of magnetic levitation.
  • the device consists of a flow channel (1 mm in height and width) held between two custom-designed rare earth magnets for gentle and rapid separation of different cell states in a continuous flow operation.
  • aluminum-coated mirrors are placed at each side of the microchannel and a camera images the cells as they levitate and move through the flow channel.
  • the system is driven by two syringe pumps that withdraw the sample into the flow-based magnetic levitation system from droplet of paramagnetic solution (30mM GD+ in KSOM media) immersed in mineral oil.

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Abstract

One or more embryos are assessed based on a density of an embryo which serves as a proxy for health or viability of the embryo. An embryo in a paramagnetic medium is introduced into a levitation device including a channel that is positioned within in a controlled magnetic field. The embryo in the paramagnetic medium is received in the channel and subjected to the controlled magnetic field in order to levitate the embryo to an equilibrium height against the force of gravity. The equilibrium height of the embryo correlates to the density of the embryo which is dependent at least in part on the lipid content of the embryo. The health or viability of the embryo is assessed based on the density of the embryo.

Description

DENSITOMETRY-BASED SORTING FOR EMBRYO HEALTH CLASSIFICATION
CROSS-REFERENCE TO RELATED APPLICATION [0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/194,027 entitled "Densitometry-Based Sorting for Embryo Health Classification" filed May 27, 2021, the contents of which are incorporated by reference herein in its entirety for all purposes.
STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] This invention was made with Government support under contracts U54CA199075 awarded by the National Institutes of Health. The Government has certain rights in the invention.
TECHNICAL FIELD
[0003] This disclosure relates to the separation of embryos using densitometry-based sorting.
BACKGROUND
[0004] Human embryonic development is inherently characterized by low success rate and by low embryo quality, with some estimates that half of human embryos are developmental^ abnormal and more than half of IVF embryos fail to reach the blastocyst stage. For couples undergoing IVF, identifying embryos with the highest developmental potential maximizes the chances of pregnancy and birth while minimizing the number of oocytes retrieved, and embryos transferred and reducing the chances of multiplets. Therefore, more oocytes are retrieved than needed, resulting in less than 5% of all retrieved oocytes develop to live born babies. Overall, this indicates the importance of improving the current methods of embryo selection. SUMMARY
[0005] Poor outcomes of pregnancy in obese mothers are clinically recognized, but the underpinning causes are still largely not understood. To dissect the effect of obesity on embryonic development, we investigated preimplantation and postimplantation embryos obtained from females with high fat diet (HFD) induced obesity. We observed poor oocyte and preimplantation embryo quality, and retardation of development after in vitro fertilization. Transcriptomic analysis revealed disruption in genes involved in metabolism, and energy sensing / AMPK pathway. Similar changes were confirmed in a complementary genetic model of obesity (ob/ob). Lastly and importantly, for the first time, we measured preimplantation embryo density through a non-invasive densitometry device and demonstrate that density of preimplantation embryos is a predictor of fetal growth, establishing a proof of concept that densitometry can be used as a novel noninvasive method to select healthier E18.5 conceptuses and avoid conceptuses with fetal growth retardation in obese mothers.
[0006] According to one aspect, one or more embryos are assessed based on a density of an embryo which serves as a proxy for health or viability of the embryo. An embryo in a paramagnetic medium is introduced into a levitation device including a channel that is positioned within in a controlled magnetic field. The embryo in the paramagnetic medium is received in the channel and subjected to the controlled magnetic field in order to levitate the embryo to an equilibrium height against the force of gravity. The equilibrium height of the embryo correlates to the density of the embryo which is dependent at least in part on the lipid content of the embryo. The health or viability of the embryo is assessed based on the density of the embryo.
[0007] In some forms of the method, the method may include, either before levitating the embryo and/or periodically between uses to confirm accurate readings, calibrating the levitation device prior to the step of introducing the embryo in the paramagnetic medium into the levitation device. The calibration step may involve establishing a correlation between equilibrium levitation heights of reference objects having known densities in the paramagnetic and their densities such that, when the embryo in a paramagnetic medium is received in the channel, the levitation height of the embryo that is observed can be correlated to the density of the embryo (which serves as a proxy for the health or viability of the embryo in the assessing step). In one form, the reference objects may be polyethene beads of known densities.
[0008] In some forms of the method, only a single embryo may be evaluated at a time in the levitation device. In this way, embryos may be sequentially introduced and individually assessed. However, it is also contemplated that embryos may be introduced in sequence to perform a plurality of assessments.
[0009] In some forms, the levitation device may include one or more magnets positioned relative to the channel to provide the controlled magnetic field. These may include one magnet or two magnets placed on opposite sides of the channel. It is contemplated that, relative to the direction of gravity, the channel may be generally horizontal, with the magnets positioned above and/or below the channel. It is further contemplated that the channel in question may be a microchannel or microcapillary.
[0010] In some forms, the method may further include the step of placing the embryo in the paramagnetic medium before introducing the paramagnetic medium and embryo into the levitation device.
[0011] In some forms, the method may further include the step of sorting the embryos based on their equilibrium height. The equilibrium height of a respective embryo may be a function at least in part the intrinsic cellular density based on lipid content. According to such forms, the method may further include the steps of setting a threshold height and sorting a first group of the embryos having respective equilibrium heights above the threshold height from a second group of the embryos having respective equilibrium heights below the threshold height. The channel in which levitation occurs (i.e., the levitation channel) may be in fluid communication with both an upper collection channel and a lower collection channel at an outlet end of the channel. The step of sorting the embryos based on their equilibrium height may involve collecting the first group of the embryos having respective equilibrium heights above the threshold height in an upper collection channel (upper relative to the direction of gravity) and collecting the second group of the embryos having respective equilibrium heights below the threshold height in a lower collection channel (lower relative to the direction of gravity). In some instances, the upper channel and the lower channel may both be connected to respective pumps. The respective pump for the upper channel can be independent of the respective pump for the lower channel such that just one of the pumps is operated at a particular moment in time. In some forms, the respective pumps may be part of a dual syringe pump device. In some forms, the method may further involve, immediately after levitating of a respective embryo in the channel, determining whether the respective embryo in the channel is above or below the threshold height by visually imaging the respective embryo in the channel and then operating only one of the respective pumps to collect the respective embryo in the upper collection chamber or in the lower collection chamber based on the equilibrium height of the respective embryo relative to the threshold height. The visually imaging may be live imaging (for example, real time imaging) of the respective embryo in the channel.
[0012] In some forms, the channel may be a capillary.
[0013] In some forms, the method may further involve the step of transplanting an embryo after assessment into a mother.
[0014] These and still other advantages of the invention will be apparent from the detailed description and drawings. What follows is merely a description of some preferred embodiments of the present invention. To assess the full scope of the invention, the claims should be looked to as these preferred embodiments are not intended to be the only embodiments within the scope of the claims.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1A-1C provides a characterization of High Fat Diet ("HFD") female mice and their oocyte qualities. FIG. 1A includes [left panel] pictures of representative females and [right panel] female weights of control (n=14) and HFD mice (n=14). FIG. IB shows oocyte numbers extracted per female. FIG. 1C provides [left panel] number of deformed oocytes per female from control (n=12) and HFD mice (n=12), and representative images of [upper right panel] normal (N) and [lower right panel] deformed (D) oocytes. Data are presented as means ± standard deviation of three independent experiments (oocytes, n = 479 for Control and n = 371 for HFD). Mann Whitney two-tailed test. * =< 0.05, **=<0.01, ***=<0.002, ***=<0.001, ****=<0.0001. FIG. ID shows High Fat Diet content as % kcal. FIG. IE shows body fat percentage as measured by total abdominal fat ratio to total weight. FIG. IF presents the blood metabolic panel of HFD (n=3) and control (n=3) females.
[0016] FIG. 2A presents [left panel] pictures of representative females and [right panel] female weights of control (n=12) and ob/ob (n=12) mice. FIG. 2B presents the blood metabolic panel of ob/ob (n=3) and control (n=3) females. FIG. 2C presents the oocyte numbers extracted per female of control and ob/ob mice. FIG. 2D shows the number of deformed oocytes per female from control (n=12) and ob/ob (n=12) mice, and representative images of normal (N) and deformed (D) oocytes (oocytes, n = 546 for Control and n = 172 for ob/ob). Throughout, the data are presented as means ± standard deviation of three independent experiments.
Mann Whitney two-tailed test. * =< 0.05, **=<0.01, ***=<0.001, ****=<0.0001.
