scholarly journals Mitochondrial DNA variants segregate during human preimplantation development into genetically different cell lineages that are maintained postnatally

2021 ◽  
Author(s):  
Joke Mertens ◽  
Marius Regin ◽  
Neelke De Munck ◽  
Edouard Couvreu de Deckersberg ◽  
Florence Belva ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Human Development ◽  
Inner Cell Mass ◽  
Single Cells ◽  
Cell Mass ◽  
Embryonic Cell ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Asymmetric Distribution ◽  
Mtdna Variants

Humans present remarkable mitochondrial DNA (mtDNA) variant mosaicism, not only across tissues but even across individual cells within one person. The timing of the first appearance of this mosaicism has not yet been established. In this study, we hypothesized it occurs during preimplantation development. To investigate this, we deep-sequenced the mtDNA of 254 oocytes from 85 donors, 158 single blastomeres of 25 day-3 embryos, 17 inner cell mass and trophectoderm samples of 7 day-5 blastocysts, 142 bulk DNA and 68 single cells of different adult tissues. We found that day-3 preimplantation embryos already present blastomeres that carry variants unique to that cell, showing that the first events of mtDNA mosaicism happen very early in human development. We classified the mtDNA variants based on their recurrence or uniqueness across sibling oocytes and embryos, and between single cells and samples from the same embryos or adult individuals. Variants that recurred across samples had higher heteroplasmic loads and more frequently resulted in synonymous changes or were located in non-coding regions than variants that were unique to one oocyte or single embryonic cell. These differences were maintained through developmental stages, suggesting that the mtDNA mosaicism arising in preimplantation development is maintained into adulthood. Further, the results support a model in which close clustering of mitochondria carrying specific mtDNA variants in the ooplasm leads to asymmetric distribution of these mitochondria throughout the cell divisions of the preimplantation embryo, resulting in the appearance of the first form of mtDNA mosaicism in human development.

Functional roles of the chromatin remodeler SMARCA5 in mouse and bovine preimplantation embryos

2021 ◽  
Author(s):  
Yan Shi ◽  
Panpan Zhao ◽  
Yanna Dang ◽  
Shuang Li ◽  
Lei Luo ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Rna Seq ◽  
Primitive Endoderm ◽  
Functional Roles ◽  
Morula Stage ◽  
Species Specific

Abstract Upon fertilization, extensive chromatin reprogramming occurs during preimplantation development. Growing evidence reveals species-dependent regulations of this process in mammals. ATP-dependent chromatin remodeling factor SMARCA5 (also known as SNF2H) is required for peri-implantation development in mice. However, the specific functional role of SMARCA5 in preimplantation development and if it is conserved among species remain unclear. Herein, comparative analysis of public RNA-seq datasets reveals that SMARCA5 is universally expressed during oocyte maturation and preimplantation development in mice, cattle, humans and pigs with species-specific patterns. Immunostaining analysis further describes the temporal and spatial changes of SMARCA5 in both mouse and bovine models. siRNA-mediated SMARCA5 depletion reduces the developmental capability and compromises the specification and differentiation of inner cell mass in mouse preimplantation embryos. Indeed, OCT4 is not restricted into the inner cell mass and the formation of epiblast and primitive endoderm disturbed with reduced NANOG and SOX17 in SMARCA5-deficient blastocysts. RNA-seq analysis shows SMARCA5 depletion causes limited effects on the transcriptomics at the morula stage, however, dysregulates 402 genes, including genes involved in transcription regulation and cell proliferation at the blastocyst stage in mice. By comparison, SMARCA5 depletion does not affect the development through the blastocyst stage but significantly compromises the blastocyst quality in cattle. Primitive endoderm formation is greatly disrupted with reduced GATA6 in bovine blastocysts. Overall, our studies demonstrate the importance of SMARCA5 in fostering the preimplantation development in mice and cattle while there are species-specific effects.

scholarly journals Connexin trafficking and the control of gap junction assembly in mouse preimplantation embryos

