Here, we describe a method for warming vitrified human blastocysts, culturing them through the implantation period in vitro, digesting them into single cells and collecting early trophoblast cells for further investigation.
Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a critical yet enigmatic biological event that has been historically difficult to study due to technical and ethical limitations. Implantation is initiated by the development of the trophectoderm to early trophoblast and subsequent differentiation into distinct trophoblast sublineages. Aberrant early trophoblast differentiation may lead to implantation failure, placental pathologies, fetal abnormalities, and miscarriage. Recently, methods have been developed to allow human embryos to grow until day 13 post-fertilization in vitro in the absence of maternal tissues, a time-period that encompasses the implantation period in humans. This has given researchers the opportunity to investigate human implantation and recapitulate the dynamics of trophoblast differentiation during this critical period without confounding maternal influences and avoiding inherent obstacles to study early embryo differentiation events in vivo. To characterize different trophoblast sublineages during implantation, we have adopted existing two-dimensional (2D) extended culture methods and developed a procedure to enzymatically digest and isolate different types of trophoblast cells for downstream assays. Embryos cultured in 2D conditions have a relatively flattened morphology and may be suboptimal in modeling in vivo three-dimensional (3D) embryonic architectures. However, trophoblast differentiation seems to be less affected as demonstrated by anticipated morphology and gene expression changes over the course of extended culture. Different trophoblast sublineages, including cytotrophoblast, syncytiotrophoblast and migratory trophoblast can be separated by size, location, and temporal emergence, and used for further characterization or experimentation. Investigation of these early trophoblast cells may be instrumental in understanding human implantation, treating common placental pathologies, and mitigating the incidence of pregnancy loss.
Human implantation and the emergence of the early placenta are historically difficult to investigate and remain largely unknown because human tissues are inaccessible at this stage when pregnancy is clinically undetectable. Animal models are inadequate, as human placentation has its own unique features compared to other eutherian mammals. For example, human placenta invades deeply into the decidua with some trophoblast cells reaching at least the inner third of the uterine myometrium while other cells remodel the uterine spiral arteries. Even our closest evolutionary ancestors, the non-human primates, show differences in placental morphology and trophoblast interactions with the maternal decidual tissues1,2,3. Obtaining pre-implantation human embryos in vitro was not possible until in the 1980’s when clinical human in vitro fertilization (IVF) started as a routine practice for treating infertility4. Now, human blastocysts can be grown in vitro to allow for the selection of more viable embryos for transfer, as well as enable safe genetic testing. Improvement in embryo culture techniques, as well as the increasing use of IVF has yielded many surplus blastocysts which remains after patient’s treatment cycles have been completed. With patient consent, IRB approval, and with certain restrictions, these blastocysts may be utilized for research studies. They have become an invaluable resource that were used for the derivation of human embryonic stem cells5, understanding the transition of inner cell mass to embryonic stem cells6,7, and more recently, have been successfully cultured until day (D) 13 to remodel human implantation8,9. By utilizing recently developed single cell omics approaches, the access to these implantation stage human embryo tissues has offered unique opportunities to describe the molecular mechanisms that regulate this highly dynamic cell differentiation process, which were previously impossible to explore10,11,12,13.
Here, we describe the methods used in our recent publication characterizing the dynamics of trophoblast differentiation during human implantation12. This protocol includes the warming of vitrified blastocysts, extended embryo culture up to D12 post IVF, enzymatic digestion of the embryo into single cells, and cell collection for downstream assays (Figure 1). This extended culture system supports peri-implantation stage human embryo development without maternal input and recapitulates trophoblast differentiation that appears consistent with the observations made from histological specimens many years ago14,15,16,17. During implantation, the trophoblast population is comprised of at least two cell types: the mononucleated progenitor-like cytotrophoblast (CTB) and the terminally differentiated, multinucleated syncytiotrophoblast (STB). Upon trypsin digestion, the CTB are small, round cells that are morphologically indistinguishable from other cell lineages (Figure 2A, left panel). Separation of CTB from other cell lineages, such as epiblast and primitive endoderm, can be achieved by their distinct transcriptomic profiles revealed by single cell RNA sequencing. Syncytiotrophoblast cells can be easily identified as irregular shaped structures that are significantly larger than the other cell types and mainly located at the periphery of the embryo (Figure 2A, middle panel; Figure 2B, left panel). Migratory trophoblast cells (MTB) are another trophoblast sublineage found during embryo extended culture and can be recognized as seemingly moving away from the main body of the embryo (Figure 2A, right panel, Figure 2B, right panel). Migratory trophoblast, although expressing many of the same markers as extra villous trophoblast (EVT), should not be referred to as EVT, since the villous structures in the placenta have not emerged at this very early stage of development.