[0017] FIGS. 3A-3G provides a characterization of oocytes and embryonic development of control and HFD females. FIG. 3A is a schematic of the in vitro generation of embryos from female mice. FIG. 3B illustrates [left panel] blastocyst and morula ratio at 72h post IVF (pIVF) in control (n=364) and HFD embryos (n=203) and [right panel] representative images of control and HFD embryos. FIG. 3C shows [left panel] confocal immunofluorescent images of control and HFD blastocysts stained with NucView488 for apoptotic cells (scale: 50um) and [right panel] apoptotic cell quantification per control (n=23) and HFD (n=23) blastocysts. FIG. 3D shows post-implantation characterization of fetuses (control, n=27 and HFD, n=20) at E18.5 after natural mating with representative images of E18.5 fetuses. FIG. 3E compares E18.5 heights and FIG. 3F compares weights from control and HFD females. FIG. 3G compares the placenta/fetus ratio from HFD and control females. Data are presented as means ± standard deviation of three independent experiments. Mann Whitney two-tailed test. * =< 0.05, **=<0.01, ***=<0.001, ****=<0.0001.
[0018] FIGS. 4A-4J provides a characterization of oocytes and preimplantation embryos from control and ob/ob females. FIG. 4A compares fertilization efficiency of control and HFD eggs, as measured by 2C stage 24h pIVF. FIG. 4B compares percent of dead blastocysts at E3.5 from control and HFD mice. FIG. 4C compares average fetus weight per litter of the control (control female, n=5) versus HFD (HDF female, n=5) groups. FIG. 4D compares placental weights of control (n=12) and HFD (n=10) fetuses. FIG. 4E compares the number of fetuses per litter. FIG. 4F is a schematic of the in vitro generation of embryos from female control and ob/ob mice. FIG. 4G compares fertilization efficiency of control (n=392) and ob/ob (n=164) eggs, as measured by 2C stage 24h pIVF. FIG. 4H compares [left panel] blastocyst and morula ratio at 72h post IVF (pIVF) in control (n=345) and ob/ob (n-143) embryos and [right panel] provides representative images of control and ob/ob embryos. FIG. 41 compares percent of dead blastocysts at E3.5 from control and ob/ob mice. FIG. 4J illustrates [left panel] confocal immunofluorescent images of control and ob/ob blastocysts stained with NucView488 for apoptotic cells (scale 50um) and [right panel] presents apoptotic cell quantification per control (n=42) and ob/ob (n=32) blastocysts. Data are presented as means ± standard deviation of three independent experiments. Mann Whitney two-tailed test. * =< 0.05, **=<0.01, ***=<0.001, ****=<0.0001.
[0019] FIGS. 5A-5G provides transcriptomic characterization of 4-cell stage embryos from control and obese embryos. FIG. 5A is a schematic of events and stages of preimplantation embryos (ZGA- zygotic genome activation). FIG. 5B provides principal component analysis of mRNA expression showing HFD embryos clustered away from Control embryos. FIG. 5C illustrates hierarchical clustering using Euclidean distance with average linkage of all expressed genes from Control and HFD embryos. FIG. 5D provides volcano plots showing upregulated differentially expressed genes (DEG) in green [rightmost dashed box], and downregulated DEGs is in red [leftmost dashed box]. Insignificant genes are in black and primarily outside of the dashed boxes. FIG. 5E provides the gene ontology analysis of Control and HFD DEGs. FIG. 5F provides functional annotation analysis with UniProt Keywords with DEGs. FIG. 5G is a heatmap of expression of genes involved in AMPK pathway.
[0020] FIG. 6A provides an analysis of differentially expressed genes (DEG). The leftmost column of FIG. 6A provides a heatmap of Log2 of 92 DEGs with hierarchical clustering of genes and different treatments. The rightmost four columns of FIG. 6A provide a heatmap of Log2 of genes from gene ontology groups. The bottommost column of FIG. 6A is a heatmap of Log2 expression of Gpx7 and Entpd6. FIG. 6B illustrates principal component analysis showing ob/ob embryos clustered away from HFD and Control embryos. FIG. 6C shows volcano plots showing upregulated differentially expressed genes (DEG) in green [right box], and downregulated DEGs is in red [left box]. Insignificant genes are in black [all others] FIG. 6D provides a Venn diagram of shared DEGs between HFD and ob/ob embryos and a Pearson correlation analysis and heat map of 40 genes. Expression patterns of 42 shared genes. Red are upregulated and blue are downregulated genes compared to control embryos. FIG. 6E shows gene ontology analysis of ob/ob DEGs. FIG. 6F provides a heatmap of genes involved in AMPK signaling pathway that are downregulated in ob/ob embryos. FIG. 6G shows relative expressions of Gadd45 genes in HFD and ob/ob embryos. Data are presented as means ± standard deviation replicate experiments. Mann Whitney two-tailed test. * =< 0.05, **=<0.01, ***=<0.001, ****=<0.0001.
[0021] FIGS. 7A-7D illustrate downregulation of AMPK activity and metabolic disruption in HFD embryos. FIG. 7A provides immunofluorescent images of BODIPY 493/503 staining of [left panel] control and [middle panel] HFD 4Cell embryos, and [right panel] quantification of BODIPY 493/503 signal per embryo (control, n=22 and HFD, n=22). FIG. 7B provides [left panel] quantification of MitoView™ 633 signal as a measure of relative mitochondrial potential per embryo in control and HFD embryos [right panels] (control, n=21 and HFD, n=21). FIG. 7C provides [left panel] immunofluorescent images of phosphorylated AMPK [top row], actin [middle row], and DAPI [bottom row] stainings of control [left column] and HFD 4C [right column] embryos, and [right panel] quantification of pAMPK/actin signal per embryo (control, n=21, HFD, n=21). FIG. 7D compares fatty acid uptake per embryo over time in control and HFD embryos (control, n=19, HFD, n=19). Data are presented as means ± standard deviation of three independent experiments. Mann Whitney two-tailed test. * =< 0.05, **=<0.01, ***=<0.001, ****=<0.0001.
[0022] FIGS. 8A-8D illustrate downregulation of AMPK activity and metabolic disruption in HFD embryos. FIG. 8A provides immunofluorescent images of BODIPY 493/503 staining of [left panel] control and [middle panel] ob/ob 4Cell embryos, and [right panel] quantification of BODIPY 493/503 signal per embryo (control, n=22 and ob/ob, n=22). FIG. 8B provides [left panel] quantification of MitoView™ 633 signal as a measure of relative mitochondrial potential per embryo in control and ob/ob embryos (control, n=22 and ob/ob, n=22) [right panels]
FIG. 8C provides immunofluorescent images of phosphorylated AMPK [top row], actin [middle row], and DAPI [bottom row] of control [left column] and ob/ob [right column] 4C embryos, and [right panel] quantification of pAMPK/actin signal per embryo (control, n=21 and ob/ob, n=21). FIG. 8D compares fatty acid uptake per embryo over time in control and HFD embryos (control, n=19 and ob/ob, n=19). Data are presented as means ± standard deviation of three independent experiments. Mann Whitney two-tailed test. * =< 0.05, **=<0.01, ***=<0.001, ****=<0.0001.
[0023] FIGS. 9A-9C illustrate downregulation of AMPK activity and metabolic disruption in HFD embryos. FIG. 9A provides [left panels] immunofluorescent images of Gadd45a staining in control and HFD 4C embryos, and [right panel] quantification of Gadd45a signal per embryo (control, n=21 and HFD, n=23). FIG. 9B provides [left panels] immunofluorescent images of pH2A.X staining in control and HFD 4C embryos, and [right panels] quantification of pH2A.X signal per embryo (n=24). FIG. 9C provides [left panels] immunofluorescent images of NucView488 and Gadd45a staining of control and HFD blastocysts and [right panels] further quantification of NucView488 and Gadd45a positive cells per blastocyst. The leftmost plot are control embryos (n=22), whereas the rightmost plot are HFD embryos (n=21). Mann Whitney two-tailed test. * =< 0.05, **=<0.01, ***=<0.001, ****=<0.0001.
[0024] FIGS. 10A-10C illustrate downregulation of AMPK activity and metabolic disruption in ob/ob embryos. FIG. 10A provides [left panels] immunofluorescent images of Gadd45a staining in control and ob/ob 4C embryos, and [right panel] quantification of Gadd45a signal per embryo (control, n=24 and ob/ob, n=21). FIG. 10B provides [left panels] immunofluorescent images of pH2A.X staining in control and ob/ob 4C embryos, and [right panels] quantification of pH2A.X signal per embryo (control, n=24 and ob/ob, n=23). FIG. IOC provides [left panels] immunofluorescent images of NucView488 and Gadd45a staining of control and ob/ob blastocysts (control, n=25 and ob/ob, n=26) and [right panels] quantification of NucView488 and Gadd45a positive cells per blastocyst. The leftmost plot are control embryos, whereas the rightmost plot are ob/ob embryos. Mann Whitney two-tailed test. * =< 0.05, **=<0.01, ***=<0.001, ****=<0.0001.