Development ◽  
1993 ◽  
Vol 117 (4) ◽  
pp. 1355-1367 ◽  
Author(s):  
P.A. De Sousa ◽  
G. Valdimarsson ◽  
B.J. Nicholson ◽  
G.M. Kidder
Keyword(s):  
Gap Junction ◽  
Plasma Membranes ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Brefeldin A ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Cell Fractionation ◽  
Cell Stage ◽  
Junction Protein

Gap junction assembly in the preimplantation mouse embryo is a temporally regulated event, beginning a few hours after the third cleavage during the morphogenetic event known as compaction. Recently, we demonstrated that both mRNA and protein corresponding to connexin43, a gap junction protein, accumulate through preimplantation development beginning at least as early as the 4-cell stage. Using an antibody raised against a synthetic C-terminal peptide of connexin43, this protein was shown to assemble into gap junction-like plaques beginning at compaction (G. Valdimarsson, P. A. De Sousa, E. C. Beyer, D. L. Paul and G. M. Kidder (1991). Molec. Reprod. Dev. 30, 18–26). The purpose of the present study was to follow the fate of nascent connexin43 during preimplantation development, from synthesis to plaque insertion, and to learn more about the control of gap junction assembly during compaction. Cell fractionation and reverse transcription-polymerase chain reaction were employed to show that connexin43 mRNA is in polyribosomes at the 4-cell stage, suggesting that synthesis of connexin43 begins at least one cell cycle in advance of when gap junctions first form. The fate of nascent connexin43 was then followed throughout preimplantation development by means of laser confocal microscopy, using two other peptide (C-terminal)-specific antibodies. As was reported previously, connexin43 could first be detected in gap junction-like plaques beginning in the 8-cell stage, at which time considerable intracellular immunoreactivity could be seen as well. Later, connexin43 becomes differentially distributed in the apposed plasma membranes of morulae and blastocysts: a zonular distribution predominates between outside blastomeres and trophectoderm cells whereas plaque-like localizations predominate between inside blastomeres and cells of the inner cell mass. The cytoplasmic immunoreactivity in morulae was deemed to be nascent connexin en route to the plasma membrane since it could be abolished by treatment with cycloheximide, and redistributed by treatment with monensin or brefeldin-A, known inhibitors of protein trafficking. Treatment of uncompacted 8-cell embryos with either monensin or brefeldin-A inhibited the appearance of gap junction-like structures and the onset of gap junctional coupling in a reversible manner. These data demonstrate that the regulated step in the onset of gap junction assembly during compaction is downstream of transcription and translation and involves mobilization of connexin43 through trafficking organelles to plasma membranes.

scholarly journals Spatio-temporal analysis of human preimplantation development reveals dynamics of epiblast and trophectoderm

2019 ◽  
Author(s):  
Dimitri Meistermann ◽  
Sophie Loubersac ◽  
Arnaud Reignier ◽  
Julie Firmin ◽  
Valentin Francois Campion ◽  
...  
Keyword(s):  
Spatial Information ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Temporal Analysis ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Human Lineage ◽  
Lineage Specification ◽  
Sequence Of Events ◽  
Spatio Temporal

AbstractRecent technological advances such as single-cell RNAseq1-3and CRISPR-CAS9-mediated knock-out4have allowed an unprecedented access into processes orchestrating human preimplantation development5. However, the sequence of events which occur during human preimplantation development are still unknown. In particular, timing of first human lineage specification, the process by which the morula cells acquire a specific fate, remains elusive. Here, we present a human preimplantation development model based on transcriptomic pseudotime modelling of scRNAseq biologically validated by spatial information and precise time-lapse staging. In contrast to mouse, we show that trophectoderm (TE) / inner cell mass (ICM) lineage specification in human is only detectable at the transcriptomic level at the blastocyst stage, just prior to expansion. We validated the expression profile of novel markers enabling precise staging of human preimplantation embryos, such as IFI16 which highlights establishment of epiblast (EPI) and NR2F2 which appears at the transition from specified to mature TE. Strikingly, mature TE cells arise from the polar side, just after specification, supporting a model of polar TE cells driving TE maturation. Altogether, our study unravels the first lineage specification event in the human embryo and provides a browsable resource for mapping spatio-temporal events underlying human lineage specification.