Experimentally, we were able to collect small CTB and large STB that are easily distinguishable after embryos are digested into single cells at D8, D10, and D12 (Figure 2A,B). Migratory trophoblasts arise at the later stages of extended embryo culture and can be collected at D12 before enzymatic digestion of the whole embryo (Figure 2A,B). By phenotypically separating these three trophoblast sublineages before single cell analysis, we can identify specific transcriptomic markers and define the biological role of each cell type. Cytotrophoblast are highly proliferative and act as progenitor cells in supplying the STB and MTB differentiated lineages10,11,12,13. Syncytiotrophoblast are involved in producing placental hormones to maintain pregnancy and may be also responsible for the embryos burrowing into the endometrium10,11,12,13. Migratory trophoblast has even stronger features of an invasive, migratory phenotype and are likely responsible for deeper and more extensive colonization of the uterine endometrium10,11,12,13. After defining the transcriptomic signature of each cell type, clustering analyses have also revealed two additional subsets of cells that were morphologically indistinguishable from CTB and had transcriptomes with features of STB and MTB, respectively12. These intermediate stage cells are likely in the process of differentiating from CTB to either the MTB or STB sublineages and would have been overlooked if embryos were blindly digested and cells were separated by transcriptome alone.
The protocol described here utilizes a two-dimensional (2D) culture system and may not be optimal for supporting three-dimensional (3D) structural development, as suggested by a recent publication describing a newly developed 3D culture system13. Nevertheless, in this 2D system the differentiation of early trophoblast seems to be consistent with observations made from in vivo specimens14,15,16,17. This protocol may also be easily adapted for the use in the recently described 3D culture system13 with minimal changes. All steps are carried out with a handheld micromanipulation pipette with commercially available disposable tips or a mouth pipette from a finely pulled glass pipette attached to rubber tubing, a filter, and a mouthpiece.
All human embryos have been donated with consent for use in research. Protocols for extended human embryo culture have been approved by the Western Institutional Review Board (study no. 1179872) and follow international guidelines. Any use of human embryos must be reviewed by the appropriate ethics and governing bodies associated with the research institution using this protocol.
1. Preparation
2. Warming vitrified D5 human embryos
NOTE: Other manufacturers may have slightly different protocols according to their own vitrification technology. See manufacturer’s instructions for use when applicable.
3. Recovery of warmed embryos
4. Zona removal
5. Blastocyst extended culture
6. Optional collection of spent media
7. Optional fixation for immunofluorescence
8. Single cell digestion with Trypsin
NOTE: Fresh (not fixed) embryos are used for single cell digestion.
9. Single cell selection and sample collection
Healthy embryos exhibited continued proliferation over the course of extended culture (Figure 2B). Abnormal embryos began to retract from their outer edges and disintegrate (Figure 2C). From our experience, approximately 75% of embryos were attached to the bottom of the fibronectin coated dish at 48 h and the attachment increased to approximately 90% by 72 h in culture. The success of embryo attachment may be largely impacted by the initial quality of the blastocysts. Embryos not attached by 72 h likely will not survive.
At D8 post fertilization (D3 of extended culture), most cells in the embryos were CTB that were positive for trophoblast marker GATA3 (Figure 3A). Cytotrophoblasts had already began to differentiate into multinucleated STB on the periphery of the embryo (Figure 2B, left panel, dotted line). These STB had a sheet-like appearance and were stained positive for the human chorionic gonadotropin subunit beta (hCGB; Figure 3A, center panel). The embryos quickly grew, between D8 and D10, suggesting a rapid cell proliferation of CTB during this period. At D10, the formation of CGB positive MTB was at a maximum, which could be confirmed by the upsurge of hCG production at this time (Figure 3B). Migratory trophoblasts also began to emerge and migrated away from the embryo body and were stained positive for the EVT marker, HLA-G (Figure 3A, right panel). By D12, STB differentiation was in decline and MTB production became more prominent, suggesting a shift of emphasis from hormone production on D10 to cell migration on D12. These changes were observed by time-lapse video obtained during this peri-implantation period (Movie 1). Our single cell RNA sequencing data also reflected such dynamic changes in cell function. Together, these data suggest early trophoblast differentiation and the emergence of the early placenta is a dynamic process, during which the embryo can prioritize highly specialized cell functions at very specific time points to achieve successful implantation.