[0025] FIGS. 11A-11C illustrate density measurements of 4Cell stage embryos from HFD mothers. FIG. 11A shows [left panel] magnetic levitation representative images of oocyte and preimplantation embryo stages and [right panel] quantifications of density measurements. FIG. 11B is an illustration of magnetic density device and experimental procedure. FIG. 11C includes representative image of control (C) and HFD (H) 4cell stage embryos in density measurement capillary [left] and quantification of embryonic density (control, n=39 and HFD, n=39) [right]. Data are presented as means ± standard deviation of three independent experiments. Mann Whitney two-tailed test. * =< 0.05, **=<0.01, ***=<0.001, ****=<0.0001. [0026] FIG. 12A shows density measurement of polyethylene beads (1.031 g-mL-1, 1.064 g-mL-1, and 1.089 g-mL-1) in the magnetic levitation platform with [left panel] representative images of levitated beads and [right panel] the relationship between the beads and levitation heights in 30mM Gd+ concentration. FIG. 12B includes representative images of magnetic levitations of blastocysts with large and small diameters. FIG. 12C shows the relationship between the blastocyst sizes and density measurements. FIG. 12D shows [left panel] representative images of control and density measured embryo and [right panel] percentage of blastocyst formation between control and density measured embryos. FIG. 12E shows [left panel] representative images of magnetic levitation of 4Cell embryos from control and ob/ob mice and [right] quantifications of density measurements of 4Cell embryos from ob/ob mice. Data are presented as means ± standard deviation of three independent experiments. Mann Whitney two-tailed test. * =< 0.05, **=<0.01, ***=<0.001, ****=<0.0001.
[0027] FIGS. 13A-13F illustrate transplantation of density sorted embryos into foster mothers and post-implantation embryo assessment. FIG. 13A provides an illustration of workflow of density sorting by density and transplanting into foster mothers. FIG. 13B shows density sorted HFD 4 Cell embryos (n=156 embryos total sorted) with [top panel] representative images of 4Cell embryos in densitometry capillary and [bottom panel] quantitative density measurements of 4Cell embryos in which embryos were separated into two cohorts, high density and low density, based on the average density measurement.
FIG. 13C illustrates [top panel] representative images of E18.5 fetuses (n=64) and [bottom panel] quantification of E18.5 heights and weights from low- and high-density embryos, and average E18.5 weight per litter. FIG. 13D shows quantification of E18.5 placenta of low- and high-density embryos per litter (n=10 females). FIG. 13E shows E18.5 placental weights of highl and low- density embryos. FIG. 13F shows placenta to fetus ratio of high and low-density embryos. Data are presented as means ± standard deviation of three independent experiments. Mann Whitney two-tailed test. * =< 0.05, **=<0.01, ***=<0.001, ****=<0.0001.
[0028] FIG. 14A-14C provides an evaluation of low- and high-density embryos after sorting. FIG. 14A provides lipid staining with Bodipy493/503 of 4Cell embryos from HFD mice sorted by densitometry device. FIG. 14B provides single embryo fluorescent intensity quantification of high density and low-density sorted embryos. FIG. 14C provides blastocysts and morula ratio at 72h post IVF of low-density and high-density cohorts. FIG. 14D shows efficiency of transplantation of high density and low density embryos. FIG. 14E shows litter sizes of high and low density cohorts. FIG. 14F shows average placenta/fetus ratio per litter in high- and low- density embryos. Data are presented as means ± standard deviation of three independent experiments. Mann Whitney two-tailed test. * =< 0.05, **=<0.01, ***=<0.001, ****=<0.0001. [0029] FIGS. 15A-15E provide an automated density-based embryo sorting device. FIG. 15A provides an illustration of workflow. FIG. 15B shows the real-time observation of single embryos sorted by density in which an embryo with lower density was sorted to Top Syringe (Sorted Up) and an embryo with higher density was sorted to Bottom Syringe (Sorted Down). FIG. 15C shows [top panel] representative images of levitated 4 Cell embryos in which the dotted line denotes separation height and [bottom panel] density quantification of 4 Cell embryos sorted down and sorted up. FIG. 15D shows lipid staining with Bodipy493/503 of 4Cell sorted down and sorted up embryos. FIG. 15E shows [left and middle panel] representative images of embryos and [right panel] blastocyst and morula ratio at 72h post IVF of low-density and high-density cohorts. Data are presented as means ± standard deviation of three independent experiments. Mann Whitney two-tailed test. * =< 0.05, **=<0.01, ***=<0.001, ****=<0.0001.
DETAILED DESCRIPTION
[0030] Obesity affects half of all US women in reproductive age. Adverse effects of obesity on female reproduction are well recognized. In order to address infertility, women rely on IVF and obese women have low IVF success rates compared to normal weight women. Unlike pregnancies from natural conception, ART pregnancies are more likely to result in multiple- infant births (twins, triplets or more). 17% of all twin births and 32% of all triplets and higher- order births in the US resulted from conception by IVF. Multiple-infant births pose health risks not only to mothers, but also to infants, including higher rates of caesarean sections, premature birth, low birth weight, and infant disability or death.
[0031] These observations emphasize the need for the development of novel approaches of embryo selection for transfer during IVF. Embryo selection requires noninvasive methods to keep embryos intact for later transfer, therefore novel magnetic densitometry device can be potentially used as a non-invasive approach for ART in humans.
[0032] While the low quality of the embryos and the poor developmental rates may be human-specific characteristics, the underpinning mechanisms have yet to be clarified. Other factors like obesity may even further contribute to poor fertility outcomes. Obesity is one of the major health issues affecting population globally and is increasingly becoming a pandemic of monumental proportions. In fact, half of all US women of childbearing age are either overweight or obese, and obese women undergoing IVF have lower chances of fertilization, clinical pregnancy and live birth rates.
[0033] Low reproductive outcomes in obese mice are linked to oocytes' and embryos' intrinsic aberrations in DNA methylation, mitochondria and endoplasmic reticulum. Lipotoxicity from dietary excess during obesity is the main risk factor for viability of oocyte and embryos. Lipotoxicity is accumulation of lipids in cells other than adipocytes due to the elevated lipids in the bloodstream. Elevated free fatty acid is associated with low qualities in oocyte- preimplantation embryos. Moreover, elevated lipid content is observed in arrested embryos and blastocysts from obese mothers. Disruption in metabolic homeostasis is also seen in preimplantation embryos from obese patients, where glucose, amino acid and lipid metabolism is disturbed. Despite the biological and clinical significance, the underlying mechanisms of these changes are still ambiguous and poorly understood.
[0034] The importance of metabolism to embryonic viability was recognized decades ago. The metabolic signatures of embryos have been proposed as a method for selecting of the most viable embryos. However, such prediction approaches utilize indirect measurements performed on culture media. Moreover, the embryonic metabolic signatures do not correlate with the embryo grading system. Therefore, there is an unmet need for direct measurements of embryonic metabolism for assessing the viability for ART-derived embryos.
[0035] Even though there have been good improvements in establishing and understanding the negative effects of obesity on preimplantation embryos, there is still need in improving IVF success in obese patients to lower health risk, and social, emotional, and financial burden to patients.
[0036] To study obesity in mice, two categories of mouse models are investigated: first, mouse models with genetic mutations (ob/ob, Tallyho etc.) and second, genetically intact animals exposed to obesogenic environments (Diet induced model). Genetic mutation in leptin mouse ob/ob is one of the most studied models of obesity and it has been recognized since 1950s. ob/ob mice are morbidly obese and experience hyperphagia, glucose intolerance, and are infertile. However, ob/ob female infertility was concluded only by absence of pregnancy after natural mating, and the underpinning cause (maternal versus embryonic) is not known. [0037] Diet induced obesity (DIO) is thought to better mimic the state of common obesity in humans. However, DIO is not standardized and generally it is achieved by 3-16 weeks of feeding with 45-65% fat calorie diet (High Fat Diet or "HFD"). High fat diet has been shown to negatively impact reproduction in primates and mice. However, due to the variability in approaches, the effects of maternal obesity can be inconsistent as well.