Berberine alleviates LPS-induced apoptosis, oxidation, and skewed lineages during mouse preimplantation development

2022 ◽  
Author(s):  
Xiaosu Miao ◽  
Wei Cui
Keyword(s):  
Inner Cell Mass ◽  
Polycystic Ovary ◽  
Cell Mass ◽  
Cell Lineage ◽  
Culture Conditions ◽  
Preimplantation Development ◽  
Embryonic Cells ◽  
Preimplantation Embryo Development ◽  
New Treatment ◽  
Induced Apoptosis

Abstract Female infertility is a heterogeneous disorder with a variety of complex causes, including inflammation and oxidative stress, which are also closely associated with the pathogenesis of Polycystic Ovary Syndrome (PCOS). As a new treatment for PCOS, berberine (BER), a natural compound from Berberis, has been clinically applied recently. However, the mechanisms underlying the association between BER and embryogenesis are still largely unknown. In this study, effects of BER on preimplantation development was evaluated by using both normal and inflammatory culture conditions induced by lipopolysaccharide (LPS) in the mouse. Our data first suggest that BER itself (25 nM) does not affect embryo quality or future developmental potency, moreover, it can effectively alleviate LPS-induced embryonic damage by mitigating apoptosis via ROS−/caspase-3-dependent pathways and by suppressing pro-inflammatory cytokines via inhibition of NF-κB signaling pathway during preimplantation embryo development. In addition, skewed cell lineage specification in inner cell mass (ICM) and primitive endoderm (PE) caused by LPS can also be successfully rescued with BER. In summary, these findings for the first time demonstrate the non-toxicity of low doses of BER and its anti-apoptotic and anti-oxidative properties on embryonic cells during mammalian preimplantation development.

A novel H+ permeability dominating intracellular pH in the early mouse embryo

Development ◽  
1993 ◽  
Vol 118 (4) ◽  
pp. 1353-1361
Author(s):  
J.M. Baltz ◽  
J.D. Biggers ◽  
C. Lechene
Keyword(s):  
Plasma Membrane ◽  
Intracellular Ph ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Types ◽  
Preimplantation Embryos ◽  
Developmentally Regulated ◽  
Cell Stage ◽  
K Concentration ◽  
Early Mouse

Most cell types are relatively impermeant to H+ and are able to regulate their intracellular pH by means of plasma membrane proteins, which transport H+ or bicarbonate across the membrane in response to perturbations of intracellular pH. Mouse preimplantation embryos at the 2-cell stage, however, do not appear to possess specific pH-regulatory mechanisms for relieving acidosis. They are, instead, highly permeable to H+, so that the intracellular pH in the acid and neutral range is determined by the electrochemical equilibrium of H+ across the plasma membrane. When intracellular pH is perturbed, the rate of the ensuing H+ flux across the plasma membrane is determined by the H+ electrochemical gradient: its dependence on external K+ concentration indicates probable dependence on membrane potential and the rate depends on the H+ concentration gradient across the membrane. The large permeability at the 2-cell stage is absent or greatly diminished in the trophectoderm of blastocysts, but still present in the inner cell mass. Thus, the permeability to H+ appears to be developmentally regulated.

Interactions of blastomeres suggest changes in cell surface adhesiveness during the formation of inner cell mass and trophectoderm in the preimplantation mouse embryo

Development ◽  
1982 ◽  
Vol 70 (1) ◽  
pp. 133-152
Author(s):  
Susan J. Kimber ◽  
M. Azim ◽  
H. Surani ◽  
Sheila C. Barton
Keyword(s):  
Inner Cell Mass ◽  
Single Cells ◽  
Cell Mass ◽  
Cell Stage ◽  
Trophectoderm Cells ◽  
Adhesive Surface ◽  
Preimplantation Mouse Embryo ◽  
Divergent Differentiation ◽  
Preimplantation Mouse ◽  
The Difference