Figure 1: Procedural schematic of extended culture and single cell sample collection.
Workflow of warming vitrified embryos, zona pellucida removal, extended embryo culture, enzymatic digestion, and isolation of cytotrophoblast (CTB), syncytiotrophoblast (STB), and migratory trophoblast (MTB). This figure has been modified from West et al.12. Please click here to view a larger version of this figure.
Figure 2: Morphologies of trophoblast cells and embryos during extended culture.
(A) Representative images of different trophoblast cell types after enzymatic digestion with trypsin. Small CTB on the left, large STB in the middle, and MTB on the right. (B) Representative images of healthy embryos at D8 (left), D10 (middle) and D12 (right). Dashed lines in left panel outline presumably proliferative CTB population whereas dotted line outlines the flattened STB. Pink circles in the right panel outline MTB that were remotely located from the embryo. (C) Representative images of abnormal embryos beginning to retract and disintegrate at D8 (left), D10 (middle), and D12 (right). Scale bars = 200 µm. Some images have been adapted from West et al.12. Please click here to view a larger version of this figure.
Figure 3: Representative images of trophoblast marker expression and hCG production during extended culture.
(A) Representative immunofluorescence images of embryos showing expression of a CTB marker GATA3 at D10 (left), STB marker CGB at D10 (middle), and MTB marker HLA-G at D12 (right). Scale bars= 200 µm. (B) Representative hCG concentration (mIU/mL) over the course of extended culture from D8 to D12 (mean± SEM, n = 3). Statistical analysis was performed with One-Way ANOVA followed by Tukey’s test (P < 0.05). These images have been adapted from West et al.12. Please click here to view a larger version of this figure.
Movie 1: Time-lapse video of a human embryo in extended culture from D8 to D12. The video demonstrates the collapse of the blastocoel, the formation of the STB (indicated by the green circles), and then eventual differentiation and migration of MTB (indicated by the orange circles). This has been adapted from West et al.12. Please click here to download this video.
The development of the protocol to culture human embryos through implantation has allowed scientists to explore a previously uncharted time in development8,9. Here, we use an extended culture system to culture human embryos and study early trophoblast differentiation before the formation of villous placenta. The methods described here allow us to collect different TB sublineages for use in downstream single-cell analysis. This work allows the scientific community a chance to understand this critical and enigmatic period in human development, and may open new opportunities for therapeutic treatments for miscarriage or other placental pathologies such as pre-eclampsia, as well as provide new insights about early human development.
This protocol requires skills in quickly manipulating embryos during embryo warming, zona removal and plating to minimize embryo stress prior to extended culture. Minimizing media evaporation and thus an increase in medium osmolality by limiting the time of exposure during media exchange is essential. Taking care to use proper aseptic technique when exchanging medium and moving dishes will help minimize contamination. Stabilizing the dish when in use to minimize any mechanical disturbance once embryos have attached is also important. Embryos that become stressed will start to recede their syncytial projections and begin to apoptose. It is also critical to avoid allowing the meniscus of the culture media to touch the surface of the embryo, and to minimize any oil in the wells. Either of these scenarios may destroy the embryo.
Culturing human embryos beyond the blastocyst stage is a rapidly evolving field. A recent study13 demonstrated that a novel culture system which contains 10% of an extracellular matrix protein mixture and a medium with additional supplementation of sodium pyruvate, lactate, and rho-associate protein kinase (ROCK) inhibitor is advantageous in modeling in vivo 3D embryonic architectures and yielded significantly more viable embryos at later stage of extended culture compared to the extended culture system described here. Validation of this new system to demonstrate its reproducibility will be required. We believe the procedures described here can be adapted to this new 3D system with minor changes and may, therefore, benefit the advancement in methodology in this field.