[0038] Here, to characterize the effects of maternal obesity on preimplantation and postimplantation embryos, two distinct mouse models of material obesity were investigated. Oocytes were collected from HFD and control females and they were in vitro fertilized (IVF) with sperm from males fed with a normal diet. The preimplantation development and metabolic state from obese mothers were first characterized. To investigate potential cellular pathways affected in embryos from HFD mice, transcriptional profiles at late 4 cell stage embryos were analyzed and disruption in metabolic genes was shown. Abnormal metabolism with downregulated AMPK, and elevated lipid content in embryos from HFD mothers was confirmed. A similar disruption in metabolism in embryos from ob/ob females, a genetic model of obesity, was also observed. To measure lipid content in live embryos, a novel single-cell magnetic levitation densitometry device (MagDense) was used and observed declines in embryonic density from HFD mothers, which correlated with elevated lipid content. Embryos were then sorted based on their density to separate embryos with high lipid content from the ones with low lipid content. Embryo transfer experiments showed that selection of embryos with low levels of lipids (higher density) significantly correlates with normal-sized E18.5 conceptuses from obese mice as measured by fetal weight and heights. On the contrary, embryos with increases amounts of cellular lipids (low density) show growth delay and retardation. Lastly, an automated embryo sorting device based on density measurements was devised that allows automated sorting of embryos based on their density measurements. The results indicate lipid levels in embryos can be a proxy to screen for the healthier normal sized E18.5 conceptuses in obese mice. Our findings can be valuable to improve success rate of ART for obese patients in humans.
[0039] Low embryonic qualities were able to be confirmed from obese mothers, including increased apoptosis, slow developmental rates. Slow development has been shown previously with preimplantation embryos from obese female mice. Post implantation embryos after natural mating in HFD mothers showed smaller fetal sizes and placental to fetus ratio was increased. Similar results were shown previously with different mice backgrounds. In humans, high maternal BMI is associated with increased placenta to fetus ratio as well. Interestingly, similar increase in placenta to fetus ratio was observed in normal weight women with low birthweight infants in clinical prospective studies, suggesting similarity between obese women and normal weight women with low birthweight infants. RNA-seq analysis indicated disruption in metabolic processes, and disruption of AMPK associated genes in embryos from HFD and ob/ob mice, including Gadd45a, which was downregulated in obese embryos.
[0040] Gadd45a is associated with early lineage commitments in preimplantation embryos, and knockout of Gadd45a/b genes in preimplantation embryos caused lethality at 4-cell stage. Overall, this indicates that Gadd45a plays important role in preimplantation development, and this necessitates deeper examination of its role in embryos under maternal obesity. As Gadd45a is regulated by AMPK via FOX03 signaling pathway, AMPK and Gadd45a reduction in obese embryos can potentially be activated by small molecule drugs, such as resveratrol and metformin. An accumulation of lipids in embryos from mice was also observed, which was also seen in embryos from ob/ob mice. Importantly, lipid accumulation has been observed in human embryos as well, suggesting its clinical relevance. Since lipid accumulation is associated with low embryonic quality, lipid accumulation in obese embryos was used as a proxy for low embryo quality. To noninvasively assess embryonic lipid accumulation, the embryonic density was measured using a novel magnetic densitometry device (MagDense, see for example,
FIG. 15A). Low density was shown to be correlated with higher lipid content in embryos. For the first time, single embryo densities was able to be measured using the MagDense device and density measurements used as a proxy for embryonic quality in embryos from fetal growth retardation obese mothers.
[0041] Ultimately, by sorting embryos based on their density at 4-cell stage, the qualities of enriched normal-sized conceptuses after implantation could be predicted. Importantly, the transplantation was conducted into wild-type normal weight foster mothers, indicating that there are embryo-intrinsic failure factors in low-density embryos that are retained even if the postimplantation development occurs in a normo-dietary environment. This disclosure shows a proof-of-principle for density measurements as a novel approach for non-invasive assessment of embryonic qualities of embryos before implantation.
[0042] Human reproduction is a complex and highly inefficient process. Reliance of American couples on ART almost doubled in the past decade, with approximately 2% of all infants born in the United States conceived using ART in 2016. Therefore, the MagDense densitometry device can be used for singleton embryo selection during IVF. In the future, the non-invasive, MagDense densitometry device can be potentially applied to other scenarios where IVF is correlated with poor developmental outcomes, including age related infertility.
The densitometry approach can be used to non-invasively assess the lipid content of human embryos before implantation, as the lipid content was correlated with preimplantation embryonic arrest. EXAMPLES
[0043] Below are examples of specific experiments and embodiments involving exemplary aspects of the disclosure. The examples are offered for illustrative purposes only and are not intended to limit the scope of the present invention in any way.
[0044] Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for. The methods and materials as well as explanation of the statistical analysis follows the numbered examples.
Example I: Characterization of oocytes from obese females
[0045] To evaluate effects of obesity on preimplantation embryo development, obesity was induced in CDl(ICR) females with high fat diet with 49 % kcal of fat and 20 % kcal of fructose (designated as "HFD") (FIG. ID). Fructose was included in the diet because it has been shown to have more severe weight-gaining effect on mice. Female mice were fed with HFD for 16 weeks, and oocytes were extracted to perform IVF with sperm from non-obese CD1 males fed with control diet. Female mice gained considerable weight at week 16 (FIG. ID) and increased body fat (FIG. IE) and elevated blood metabolic panel (FIG. IF). HFD female mice had lower number of oocytes (FIGS. 1A and IB) and significantly higher percentage of deformed oocytes per female (FIG. 1C) compared to control females of the same age fed with a regular diet.
[0046] To confirm the findings from HFD embryos, B6.Cg-Lepob/J (ob/ob) females were also investigated, which have a spontaneous mutation of leptin gene ob/ob mice are characterized by obesity, hyperphagia, and infertility ob/ob females gained considerable weight (FIG. 2A), and their blood metabolic panel was elevated (FIG. 2B). ob/ob females were able to generate Mil oocytes upon superovulation, but the oocyte number was lower (FIG. 2C) and the percentage of deformed oocytes was increased (FIG. 2D) compared to aged-matched WT mice, indicating that ob/ob females generated Mil oocytes of much lower quality compared to control females as seen in HFD mice. Example II: Characterization of preimplantation embryos from obese females [0047] Preimplantation embryonic development was then examined by performing in vitro fertilization (IVF) of oocytes with HFD mice with sperm from CDl(ICR) males fed with normal diet (FIG. BA). Morphologically normal oocytes from HFD mother had similar fertilization efficiency compared to control (FIG. 4A), however, slow developmental rate at the blastocyst stage was observed in HFD embryos. In fact, at 72 hours post fertilization only ~40% turned to blastocysts in HFD embryos compared to ~85% in control embryos (FIG. 3B). Analysis of blastocysts showed increased apoptosis (FIG. 3C), compared to control embryos. However, the percentage of dead embryos at blastocyst stage was not significantly different (FIG. 4B). Next, embryonic development after implantation was evaluated by natural mating of HFD females with control males. Previously maternal obesity resulted in fetal growth retardation which was measured by fetal length and weights and has been associated with congenital malformation and brain defects. The most drastic growth retardation in obese mice was observed at E18.5. Here, the fetuses at E18.5 were evaluated and smaller fetal sizes (FIG. 3D) were measured in heights (FIG. 3E) and weights (FIG. 3F). Moreover, the average weight of fetuses in a litter was smaller as well (FIG. 4C). Even though there was no significant difference in placental weights (FIG. 4D), the ratio of placenta to embryo was elevated in HFD embryos compared to controls (FIG. 3G). Moreover, the litter sizes were smaller in HFD mice compared to control mice (FIG.4E).
[0048] Again, the findings were sought to be confirmed with ob/ob mice as well ob/ob female infertility is exhibited in the absence of pregnancy when mated with inbred C57BL/6J males, however, whether ob/ob females can generate viable preimplantation development remains to be elucidated (FIG. 4F). After performing IVF with sperm from C57BL/6 males, morphologically normal oocytes from ob/ob females had similar fertilization efficiency compared to oocytes from control females (FIG. 4G). Interestingly, as with embryos from HFD mice, the preimplantation developmental rate was slower in embryos from ob/ob females (FIG. 4H), where only ~20% turned to blastocysts at 72 hours post fertilization compared to ~70% in control embryos. [0049] Furthermore, at blastocyst stage there were significantly more aberrant and dead embryos in ob/ob cohort (FIG. 41). Moreover, unlike embryos from HFD mice, blastocysts from ob/ob mothers have more apoptotic cells (FIG. 4J) compared to control embryos, indicating increased cell death. Natural mating of ob/ob females with C57BL/6J males did not lead to pregnancy, confirming the infertility of ob/ob females.
[0050] Altogether this data indicates that obesity negatively affects both preimplantation and post-implantation development.