Whole 8-cell morulae can be aggregated with isolated inner cell masses from blastocysts. On examining semithin light microscope sections of such aggregates we found that cells of the morula changed shape and spread over the surface of the ICM, thus translocating it to the inside of the aggregate. Using single cells from 8-cell embryos in combination with single cells from other stage embryos or isolated ICMs we show that 1/8 blastomeres spread over other cells providing a suitably adhesive surface. The incidence of spreading is high with inner cells from 16-cell embryos (56 %) and 32-cell embryos (62%) and isolated inner cell masses (64%). In contrast, the incidence of spreading of 1/8 blastomeres is low over outer cells from 16-cell embryos (26%) and 32-cell embryos (13%). Blastomeres from 8-cell embryos do not spread over unfertilized 1-cell eggs, 1/2 or 1/4 cells or trophectoderm cells contaminating isolated ICMs. When 1/8 cells are aggregated in pairs they flatten on one another (equal spreading) as occurs at compaction in whole 8-cell embryos. However, if 1/8 is allowed to divide to 2/16 in culture one of the cells engulfs the other (51-62/ pairs). Based on the ideas of Holtfreter (1943) and Steinberg (1964,1978) these results are interpreted to indicate an increase in adhesiveness at the 8-cell stage as well as cytoskeletal mobilization. Following the 8-cell stage there is an increase in adhesiveness of inside cells while the outside cells decrease in adhesiveness. The difference in adhesiveness between inside and outside cells in late morulae is probably central to the divergent differentiation of (inner) ICM and (outer) trophectoderm cell populations.

scholarly journals Three-Dimensional Live Imaging of Bovine Preimplantation Embryos: A New Method for IVF Embryo Evaluation

2021 ◽  
Vol 8 ◽  
Author(s):  
Yasumitsu Masuda ◽  
Ryo Hasebe ◽  
Yasushi Kuromi ◽  
Masayoshi Kobayashi ◽  
Kanako Urataki ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Three Dimensional ◽  
Preimplantation Embryos ◽  
Infrared Light ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Developmental Potential

Conception rates for transferred bovine embryos are lower than those for artificial insemination. Embryo transfer (ET) is widely used in cattle but many of the transferred embryos fail to develop, thus, a more effective method for selecting bovine embryos suitable for ET is required. To evaluate the developmental potential of bovine preimplantation embryos (2-cell stage embryos and blastocysts), we have used the non-invasive method of optical coherence tomography (OCT) to obtain live images. The images were used to evaluate 22 parameters of blastocysts, such as the volume of the inner cell mass and the thicknesses of the trophectoderm (TE). Bovine embryos were obtained by in vitro fertilization (IVF) of the cumulus-oocyte complexes aspirated by ovum pick-up from Japanese Black cattle. The quality of the blastocysts was examined under an inverted microscope and all were confirmed to be Code1 according to the International Embryo Transfer Society standards for embryo evaluation. The OCT images of embryos were taken at the 2-cell and blastocyst stages prior to the transfer. In OCT, the embryos were irradiated with near-infrared light for a few minutes to capture three-dimensional images. Nuclei of the 2-cell stage embryos were clearly observed by OCT, and polynuclear cells at the 2-cell stage were also clearly found. With OCT, we were able to observe embryos at the blastocyst stage and evaluate their parameters. The conception rate following OCT (15/30; 50%) is typical for ETs and no newborn calves showed neonatal overgrowth or died, indicating that the OCT did not adversely affect the ET. A principal components analysis was unable to identify the parameters associated with successful pregnancy, while by using hierarchical clustering analysis, TE volume has been suggested to be one of the parameters for the evaluation of bovine embryo. The present results show that OCT imaging can be used to investigate time-dependent changes of IVF embryos. With further improvements, it should be useful for selecting high-quality embryos for transfer.

