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ORIGINAL RESEARCH
published: 18 September 2019
doi: 10.3389/fimmu.2019.02234
Frontiers in Immunology | www.frontiersin.org 1September 2019 | Volume 10 | Article 2234
Edited by:
Mats Bemark,
University of Gothenburg, Sweden
Reviewed by:
Gerard Chaouat,
INSERM U976 Immunologie,
Dermatologie, Oncologie, France
Jean F. Regal,
Medical School, University of
Minnesota, United States
*Correspondence:
Martin P. Reichhardt
martin.reichhardt@path.ox.ac.uk
†These authors
share senior-authorship
Specialty section:
This article was submitted to
Mucosal Immunity,
a section of the journal
Frontiers in Immunology
Received: 17 June 2019
Accepted: 03 September 2019
Published: 18 September 2019
Citation:
Reichhardt MP, Lundin K, Lokki AI,
Recher G, Vuoristo S, Katayama S,
Tapanainen JS, Kere J, Meri S and
Tuuri T (2019) Complement in Human
Pre-implantation Embryos: Attack and
Defense. Front. Immunol. 10:2234.
doi: 10.3389/fimmu.2019.02234
Complement in Human
Pre-implantation Embryos: Attack
and Defense
Martin P. Reichhardt 1,2
*, Karolina Lundin 3, A. Inkeri Lokki 2,4 , Gaëlle Recher 5,
Sanna Vuoristo 3, Shintaro Katayama 6, Juha S. Tapanainen 3,7, Juha Kere 6, 8,9 ,
Seppo Meri 2,4,10† and Timo Tuuri3†
1Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom, 2Department of Bacteriology and
Immunology, University of Helsinki, Helsinki, Finland, 3Department of Obstetrics and Gynecology, Helsinki University Hospital,
University of Helsinki, Helsinki, Finland, 4Translational Immunology Research Program, Research Programs Unit, University of
Helsinki, Helsinki, Finland, 5Institut d’Optique Graduate School, CNRS - Université de Bordeaux, Talence, France,
6Department of Biosciences and Nutrition, Karolinska Institutet, Solna, Sweden, 7PEDEGO Research Unit, Department of
Obstetrics and Gynecology, University of Oulu and Oulu University Hospital, Medical Research Center, Oulu, Finland, 8School
of Basic and Medical Biosciences, King’s College London, London, United Kingdom, 9Stem Cells and Metabolism Research
Program, Folkhälsan Institute of Genetics, University of Helsinki, Helsinki, Finland, 10 Department of Biomedical Sciences,
Humanitas University, Milan, Italy
It is essential for early human life that mucosal immunological responses to developing
embryos are tightly regulated. An imbalance of the complement system is a common
feature of pregnancy complications. We hereby present the first full analysis of
the expression and deposition of complement molecules in human pre-implantation
embryos. Thus, far, immunological imbalance has been considered in stages of
pregnancy following implantation. We here show that complement activation against
developing human embryos takes place already at the pre-implantation stage. Using
confocal microscopy, we observed deposition of activation products on healthy
developing embryos, which highlights the need for strict complement regulation.
We show that embryos express complement membrane inhibitors and bind soluble
regulators. These findings show that mucosal complement targets human embryos,
and indicate potential adverse pregnancy outcomes, if regulation of activation fails.
In addition, single-cell RNA sequencing revealed cellular expression of complement
activators. This shows that the embryonic cells themselves have the capacity to express
and activate C3 and C5. The specific local embryonic expression of complement
components, regulators, and deposition of activation products on the surface of embryos
suggests that complement has immunoregulatory functions and furthermore may impact
cellular homeostasis and differentiation at the earliest stages of life.
Keywords: reproductive immunology, mucosal immunology, complement, embryo, development, pre-implantation
INTRODUCTION
The complement system is a part of the innate immune defense system, primarily involved in
anti-microbial defense, clearance of debris, and immune regulation. This multi-lineage enzymatic
cascade functions as one of the earliest initiators of inflammation and a potent inducer of adaptive
immune responses. It may be initiated through the classical and lectin pathways, driven by
Reichhardt et al. Complement in Human Embryos
pattern-recognition (e.g., via C1q), or through the auto-
activation of the alternative pathway. All three pathways converge
at the activation of C3 to C3a and C3b, with the subsequent
activation of C5 to C5a and C5b, and finally assembly of the
pore-forming membrane attack complex (MAC, C5b-C9). To
avoid complement attack against self-tissue, human surfaces
express and/or recruit a number of membrane-bound and soluble
regulators (1,2).
Pregnancy involves, for the mother, a state of induced
local immunological tolerance to the growing embryo. Strict
immune regulation localized at the feto-maternal interface is
therefore essential for a healthy outcome for both mother and
child (3,4). Increased complement activation is observed in
certain autoimmune diseases, but also during pregnancy (5,6).
Thus, the role of complement dysregulation has emerged as an
important contributing factor to pregnancy complications and
infertility, e.g., in pre-eclampsia and recurring miscarriage (7–
9). Mouse-models of pregnancy hypertension show increased
complement deposition already during implantation (10). These
findings highlight the importance of certain complement
regulators identified on human oocytes and embryos (11,
12). Understanding how the clearance-function of complement
is regulated in the context of tolerating the “semiallograft”
embryo during human pregnancy is therefore of paramount
importance (13).