Example III: Transcriptomic analysis of embryos from HFD mothers revealed disruption in metabolic genes and genes involved in energy regulation
[0051] Establishment of embryonic transcriptome is vital for the development of embryos. Embryonic transcriptome is established by a sequence of well-regulated events, including maternal mRNA degradation and chromatin remodeling, that are initiated with fertilization and almost completed by the 4-cell stage. Therefore, to evaluate the transcriptomes of embryos from HFD mothers, 4 cell stage embryos were analyzed where embryonic state has been established (FIG. 5A). Principal component analysis showed that 4 cell embryos from HFD (HFD 4C embryos) and control mothers seem to be separated more on Principal Component 1 (PCI) (FIG. 5B), which is defined by 66% of genes rather than Principle Component 2 (PC2) defined by 24% of genes. Moreover, the hierarchical clustering analysis showed HFD 4C embryos clustered separately from control embryos (FIG. 5C). Differential gene expression analysis of HFD 4C embryos and control embryos showed only 92 genes (FIG. 5D, FIG. 6 leftmost column). Interestingly, gene ontology analysis of these 92 genes showed metabolic processes as top hit (FIG. 5E, FIG. 6). Functional annotation analysis with UniProt Keywords revealed top two hits as Glycogen and Carbohydrate metabolisms (FIG. 5F). Furthermore, cluster of orthologous groups analysis revealed multiple proteins that are involved in metabolism and biogenesis, including carbohydrate transport and metabolism, secondary metabolites biosynthesis, transport, and catabolism (Supplementary Table 1, below). Supplementary Table 1: Genes identified by COG analysis and their functional ontology.
Figure imgf000020_0001
[0052] Due to small number of genes, the analysis of gene pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG) did not reveal any significantly represented pathways among 92 genes. However, with KEGG analysis we identified 28 metabolism associated genes. Even though these genes were not part of significantly represented pathways, they are listed in Supplementary Table 2, below. Gpx7 and Entpd6 were downregulated in HFD embryos (FIG. 6, center bottom). Gpx7 (Glutathione Peroxidase 7) is part of glutathione metabolism and detects intracellular reactive oxygen species (ROS) to maintains redox homeostasis in cells.
Figure imgf000021_0001
[0053] Previously ROS has been shown to be elevated in eggs from obese mice. Entpd6 (ectonucleoside triphosphate diphosphohydrolase 6) is part of purine and pyrimidine metabolism and it is associated with obesity in humans.
[0054] Since AMP-activated protein kinase (AMPK), a master regulator of cellular energy homeostasis was also downregulated in obese mice, the genes implicated in AMPK pathway were investigated. Four genes involved in AMPK pathway among DEGs were identified: Idua, Ppp2r2c, Eef2k, Gadd45a (Supplementary Table 3, below).
Supplementary Table 3. Genes involved in AMPK pathway.
Genes involved so the AMPK signaling pathway
Figure imgf000022_0001
[0055] Idua (alpha-L-iduronidase) is part of glycosaminoglycan biosynthesis pathway and modulated by AMPK pathway, where upon downregulation of AMPK, the expression of Idua significantly increased. Interestingly, in HFD 4C embryos, similar overexpression of Idua was seen (FIG. 5G). Ppp2r2c (protein phosphatase 2, regulatory subunit B, gamma), subunit of PP2A, is another gene implicated in AMPK pathway. PP2A is a heterotrimeric serine/threonine phosphatase that has been shown to inactivate AMPK. Interestingly, the expression of Ppp2r2c is also upregulated in HFD 4C embryos (FIG. 5G), which potentially can play role in inactivation of AMPK in embryos from obese mice. Eef2k (eukaryotic elongation factor 2 kinase) is a key regulator of protein synthesis in mammalian cells, which in turn is activated by AMPK. Eef2k downregulated in HFD 4C embryos was observed (FIG. 5G), which correlates with AMPK downregulation in HFD 4C embryos. Lastly, Gadd45a downregulated in HFD 4C embryos was seen as well (FIG. 5G). Gadd45a (Growth Arrest And DNA Damage Inducible Alpha), a protein activated by AMPK via FOXO signaling pathway. Overall, transcriptional analysis showed that metabolic genes and genes involved in energy regulation were disrupted in embryos from HFD mice.
Example IV: Embryos from ob/ob mothers have similar transcriptomic disruptions to embryos from HFD females
[0056] To confirm the transcriptomic findings from HFD embryos, RNAseq was performed on ob/ob embryos as well and compared it to control embryos. Since ob/ob mice are genetically modified and have different genetic background (C57BL/6 J) than control and HFD embryos (Crl:CDl(ICR)), PC analysis showed ob/ob embryos cluster away from both HFD and control embryos (FIG. 6B) and we identified more DEGs (n=1888) compared to DEG (n=93) from HFD and Control comparison (FIG. 6C). However, 45% of DEGs from HFD embryos are shared with ob/ob embryos (FIG. 6D) indicating to the similarities between HFD and ob/ob embryos. Moreover, Pearson correlation analysis of these shared genes showed ob/ob and HFD embryos clustered separately from control embryos (FIG. 6D).
[0057] GO analysis indicated similar metabolic processes compared to HFD-control comparisons (FIG. 6E). Analysis of gene pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that AMPK signaling pathway was also downregulated in ob/ob embryos (FIG. 6F). Moreover, Gadd45a (Growth Arrest and DNA Damage Inducible Alpha) was downregulated in ob/ob 4C embryos (FIG. 6G). with additional downregulation of two other members in ob/ob embryos (Gadd45b and Gadd45g). Overall, transcriptional analysis of ob/ob embryos showed that metabolic genes and genes involved in energy regulation were disrupted in embryos from ob/ob females pointing to similarities to HFD embryos.
Example V: Immunofluorescent imaging revealed potential role o/Gadd45a in apoptotic cells at blastocysts from obese females
[0058] Gadd45a have been shown to be important in normal preimplantation embryo development and it is associated with early lineage commitments in preimplantation embryos, and knockout of GADD45a/ genes in preimplantation embryos caused lethality at 4 cell stage. Gadd45a has not been investigated in the context of obesity in preimplantation embryos, therefore it was decided to explore its role in preimplantation embryos from obese mice. To confirm downregulation of Gadd45a observed in RNA-seq analysis, protein levels of Gadd45a was investigated by immunofluorescence and confirmed Gadd45a downregulation in 4 cell embryos from HFD mothers (FIG. 9A) and ob/ob mothers (FIG. 10A). As the similar downregulation of Gadd45a in ob/ob embryos has been seen (FIG. 6G), and the investigation of protein levels by immunofluorescence imaging sufficient was deemed sufficient for confirming RNAseq results seen in HFD embryos and the investigation with RT PCR is redundant. Moreover, the RT PCR results have been shown to not correlate to protein levels previously. Gadd45a (Growth Arrest and DNA Damage Inducible Alpha) plays an important role in DNA damage response and regulates apoptosis, and DNA damage and apoptosis is mostly observed in blastocysts stage of preimplantation embryos, thus, we sought to investigate the role of Gadd45a in apoptosis in blastocysts. Increased apoptosis in blastocysts from obese mothers was observed (see FIG. 9C, FIG. 4J), therefore the DNA damage in obese blastocysts was assessed and increased levels of DNA damage observed as measured by phospho-yH2A.X signal (FIG. 9B). Similar increase in embryos from ob/ob mice was confirmed as well (FIG. 10B). Then we sought to investigate the relationship of Gadd45a in apoptotic cells in blastocysts with co staining NucVew488 and Gadd45a antibody. In control blastocysts, we saw Gadd45a is co expressed in apoptotic cells (FIG. 9C). In contrast, in blastocysts from HFD mothers, a dramatic decrease in Gadd45a co-expression in apoptotic cells was noticed (FIG. 9C). A similar result was obtained in ob/ob mice (FIG. IOC). Overall, these results indicate that in embryos from obese mothers, Gadd45a is downregulated in 4Cell embryos along with downregulation of Gadd45a co-expression with apoptotic cells in blastocysts.
Example VI: Embryos from obese mothers have abnormal metabolism and disruption in cellular energy homeostasis
[0059] Previous studies on mouse models of obesity showed abnormal metabolism in preimplantation embryos with elevation in lipid content and disruption in mitochondria. Similarly, human preimplantation embryos from obese patients had disruption in metabolism, including glucose, amino acid and lipid metabolisms. Disruption in metabolic genes from RNA- sequencing analysis was also seen. Therefore, it was sought to investigate metabolism of embryos from obese mice. First, the lipid content in embryos by BODIPY 493/503 neutral lipid stain was examined. Confocal imaging showed elevated levels of lipids in embryos from HFD and ob/ob mice (FIG. 7A, FIG. 8A). Next, we measured mitochondrial activity, which was significant reduced in embryos from HFD and ob/ob mice (FIG. 7B, FIG. 8B). Since, genes involved in AMPK pathway downregulated in RNA-seq analysis were seen, the levels of phosphorylated AMPK levels, the activated form of AMPK, were measured. Immunofluorescent imaging showed a significant decrease in phosphorylated AMPK levels in embryos from both HFD and ob/ob mice (FIG. 7C, FIG. 8C), which agrees with low mitochondrial activity and high lipid content. AMPK activity can modulate fatty acid uptake, therefore the nutrient uptake by measuring fatty acid uptake was also investigated. It was found that embryos from HFD and ob/ob mice have lower levels of fatty acid uptake (FIG. 7D, FIG. 8D). Overall, on top of RNA- sequencing analysis, these results indicate embryos from both obese mice have abnormal metabolism and disruption in energy homeostasis.