Regulation of desmocollin transcription in mouse preimplantation embryos

Development ◽  
1995 ◽  
Vol 121 (3) ◽  
pp. 743-753 ◽  
Author(s):  
J.E. Collins ◽  
J.E. Lorimer ◽  
D.R. Garrod ◽  
S.C. Pidsley ◽  
R.S. Buxton ◽  
...  
Keyword(s):  
Protein Expression ◽  
Molecular Mechanisms ◽  
Inner Cell Mass ◽  
Splice Variants ◽  
Cell Mass ◽  
Cumulus Cells ◽  
Early Embryo ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Cleavage Stages

The molecular mechanisms regulating the biogenesis of the first desmosomes to form during mouse embryogenesis have been studied. A sensitive modification of a reverse transcriptase-cDNA amplification procedure has been used to detect transcripts of the desmosomal adhesive cadherin, desmocollin. Sequencing of cDNA amplification products confirmed that two splice variants, a and b, of the DSC2 gene are transcribed coordinately. Transcripts were identified in unfertilized eggs and cumulus cells and in cleavage stages up to the early 8-cell stage, were never detected in compact 8-cell embryos, but were evident again either from the 16-cell morula or very early blastocyst (approx 32-cells) stages onwards. These two phases of transcript detection indicate DSC2 is encoded by maternal and embryonic genomes. Previously, we have shown that desmocollin protein synthesis is undetectable in eggs and cleavage stages but initiates at the early blastocyst stage when desmocollin localises at, and appears to regulate assembly of, nascent desmosomes that form in the trophectoderm but not in the inner cell mass (Fleming, T. P., Garrod, D. R. and Elsmore, A. J. (1991), Development 112, 527–539). Maternal DSC2 mRNA is therefore not translated and presumably is inherited by blastomeres before complete degradation. Our results suggest, however, that initiation of embryonic DSC2 transcription regulates desmocollin protein expression and thereby desmosome formation. Moreover, data from blastocyst single cell analyses suggest that embryonic DSC2 transcription is specific to the trophectoderm lineage. Inhibition of E-cadherin-mediated cell-cell adhesion did not influence the timing of DSC2 embryonic transcription and protein expression. However, isolation and culture of inner cell masses induced an increase in the amount of DSC2 mRNA and protein detected. Taken together, these results suggest that the presence of a contact-free cell surface activates DSC2 transcription in the mouse early embryo.

Specific expression of a retinoic acid-regulated, zinc-finger gene, Rex-1, in preimplantation embryos, trophoblast and spermatocytes

Development ◽  
1991 ◽  
Vol 113 (3) ◽  
pp. 815-824 ◽  
Author(s):  
M.B. Rogers ◽  
B.A. Hosler ◽  
L.J. Gudas
Keyword(s):  
Retinoic Acid ◽  
Germ Cell ◽  
Cell Fate ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Preimplantation Embryos ◽  
Specific Expression

We have previously isolated a cDNA clone for a gene whose expression is reduced by retinoic acid (RA) treatment of F9 embryonal carcinoma cells. The nucleotide sequence indicated that this gene, Rex-1, encodes a zinc-finger protein and thus may be a transcriptional regulator. The Rex-1 message level is high in two lines of embryonic stem cells (CCE and D3) and is reduced when D3 cells are induced to differentiate using four different growth conditions. As expected for a stem-cell-specific message, Rex-1 mRNA is present in the inner cell mass (ICM) of the day 4.5 mouse blastocyst. It is also present in the polar trophoblast of the blastocyst. One and two days later, Rex-1 message is found in the ectoplacental cone and extraembryonic ectoderm of the egg cylinder (trophoblast-derived tissues), but its abundance is much reduced in the embryonic ectoderm which is directly descended from the ICM. Rex-1 is expressed in the day 18 placenta (murine gestation is 18 days), a tissue which is largely derived from trophoblast. The only tested adult tissue that contains detectable amounts of Rex-1 mRNA is the testis. In situ hybridization and northern analyses of RNA from germ-cell-deficient mouse testis and stage-specific germ cell preparations suggest that Rex-1 expression is limited to spermatocytes (germ cells undergoing meiosis). These results suggest that Rex-1 is involved in trophoblast development and spermatogenesis, and is a useful marker for studies of early cell fate determination in the ICM.

