Endometrial Assembloids to Model Human Embryo Implantation In Vitro.

Understanding the process of human embryo implantation is impeded by the inability to study this phenomenon in vivo, thus limiting opportunities to gain knowledge to in vitro modeling. Previous models have relied on monolayer co-cultures, which do not replicate the complexity of endometrial tissue. Here, we detail the establishment of three-dimensional endometrial assembloids, comprising gland-like epithelial organoids in a stromal matrix. Endometrial assembloids mimic endometrial tissue structure more faithfully and can be used to study human embryo-endometrial interactions. Co-cultures of human embryos and endometrial assembloids will enhance our fundamental understanding of these processes as well as allowing us to study the mechanisms of persistent reproductive failure.

The first mitotic division of human embryos is highly error prone.

Human beings are made of ~50 trillion cells which arise from serial mitotic divisions of a single cell - the fertilised egg. Remarkably, the early human embryo is often chromosomally abnormal, and many are mosaic, with the karyotype differing from one cell to another. Mosaicism presumably arises from chromosome segregation errors during the early mitotic divisions, although these events have never been visualised in living human embryos. Here, we establish live cell imaging of chromosome segregation using normally fertilised embryos from an egg-share-to-research programme, as well as embryos deselected during fertility treatment. We reveal that the first mitotic division has an extended prometaphase/metaphase and exhibits phenotypes that can cause nondisjunction. These included multipolar chromosome segregations and lagging chromosomes that lead to formation of micronuclei. Analysis of nuclear number and size provides evidence of equivalent phenotypes in 2-cell human embryos that gave rise to live births. Together this shows that errors in the first mitotic division can be tolerated in human embryos and uncovers cell biological events that contribute to preimplantation mosaicism.

Modelling the impact of decidual senescence on embryo implantation in human endometrial assembloids.

Decidual remodelling of midluteal endometrium leads to a short implantation window after which the uterine mucosa either breaks down or is transformed into a robust matrix that accommodates the placenta throughout pregnancy. To gain insights into the underlying mechanisms, we established and characterized endometrial assembloids, consisting of gland-like organoids and primary stromal cells. Single-cell transcriptomics revealed that decidualized assembloids closely resemble midluteal endometrium, harbouring differentiated and senescent subpopulations in both glands and stroma. We show that acute senescence in glandular epithelium drives secretion of multiple canonical implantation factors, whereas in the stroma it calibrates the emergence of anti-inflammatory decidual cells and pro-inflammatory senescent decidual cells. Pharmacological inhibition of stress responses in pre-decidual cells accelerated decidualization by eliminating the emergence of senescent decidual cells. In co-culture experiments, accelerated decidualization resulted in entrapment of collapsed human blastocysts in a robust, static decidual matrix. By contrast, the presence of senescent decidual cells created a dynamic implantation environment, enabling embryo expansion and attachment, although their persistence led to gradual disintegration of assembloids. Our findings suggest that decidual senescence controls endometrial fate decisions at implantation and highlight how endometrial assembloids may accelerate the discovery of new treatments to prevent reproductive failure.

At the beginning of a human pregnancy, the embryo implants into the uterus lining, known as the endometrium. At this point, the endometrium transforms into a new tissue that helps the placenta to form. Problems in this transformation process are linked to pregnancy disorders, many of which can lead to implantation failure (the embryo fails to invade the endometrium altogether) or recurrent miscarriages (the embryo implants successfully, but the interface between the placenta and the endometrium subsequently breaks down). Studying the implantation of human embryos directly is difficult due to ethical and technical barriers, and animals do not perfectly mimic the human process, making it challenging to determine the causes of pregnancy disorders. However, it is likely that a form of cellular arrest called senescence, in which cells stop dividing but remain metabolically active, plays a role. Indeed, excessive senescence in the cells that make up the endometrium is associated with recurrent miscarriage, while a lack of senescence is associated with implantation failure. To study this process, Rawlings et al. developed a new laboratory model of the human endometrium by assembling two of the main cell types found in the tissue into a three-dimensional structure. When treated with hormones, these 'assembloids' successfully mimic the activity of genes in the cells of the endometrium during implantation. Rawlings et al. then exposed the assembloids to the drug dasatinib, which targets and eliminates senescent cells. This experiment showed that assembloids become very robust and static when devoid of senescent cells. Rawlings et al. then studied the interaction between embryos and assembloids using time-lapse imaging. In the absence of dasatinib treatment, cells in the assembloid migrated towards the embryo as it expanded, a process required for implantation. However, when senescent cells were eliminated using dasatinib, this movement of cells towards the embryo stopped, and the embryo failed to expand, in a situation that mimicks implantation failure. The assembloid model of the endometrium may help scientists to study endometrial defects in the lab and test potential treatments. Further work will include other endometrial cell types in the assembloids, and could help increase the reliability of the model. However, any drug treatments identified using this model will need further research into their safety and effectiveness before they can be offered to patients.