Example VII: Non-invasive cellular density measurements can predict embryo quality at 4 cell stage
[0060] For patients undergoing IVF, it is essential to select most viable embryos to increase the chances of pregnancy. It is generally recognized that obese women have poor IVF outcomes. The data found herein showed that preimplantation embryos from obese mothers are metabolically abnormal, are characterized by slow preimplantation developmental rate, and have increased cell death.
[0061] As we saw similar fetal growth retardation shown previously by others, HFD in female mice resulted in smaller fetal size at E18.5 (see FIG. 3E and 3F). In contrast to embryos from control mothers, 4 cell stage embryos from obese mothers have elevated lipid content in average but there is a degree of heterogeneity among embryos, indicating some embryos from obese mothers have normal levels of lipids (FIG. 7A). Similar heterogeneity in weight and heights of E18.5 fetuses from obese mothers was seen. Therefore, it was hypothesized that the amount of lipid content at the 4- cell stage could be utilized as a predictor of embryo viability and of postimplantation development.
[0062] Previous reports have shown fetuses with growth retardation have congenital malformation, including impaired development of ocular structures, abnormal ear attachment, disrupted ventricular development, and an underdeveloped choroid plexus in the brain. Here, it was thought to use fetal growth retardation in obese mice as a proxy for fetal health.
[0063] Magnetic levitation densitometry allows the measurement of cellular density at the single cell level, without compromising cellular integrity and without the use of biomarkers, antibodies or tags. The device consists of a glass microcapillary or channel held between two permanent magnets with same poles facing each other (although a magnetic field may be produced in other ways as well using fewer or more magnets and in various possible configuration). Cells are spiked or introduced into the capillary or channel with paramagnetic medium. Under the magnetic field, cells are levitated at specific heights when gravitational, buoyancy, and magnetic forces reach an equilibrium. This equilibrium mainly depends on intrinsic cellular density.
[0064] First, a standard curve of elevation height versus density was established by elevating polyethylene beads with known densities in paramagnetic medium (30mM Gd in KSOM) inside the capillary (FIG. 12A). Then, the density profiles of normal mice embryos was measured starting from Oocytes to Blastocyst stages and their unique density features were identified. Interestingly, after zygote, the density measurement increased at 2 cell stage and further decreased after 4 cell stages (FIG. 11A). The following densities were measured: Oocyte - 1.091 ±0.0016 g/ml; Zygote - 1.091 ±0.0013 g/ml; 2 Cell - 1.100 ±0.0009 g/ml; 4 Cell - 1.0855 ±0.002 g/ml; Morula - 1.0822 ±0.00062 g/ml; and Blastocyst - 1.0811 ±0.0057 g/ml which are mapped in FIG. 11A, right panel. For blastocyst stage embryos, a high degree of heterogeneity was observed (FIG. 12B) and, upon differentiating by the size of embryos, the diameters of embryos were seen have positive correlation to the density measurements (FIG. 12C). This can be due to differences in number of cells, the morphological characteristics of blastocysts, which include fluid filled blastocoel and differentiated populations of cells (ICM and Trophectoderm). [0065] Then, to confirm the viability of embryos post magnetic levitation, density of fertilized oocytes in the density solution (30mM Gd+ in KSOM) were measured and the percentage of them that reached blastocyst stage quantified. No significant difference in viability after density measurements (FIG. 12D) was observed, which indicated the density measurements did not impair blastocyst development.
[0066] Previous non-invasive embryo selection methods focused on pre-zygotic genome activation (ZGA) embryos and oocytes. ZGA is a well-regulated event, that includes maternal mRNA degradation and chromatin remodeling, without ZGA, embryos are doomed to fail. 30- 60% of IVF embryos are developmental^ arrested before blastocyst stage and characterized by mutations affecting proper ZGA. Human embryos undergo ZGA at 4-8 cell stages, while ZGA in mouse embryos occurs at Zygote and continues until the end of 2-Cell stages. Therefore, to further investigate the relationship of lipid accumulation in embryos from obese mice, it was deemed that it is important to focus our density measurements on embryos successfully finalized ZGA, which is 4-Cell stage in mice.
[0067] Lipids are buoyant macromolecules that affect cellular density. Recently, we showed that iPSC-derived cardiomyocytes with high lipid content have lower densities and these minute density differences were leveraged to separate diseased cell populations based on their intracellular lipid content by using MagDense. We also sought to utilize MagDense device to sort embryos based on their inherent magnetic levitation profiles driven by their lipid content (FIG. 13B). Since we observed elevated lipid content in embryos from HFD mothers, we assessed whether embryos are also characterized by lower density due to higher lipid content. Interestingly, we detected a significant decrease in density measurements (FIG. 11C) which is inversely correlated with the lipid content observed in HFD embryos. Average densities for control and HFD 4 Cell embryos were measured as 1.0862 ±0.002 g/ml and 1.084 ±0.002 g/ml, respectively. Since we saw increased lipid content in 4 cell embryos from ob/ob females, we tested their densities as well and saw similar low-density measurements (FIG. 12E). Average densities for control and ob/ob 4 Cell embryos were measured as 1.087 ±0.004 g/ml and 1.082 ±0.0006 g/ml, respectively. This indicates that maternal obesity (HFD and ob/ob mothers) were able to influence embryonic density characteristics as well. [0068] To test if the embryonic densities at early preimplantation stage (4 Cell stage) play significant role in further embryonic development, we sought to sort embryos based on their densities (FIG. 15A). We performed IVF with HFD oocytes and normal diet male sperms and cultured embryos in vitro. At 4 cell stage, we measured the densities at single embryo level and sorted them onto high- and low-density cohorts based on the average density measurement (FIG. 15B). Average density of 4 Cell embryos were 1.085 ±0.002 g/ml, and the average measurements of high- and low-density embryos were 1.083 ±0.001 g/ml and 1.087 ±0.001 g/ml, respectively. We then confirmed that embryos with low density have more lipid than embryos with higher density (FIG. 14A and 14B), as measured by BODIPY 493/503 neutral lipid stain. After the density measurements and density-based sorting with MagDense, we allowed embryos to reach blastocyst stage. We did not observe any difference in blastocysts formation rate between low-density and high-density cohorts (FIG. 14C). Then we transplanted blastocysts into CD1 foster mothers with normal diet and allowed for pregnancy continue until E18.5. At the end, we sacrificed mice and characterized E18.5 fetuses and placenta. We observed E18.5 embryos from low density cohorts have smaller sizes compared to high density cohort (FIG. 13C) by measuring the heights and weights. Even when comparing an average embryo weights and heights per litter, low density embryos were significantly smaller (FIG.
13D), indicating the change in weights was independent of foster mothers. However, embryo transplantation efficiency and litter sizes were not significantly different between high- and low-density embryos (FIG. 14D and 14E), indicating density metrics of embryos did not predict embryo transplant efficiency and life birth. There was no difference in placental weights (FIG. 13E), however, the ratio of placenta to fetus was increased in low density cohort (Fig. 13F). This indicates that density measurements at early preimplantation stage can predict fetal growth retardation in post implantation conceptuses in HFD mice.
Example VIII: Automated Density Based Embryo Sorting Device
[0069] Next, we thought to devise an automated density-based embryo sorting device that would allow for fast and controlled embryo sorting. In order to achieve automated sorting, we connected a flow channel to two syringe pumps that withdraw the sample into the MagDense device, and the real-time imaging is carried out to visualize and separate levitated HFD embryos into two cohorts, low density and high density (FIG. 15A). The real time evaluation of the heights was carried out by a scientist by looking at the embryo levitation on the screen of the monitor, and preset height was used as a separation height on the monitor which was established after earlier experiments at 93-pixel height. Embryos above separation height were sorted to the top syringe, and embryos below the separation height were sorted to the bottom syringe (Fig. 15B). The secondary measurement was done after-the-fact to quantify the exact density measurements for each embryo. The average density of low-density and high-density cohorts were 1.085 ±0.002 g/ml and 1.089 ±0.001 g/ml, respectively (FIG. 15C). Then we measured the lipid content by BODIPY 493/503 staining and confirmed the elevated lipid content in low density cohort (FIG. 15D). After density sorting, embryos were normally developed to blastocyst stage with no difference between up sorted and down sorted embryos (FIG. 15E). Overall, our results indicate that automated density-based embryo sorting can be used to separate embryos based on density and by lipid content.