This protocol may also be adapted to allow investigation into IVF culture media optimization. We have recently shown that in mouse, embryo development during extended culture may predict the success of fetal development after embryo transfer18. Abnormally fertilized and discarded human zygotes with 3 pronuclei (PN) can be cultured to blastocysts on D5 in different conditions that may then influence their development during extended culture. Donated D5 human blastocysts may be cultured for 48 h in novel conditions before being placed into the extended culture system for performance evaluation. Routine measurements of epiblast cell number, total cell number, outgrowth area, and hCG have allowed our laboratory to better understand how different culture media conditions may better support human pre-implantation embryo development and influence their post-implantation success.
The authors have nothing to disclose.
We would like to acknowledge the many patients at the Colorado Center for Reproductive Medicine (CCRM) that have graciously donated their embryos for research. We would also like to thank Karen Maruniak and the clinical laboratory at CCRM for their help in processing hCG samples, as well as Sue McCormick and her clinical IVF embryology team at CCRM for their help with embryo collection, storage, tracking, and donation. Funding was provided internally by CCRM.
3130 or 3110 Forma Series II water-jacketed CO2 incubator | Thermo Fisher Scientific | 13-998-078 | |
35 mm Corning Primaria tissue culture dish | VWR | 62406-038 | Case of 500 |
5 mL snap cap tube | VWR | 60819-295 | Pack of 25 |
60 mm Corning Primaria tissue culture dish | VWR | 25382-687 | Case of 200 |
6-well dish | Agtech Inc. | D18 | Pack of 1, 10, or 50 |
Acidic Tyrode's solution | Millipore Sigma | T1788 | 100 mL |
Biotix 1250 µL pipette tips | VWR | 76322-156 | Pack of 960 |
Blast, blastocyst culture media | Origio | 83060010 | 10 mL |
Dilution Solution | Kitazato | VT802 | 1 x 4 mL |
Disposable Borosilicate Glass Pasteur Pipets | Thermo Fisher Scientific | 1367820D | 5.75 in. (146mm); 720/Cs |
Dulbecco's Phosphate Buffered Saline | Millipore Sigma | D8537 | |
Embryo culture paraffin oil OvOil | Vitrolife | 10029 | 100 mL |
Eppendorf PCR tubes 0.2 mL | VWR | 47730-598 | Pack of 1,000 |
Eppendorf PCR tubes 0.5 mL | VWR | 89130-980 | Case of 500 |
Fibronectin from human plasma. Liquid .1% solution | Millipore Sigma | F0895 | 1 mg |
Gilson 1 mL Pipetteman | Thermo Fisher Scientific | F123602G | 1 Pipetteman 200-1000 µL |
Gilson 20 µL Pipetteman | Thermo Fisher Scientific | F123602G | 1 Pipetteman 2-20 µL |
Gilson 200 µL Pipetteman | Thermo Fisher Scientific | F123602G | 1 Pipetteman 50-200 µL |
G-MOPS handling media | Vitrolife | 10129 | 125 mL |
Handling media | Origio | 83100060 | 60 mL |
Ibidi 8 well chambered coverslip | Ibidi | 80826 | 15 slides per box |
IVC1/IVC2 | Cell Guidance Systems | M11-25/ M12-25 | 5-5mL aliquots |
K System T47 Warming Plate | Cooper Surgical | 23054 | |
MilliporeSigma Millex Sterile Syringe Filters with Durapore PVFD Membrane | Fisher Scientific | SLGVR33RS | Pack of 50 |
Mouth pieces | IVF Store | MP-001-Y | 100 pieces |
Oosafe center well dish | Oosafe | OOPW-CW05-1 | Case of 500 |
Quinn's Advantage SPS | Origio | ART-3010 | 12x 12 mL |
Rubber latex tubing for mouth pieces | IVF Store | IVFS-NRL-B-5 | 5 ft. |
Stereomicroscope | Nikon | SMZ1270 | |
Stripper tips | Cooper Surgical | MXL3-275 | 20/pk 275 µm |
Thawing Solution | Kitazato | VT802 | 2 x 4 mL |
The Stripper Micropipetter | Cooper Surgical | MXL3-STR | |
TrypLE Express Enzyme (1X), no phenol red | Thermo Fisher Scientific | 12604013 | 1 x 100 mL |
Tween20 | Millipore Sigma | P1379-25ML | 25 mL bottle |
VWR 1-20 µL pipette tips | VWR | 76322-134 | Pack of 960 |
VWR 1-200 µL pipette tips | VWR | 89174-526 | Pack of 960 |
Washing Solution | Kitazato | VT802 | 1 x 4 mL |