P–516 Evidence for self-correction in preimplantation embryos by comparative molecular karyotyping of blastocoele fluid, trophectoderm and inner cell mass

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
D Zhigalina ◽  
N Skryabin ◽  
O Kanbekova ◽  
V Artyukhova ◽  
A Svetlakov ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Morphological Characteristics ◽  
Preimplantation Embryos ◽  
Molecular Karyotyping ◽  
Embryonic Cells ◽  
Developmental Potential ◽  
Positive Correlation ◽  
Registration Number ◽  
Molecular Karyotype

Abstract Study question Does the molecular karyotype of the cell-free DNA (cfDNA) from the blastocyst fluid (BF) can predict the efficiency of self-correction of karyotype of preimplantation embryo? Summary answer Detection of aneuploidies in the BF potentially can point out on effective self-correction of blastocyst karyotype and consequently on high developmental potential of mosaic embryos. What is known already Correction of aneuploidies in the preimplantation embryos can be provided by several mechanisms, including apoptosis. The predominant death of aneuploid cells was demonstrated in mouse embryos (Bolton, 2016). A positive correlation was also shown between the concentration of cfDNA from the BF of human blastocyst and the morphology of the embryo, as well as between the activity of caspase–3 and the concentration of cfDNA (Rule, 2018). The incidence of failed amplification after WGA being significantly higher among euploid blastocysts (Magli, 2019). The capacity of abnormal cells extruding into the BF would be related to the embryo development potential (Gianaroli, 2019). Study design, size, duration This is a prospective observational study of thirty-one Day 5 human blastocysts. Cryopreserved blastocysts were received after treatment cycles at the IVF Center with informed consent obtained from couples. The average age of 15 women was 32.25±5 years. The morphological characteristics of blastocysts were estimated in accordance with the Gardner classification (Gardner, Schoolcraft, 1999). The procedure of BF aspiration and trophectoderm (TE) and ICM cells separation of the blastocysts was previously described (Tsuiko, 2018). Participants/materials, setting, methods WGA was performed by PicoPLEX kit (Rubicon Genomics, USA) or REPLI-g Mini kit (Qiagen) according to manufacturer’s protocols. The DNA of the BF, ICM and TE were analyzed separately using cCGH, aCGH and NGS. SurePrint G3 Human CGH Microarrays (8x60K, Agilent Technologies) were used according to the manufacturer’s recommendations. Image analysis was done using ISIS (v.5.5) (Metasystems) and Agilent CytoGenomics Software (v.3). VeriSeq™ PGS Kit - MiSeq® System (Illumina) was used for NGS. Main results and the role of chance Molecular karyotypes of all three samples - BF, ICM and TE, were obtained for 23 (74.2%) blastocysts. A correlation between the woman’s age and the number of aneuploidies in cfDNA (p = 0.0009) was found. A positive correlation may indicate that the number of aneuploidies in the embryonic cells increases with the age of a woman, however, the embryonic karyotype undergoes self-correcting through the elimination of aneuploid cells. It was noted that well-developing blastocysts (groups 4–5, according to Gardner’s classification) had fewer aneuploidies in ICM (p = 0.0141) and TE (p = 0.0436). In contrast, there was a tendency to an increase in the number of aneuploidies in the BF during blastocysts transition from stage 3 to 5 (p = 0.3542). We assessed the relationship between the number of aneuploidies in groups of blastocysts with different characteristics of ICM (groups “A” and “B” according to Gardner’s classification). These groups significantly differ in the number of aneuploidies in cfDNA (p = 0.0352), although the statistically significant differences between the number of aneuploidies in ICM (p = 0.5992) and in TE (p = 0.5934) was not detected. Thus, higher-quality embryos in terms of ICM morphology contain more abnormalities in the BF, since in this group the elimination of aneuploid cells is more efficient. Limitations, reasons for caution The number of embryos is limited in this study. More comprehensive studies are required to confirm the observed tendency. Wider implications of the findings: Aneuploid cells elimination can be a cause of increasing cfDNA concentration in the BF, which may be a marker of the viability of mosaic embryos when it is necessary to decide on mosaic embryo transfer. This study was supported by the RFBR (15–04–08265) and by the RSF (20–74–00064). Trial registration number Not applicable

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