MATERIALS AND METHODS
Mouse housing
[0070] All animal protocols were approved by the Administrative Panel on Laboratory Animal Care at Stanford University.
[0071] For high fat diet experiments, 3 weeks old CD1 mice (Crl:CDl(ICR), 022) were purchased from Charles River Laboratories. CD1 mice were randomly divided into two diet groups; one group received a HFD (TD.180431, Teklad Custom Diet, Envigo) and the other group received a Control Diet (TD.08806, Envigo) for 16 weeks. CD1 male sperm were used for in vitro fertilization procedure.
[0072] For the genetic mouse model of obesity, 4 weeks old homozygous ob/ob (B6.Cg- Lepob/J, 000632) and C57BL/6 J female mice were purchased from Jackson Laboratories. C57BL/6 J male sperm were used for in vitro fertilization procedure. Female superovulation and in vitro fertilization
[0073] Female mice were superovulated by injecting lOOul of CARD HyperOva® followed by
7.5 U of human chorionic gonadotropin (hCG, CG10-1VL, Sigma-Aldrich) 46-48 h after CARD HyperOva® treatment. Mice were euthanized by cervical dislocation 14 h after the hCG injection. Cumulus-oocyte complexes (COCs) were isolated from oviduct ampullae placed into CARD MEDIUM® (#KYD-003-EX, Cosmo Bio) drops for IVF procedure.
[0074] Sperm were isolated from the dissected epididymis of mice aged 10-20 weeks and left to capacitate for 0.5-1 h in FERTIUP® PM 0.5 ml. (#KYD-002-05-EX, Cosmo Bio). Then, dispersed spermatozoa were added to CARD MEDIUM® drops containing COCs obtained as described above. After co-incubation for a 3-4 h in a 37 °C incubator, presumptive zygotes were washed to remove cumulus cells and excess sperm. For the culture of preimplantation embryos, zygotes were transferred into preincubated KSOM medium (K0101, CytoSpring) and cultured up to the blastocyst stage at 37 °C in a humidified atmosphere of 5% C02, 5% 02 and 90% N2. Every day the developmental rate of embryos was assessed, by counting number of embryonic stages. After density measurements, the embryos were transferred into foster mothers. The embryo transfer procedure is followed from previous work. Foster mother selected were 4-5-week-old CD1 females under normal diet. To generate pseudopregnant females, proestrus stage females were selected based on the appearance of their external genital tract and sterile mated with vasectomized males. E3.5 embryos were transferred into
2.5 dpc pseudo-pregnant CD1 females. Mice were anesthetized and oviducts from both uterine horns were surgically pulled out for a transfer. 15 embryos were transferred per mouse (7-8 embryos per horn). Recipients were killed at E18.5, and fetuses were collected for evaluation.
Natural mating and postimplantation embryo collection
[0075] Male mice bedding was added into female mice cage to induce estrus cycle. After 2 days, a male was placed with 2 females. Next day, males were removed from the cage and presence of vaginal plug was checked in females. For gestational age, midnight of the day of mating was designated as day 0. On E18.5 females were euthanized, and embryos were collected, and evaluated for size. RNA sequencing of 4 cell embryos
[0076] 4 cell stage embryos were collected after 38-42h post IVF. 75-85 embryos were pooled into TRIzol Reagent (Invitrogen) and frozen until further steps. Due to high number of embryos required to extract enough RNA, only 2 replicates were used per treatment, and great caution was taken on every step of RNA extraction. RNA was extracted from embryos using the TRIzol RNA Isolation Protocol described here [Short technical reports. Modification of the TRI reagent procedure for isolation of RNA from polysaccharide- and proteoglycan-rich sources. Chomczynski P, Mackey K. s.l.: Biotechniques, 1995; 19(6):942-945], and proceeded to cDNA library prep. Before preceding to sequencing, libraries were cDNA was quantified using a Qubit 2.0 Fluorometer (Life Technologies) and sample integrity was checked with TapeStation (Agilent Technologies). Sequencing was performed on the lllumina HiSeq instrument using a 2 x 150 paired-end configurations. Reads were mapped to the Mus musculus GRCm38 reference genome using the STAR aligner v.2.5.2b. DESeq2 was used to assess differential gene expression among control, HFD and ob/ob embryos. PCA and Hierarchical clustering analysis was performed on PCAGO (https://pcago.bioinf.uni-jena.de/).
Gene list analysis of differentially expressed genes (DEG)
[0077] Gene list analysis of DEGs was carried out in DAVID bioinformatics tool (Systematic and integrative analysis of large gene lists using DAVID Bioinformatics Resources. Huang DW, Sherman BT, Lempicki RA. 1, 2009, Nature Protoc., Vol. 4, pp. 44-57). Mus musculus gene list was used as background. Under functional categories, COG_ONTOLOGY and UP_KEYWORKDS were selected. Under Gene Ontology, GOTERM_BP_l was selected. Under Pathways, KEGG_PATHWAY was selected. Then metabolism associated genes were defined as genes from metabolic process in gene ontology analysis and having metabolism in the keywords after KEGG pathway analysis. Morpheus tool from Broad Institute was used to plot heatmap of DEG and perform hierarchical clustering. Detecting apoptosis in blastocysts
[0078] Apoptotic cell detection was performed by NucView® 488 Caspase-3 Enzyme Substrate (10402-T, Biotium) according to manufacturer's protocols. 5 uM NucView® 488 substrate was added to 50ul KSOM medium and preincubated for at least 30min. When embryos reached blastocyst stage (E3.5) embryos were transferred into this preincubated NucView® 488 -KSOM medium for 30 minutes. Afterwards, the zona pellucida was removed by Acidic Tyrode's solution (CytoSpring) and embryos were fixed in 4% PFA in PBS for 20 min at 4° C. Embryos were proceeded to immunostaining and imaging.
Embryo staining and immunofluorescence microscopy
[0079] For immunostaining of preimplantation embryos, the zona pellucida was removed by Acidic Tyrode's solution (CytoSpring) and embryos were fixed in 4% PFA in PBS for 20 min at 4° C. After permeabilization in 0.2% Triton-X, 0.1% BSA in PBS for 10 min at RT blastocysts were blocked overnight in 0.1% BSA in PBS at 4° C. Embryos were then incubated with primary antibodies in blocking solution for 3-4 h at RT at following conditions: 1:200 Gadd45a (mouse, Santa Cruz), 1:200 pAMPK (rabbit, 50081S), 1:3000 Phospho-Histone H2A.X (rabbit, 9718T), 1:500 Anti- -actin (mouse, A5441). After several washes in blocking solution at RT blastocysts were incubated with secondary antibodies using 488, 594 or 680 Alexa Fluor conjugates (Invitrogen) at 1:1000 dilution, and/or with BODIPY™ 493/503 (D3922) 1:1000 dilution for 1-2 h at room temperature. Following several washes in blocking solution embryos were incubated with Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories) for 10 min. Images were acquired using a Leica SP8 White Light confocal microscope. All quantification of signals was analyzed in ImageJ software.
Fatty acid uptake assay
[0080] Fatty acid assay kit was purchased from Biovision (# K408-100), and fatty acid incorporation was measured according to manufacturer's protocols with some modifications. Instead of plate reader we used fluorescent microscope to measure fatty acid uptake per embryo. After growth in KSOM media without Phenol, the Morula stage embryos were transferred into glass bottom plate with prewarmed Uptake Assay buffer at 37°C, 20-30 embryos per 50ul droplet and were incubated for lh. Afterwards, 2x solution of quenched Uptake Reaction Mix was added to the droplets with embryos. The fluorescence was immediately measured in EVOS FL Digital Microscope. Fluorescent intensity per embryo was measured in ImageJ. Empty regions without embryos were used as a background control signal.
Mitochondrial potential assay
[0081] Mitochondrial potential measurement was performed by MitoView® 633 (70055-T, Biotium) according to manufacturer's protocols. lOOnM MitoView® 633 solution was made in KSOM medium and preincubated at 37°C for at least 30min. 4 cell stage embryos were transferred into this preincubated lOOnM MitoView® 633 - KSOM medium for 30 minutes. Afterwards, embryos were washed one time, and imaged using EVOS FL Digital Microscope. Fluorescent intensity per embryo was measured in ImageJ.