Dichorionic triamniotic triplet pregnancy with monozygotic twins discordant for trisomy 13 after preimplantation genetic screening: case report.

OBJECTIVE: To report the first dichorionic triamniotic triplet pregnancy discordant for trisomy 13 after in vitro fertilization (IVF) treatment with preimplantation genetic screening (PGS). DESIGN: Case report. SETTING: Private IVF center. PATIENT(S): A 40-year-old para 1+6 woman. INTERVENTION(S): IVF combined with PGS for chromosomes 13, 16, 18, 21, and 22, resulting in the transfer of two embryos. MAIN OUTCOME MEASURE(S): Prenatal fetal ultrasonography revealed a dichorionic triamniotic triplet pregnancy. An amniocentesis, performed at 15-weeks' gestation, confirmed that the singleton and one monozygotic twin were normal but the other monozygotic twin was trisomy 13. RESULT(S): After diagnosis and counseling, selective termination of the trisomy 13 monozygotic twin was performed at 16 weeks and 4 days. At 18 weeks and 4 days the co-twin died. A healthy boy was delivered by elective caesarean section at 36-weeks' gestation. CONCLUSION(S): Assisted reproductive techniques that breach the embryo's zona pellucida such as assisted hatching and PGS embryo biopsy increase the incidence of monozygotic twins. Due to high levels of mosaicism in human preimplantation embryos, PGS cannot ensure that embryos diagnosed as normal and selected for transfer do not contain abnormal cells. Hence, further reports of discordant monozygotic twins following PGS are expected, emphasizing the need for appropriate counseling of patients wishing to embark on an IVF/PGS treatment cycle.

Quantitative RT-PCR reveals tuberous sclerosis gene, TSC2, mRNA degradation following cryopreservation in the human preimplantation embryo.

The use of cryopreserved human embryos in gene expression studies provides an additional source to the scarce embryos available for research. To validate their use we have implemented a quantitative RT-PCR to characterize the levels of the tuberous sclerosis, TSC2 gene in fresh and frozen-thawed human embryos. Frozen embryos were thawed using two different clinical protocols. In fresh embryos 9.95 fg of TSC2 cDNA was present in the unfertilized oocyte, which was comparable to the level on day 2 of preimplantation development. On day 3 there was a significant drop (P<0.001) to 6.8 fg, followed by an increase in cDNA levels to 10.8 fg (P<0.01) on day 6 at the expanded blastocyst stage. Day 2 frozen embryos possessed 50% less (P<0.001) TSC2 mRNA in comparison to the fresh embryos using thawing protocol one (from frozen to 37 degrees C) and 25% less TSC2 mRNA (P<0.01) with thawing protocol 2 (from frozen to room temperature). After culturing day 2 frozen embryos for an additional day they showed mRNA levels comparable with fresh day 3 embryos. There was no significant difference in the levels of TSC2 mRNA between fresh and frozen day 3 human embryos with either thawing protocol. This study demonstrates that cryopreservation does affect the normal pattern of gene expression during human preimplantation development, and that intact frozen-thawed embryos are not equivalent to their non-frozen counterparts. Furthermore human embryos frozen on day 2 appear to be more susceptible to temperature change than embryos frozen on day 3.