Embryonic density measurements with MagDense device
[0082] The densities of embryos were measured as described in Durmus et al. 2015 (Magnetic levitation of single cells. Naside Gozde Durmus, H. Cumhur Tekin, Sinan Guven, Kaushik Sridhar, Ahu Arslan Yildiz, Gizem Calibasi, lonita Ghiran, Ronald W. Davis, Lars M. Steinmetz, and Utkan Demirci. 2015, PNAS) with slight modifications. Similar magnetic levitation chip was used, that consists of polymethyl methacrylate (PMMA), two N52-grade neodymium magnets with a glass microcapillary channel in between, and two mirrors for real-time imaging purposes. Embryos were levitated in a paramagnetic medium: 30 mM Gd (gadolinium-based paramagnetic medium) in KSOM media. Before loading embryos into levitation capillary, they were preincubated in 30 mM Gd KSOM medium for 3-5 minutes. Afterwards, embryos were transferred into capillary with 30 mM Gd KSOM and levitated for 10 minutes. In this unique device configuration, embryos can be levitated and separated based on their inherent density without the use of labels, antibodies or tags. The magnetic susceptibility difference between an embryo and its surrounding paramagnetic medium causes it to move away from a higher (i.e., close vicinity at the magnets) to a lower magnetic field strength site (i.e., away from the magnets) until gravitational, buoyancy and magnetic forces acting on the embryos reach an equilibrium. Embryos are levitated at a final position between the two magnets, where the magnetic force (Fmag) equals the buoyancy force (Fb). Polyethylene beads with known densities (1.031, 1.064, and 1.089 g rnL-1) were used to generate a standard curve to calculate embryo densities based on their levitation heights. Levitation heights were quantified by ImageJ software.
MagDense embryo sorting experiments
[0083] For embryo sorting within MagDense and transfer experiments, one embryo at a time was levitated and equilibrium height was recorded for the levitating embryo. Then, next embryo was levitated, and height was recorded. This cycle was continued until enough embryos were measured for future blastocyst transfer experiments. After each levitation experiment, embryos were washed in pre-calibrated KSOM medium to avoid prolonged exposure to the levitation medium. After the washing step, each embryo was placed in a droplet of KSOM in the incubator with the specific identifier, until the densities of 55-60 embryos were measured. After the desired number of embryos were measured, the average height was calculated. The embryos were separated into two cohorts: i) embryos levitating above (low density); and ii) below (high density) the average levitating height. High- and low- density embryos were then cultured separately in KSOM until blastocyst stage (E3.5).
Automated Density Based Embryo Sorting Device
[0084] Automated Density Based Embryo Sorting Device is a densitometry and imaging platform which uses the principles of magnetic levitation. The device consists of a flow channel (1 mm in height and width) held between two custom-designed rare earth magnets for gentle and rapid separation of different cell states in a continuous flow operation. For real-time imaging, aluminum-coated mirrors are placed at each side of the microchannel and a camera images the cells as they levitate and move through the flow channel. The system is driven by two syringe pumps that withdraw the sample into the flow-based magnetic levitation system from droplet of paramagnetic solution (30mM GD+ in KSOM media) immersed in mineral oil. Droplets of paramagnetic solutions under the mineral oil were pre-calibrated for 30 min at 37C in the incubator. 4-cell stage embryos were placed in this droplet with paramagnetic solution, and one embryo at a time was introduced into inlet and flowed into the capillary with syringe pumps. Once embryo was in the capillary they were allowed to equilibrate to appropriate heights and imaged for exact density quantifications post sorting. Separation height was established with the mean elevation heights of prior embryo MagDense experiments. The top outlet was connected to a pump that withdrew the solution containing the low-density embryos, while the bottom outlet was connected to a pump that withdrew the solution containing the high-density embryos.
Statistical analysis
[0085] All statistical analyses were performed using GraphPad Prism (version 8.4) for Macintosh. Statistical comparisons were made with two-tailed unpaired nonparametric Mann- Whitney test. Sample sizes were determined according to the prior publications in oocyte- embryo works (Embryonic defects induced by maternal obesity in mice derive from Stella insufficiency in oocytes. Longsen Han, Chao Ren, Ling Li, Xiaoyan Li, Juan Ge, Haichao Wang, Yi- Liang Miao, Xuejiang Guo, Kelle H. Moley, Wenjie Shu & Qiang Wang. 2018, Nature Genetics , pp. volume 50, pages 432-442. PMID:29459681 DOI:10.1038/s41588-018-0055-6.); there was no statistical method used to predetermine sample size. Data are presented as mean values ± standard deviation. Changes were considered statistically significant when P < 0.05.
[0086] It should be appreciated that various other modifications and variations to the preferred embodiments can be made within the spirit and scope of the invention. Therefore, the invention should not be limited to the described embodiments. To ascertain the full scope of the invention, the following claims should be referenced.

Claims

CLAIMS What is claimed is:
1. A method for assessing one or more embryos based on a density of an embryo which serves as a proxy for health or viability of the embryo, the method comprising: introducing the embryo in a paramagnetic medium into a levitation device including a channel that is positioned within in a controlled magnetic field, the embryo in the paramagnetic medium being received in the channel and subjected to the controlled magnetic field in order to levitate the embryo to an equilibrium height against the force of gravity, in which that equilibrium height correlates to the density of the embryo which is dependent at least in part on the lipid content of the embryo; and assessing the health or viability of the embryo based on the density of the embryo.
2. The method of claim 1, further comprising calibrating the levitation device prior to the step of introducing the embryo in the paramagnetic medium into the levitation device, the calibration step involving establishing a correlation between equilibrium levitation heights of reference objects having known densities in the paramagnetic and their densities such that, when the embryo in a paramagnetic medium is received in the channel, the levitation height of the embryo can be correlated to the density of the embryo which serves as a proxy for the health or viability of the embryo in the assessing step.
3. The method of claim 2, wherein the reference objects are polyethylene beads of known densities.
4. The method of claim 1, wherein only a single embryo is evaluated at a time in the levitation device.
5. The method of claim 1, wherein, the device includes one or more magnets positioned relative to the channel to provide the controlled magnetic field.
6. The method of claim 1, further comprising the step of placing the embryo in the paramagnetic medium before introducing the paramagnetic medium and embryo into the levitation device.
7. The method of claim 1, further comprising the step of sorting the embryos based on their equilibrium height.
8. The method of claim 7, wherein the equilibrium height of a respective embryo is a function at least in part the intrinsic cellular density based on lipid content.
9. The method of claim 7, further comprising setting a threshold height and sorting a first group of the embryos having respective equilibrium heights above the threshold height from a second group of the embryos having respective equilibrium heights below the threshold height.
10. The method of claim 9, wherein the channel is in fluid communication with both an upper collection channel and a lower collection channel at an outlet end thereof and wherein the step of sorting the embryos based on their equilibrium height involves collecting the first group of the embryos having respective equilibrium heights above the threshold height in the upper collection channel and collecting the second group of the embryos having respective equilibrium heights below the threshold height in the lower collection channel.
11. The method of claim 10, wherein the upper collection channel and the lower collection channel are both connected to respective pumps.
12. The method of claim 11, wherein the respective pump for the upper collection channel is independent of the respective collection pump for the lower channel.
13. The method of claim 11, wherein the respective pumps are part of a dual syringe pump device.
14. The method of claim 11, further comprising, immediately after levitating of a respective embryo in the channel, determining whether the respective embryo in the channel is above or below the threshold height by visually imaging the respective embryo in the channel and then operating only one of the respective pumps to collect the respective embryo in the upper collection chamber or in the lower collection chamber based on the equilibrium height of the respective embryo relative to the threshold height.
15. The method of claim 14, wherein the visually imaging is live imaging of the respective embryo in the channel.
16. The method of claim 1, wherein the channel is a capillary.
17. The method of claim 1, further comprising the step of transplanting an embryo after assessment into a mother.
PCT/US2022/031266 2021-05-27 2022-05-27 Densitometry-based sorting for embryo health classification WO2022251582A1 (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090098541A1 (en) * 2005-05-03 2009-04-16 Edwin Southern Devices and processes for analysing individual cells
WO2013044089A1 (en) * 2011-09-23 2013-03-28 President And Fellows Of Harvard College Density analysis of organisms by magnetic levitation
US20180280977A1 (en) * 2015-10-02 2018-10-04 The Board Of Trustees Of The Leland Stanford Junior University Sorting Biological and Non-Biological Moieties Using Magnetic Levitation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090098541A1 (en) * 2005-05-03 2009-04-16 Edwin Southern Devices and processes for analysing individual cells
WO2013044089A1 (en) * 2011-09-23 2013-03-28 President And Fellows Of Harvard College Density analysis of organisms by magnetic levitation
US20180280977A1 (en) * 2015-10-02 2018-10-04 The Board Of Trustees Of The Leland Stanford Junior University Sorting Biological and Non-Biological Moieties Using Magnetic Levitation

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