In addition to the manifest role of complement in immune
targeting and clearance, novel functions have been revealed
in recent years. Studies across multiple species have revealed
unexpected roles of complement molecules in fertilization,
embryonic growth, and organogenesis (14). The liver is the
main site for synthesis of complement components, however,
these novel findings have been driven by the detection of
local cell-derived complement factors and functional links
to basic cellular homeostasis and metabolism (2,9,15–18).
Complement C3 was suggested as an embryotrophic factor in
rat embryos (19).Furthermore, a number of animal models have
revealed an effect of complement on mouse embryo hatching
rate, Xenopus organogenesis as well as on rodent neuronal
development (20–24).
While our understanding of local cellular complement
activities is increasing, knowledge of complement expression
and localization at the very early stages of human life, i.e., the
pre-implantation embryonic stage, is practically non-existing.
To understand the impact of complement on embryonic
development, it is essential to investigate (i) potentially hazardous
embryonic targeting by maternal complement (complement
clearance function), as well as (ii) local embryonic production
of complement components (cellular signaling and protection
against maternal complement attack).
In an effort to map the localization of complement molecules
and understand the role of complement activation in the early
stages of human embryonic development, we here describe the
local cellular expression and surface deposition of complement
components using single cell RNA-transcriptomics and confocal
microscopy of human non-fertilized oocytes, zygotes, 4-cell
stage and 8-cell stage embryos. Current studies in the field
suggests that maternal immune tolerance toward the conceptus
is essential during and after implantation (25). The findings
presented here further support a crucial role for innate immune
mechanisms, such as complement activation, already in the pre-
implantation stage of human reproduction. Furthermore, to
the authors’ knowledge, these studies are the first to show the
expression of multiple activating and regulatory complement
components during human embryogenesis. Our findings thus
suggest that complement signaling may be essential for early
human development, as observed in other mammals.
RESULTS
Embryos Express Complement Regulators,
Activators, and Receptors
To understand the role of complement during early
embryogenesis, we analyzed single-cell transcriptomes from
embryos at different developmental stages. Included were
non-fertilized oocytes, zygotes, 4-cell stage embryos, and 8-cell
stage embryos. Using single-cell tagged reverse-transcription
sequencing (STRT), 5′- Transcript Far Ends (TFEs) were
analyzed as described previously (26). Specifically, transcripts
from the 5′-untranslated region (UTR) and upstream are reliable
templates for proteins, and these were used for the analysis
along with a number of reliable reads from the coding sequences
(CDs). The sequencing methodology only allows comparison
of two different stages within one library (26). The samples
were thus processed as three separate STRT libraries specifically
designed to compare developmental stages: (i) library L233 to
compare oocytes and zygotes; (ii) L185 to investigate the early
wave of early genome activation (EGA), comparing oocytes and
4-cell blastomeres; and (iii) L186 to study the 4-cell to 8-cell
transition, comprising the major EGA. To avoid batch-bias, these
libraries were not batch corrected. We analyzed the presence
of specific complement-related TFEs found in each of the four
stages (Figure 1).
Single-cell RNA sequencing revealed expression of a varied
set of complement related genes. Transcripts correlating
to a number of molecules known to protect cells from
complement attack were identified. These include the membrane
glycophosphoinositol (GPI)-lipid anchored complement
regulators CD55 and CD59 found at all investigated stages. In
the case that one (or more) TFEs corresponding to a certain
gene-transcript are identified, that shows mRNA transcripts
from that gene are present at the given stage. While some TFEs
for e.g., CD55 and CD59 were not identified at all investigated
states, at least one validated TFE of both CD55 and CD59 were
identified at all four developmental stages, highlighting the
cellular potential to produce these proteins. CD46 transcripts
were observed at all stages except the 8-cell stage. Furthermore,
mRNA transcripts of soluble complement inhibitors such as
C4b-binding protein (C4BP beta chain—only alternative splice-
form found), factor I and clusterin were also found at constant
levels from oocytes through to 8-cell stage embryo blastomeres.
In addition to mRNA transcripts of complement regulators
that protect the early embryos from maternal complement attack,
our data also show mRNA correlating to complement-activating
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Reichhardt et al. Complement in Human Embryos
FIGURE 1 | Expression of complement genes in developing embryos. Single-cell RNA sequencing was applied on oocytes (O), zygotes (Z), 4-cell stage, and 8-cell
stage embryos to identify the expression of complement related genes during early development. Included are reads tagged to the 5′UTR (Coding 5′-UTR) or proximal
upstream region (Coding upstream), as well as known coding sequence (Coding CDs). The numerical values indicating normalized expression levels are determined
(Continued)
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Reichhardt et al. Complement in Human Embryos
FIGURE 1 | relative to concentration of spike-in RNAs comparing two developmental stages at a time, in three individual libraries (O vs. Z, O vs. 4-cell stage, and
4-cell vs. 8-cell stages). Expression levels are averaged over n=6 to 29 embryonic cells, as indicated. White denotes no expression observed for a given TFE in a
particular library. While the data reveal the presence of non-degraded mRNA from the displayed genes, statistical analysis did not reveal significant variations in gene
expression from one developmental stage to another. For detailed functional description of identified genes see below and (1,2,27). ADIPOR1/2, adiponectin
receptor ½; C4BBP, C4 binding protein chain B; C1QBP, gC1qR/C1q globular domain-binding protein; CALR, calreticulin receptor; CFI, complement Factor I; CLU,
Clusterin; CR2, complement receptor 2; C5AR1, C5a Receptor 1; CFB, Complement factor B; CFD, Complement factor D; ITGB2, Integrin beta chain-2 (part of
complement receptor 3 and 4); MASP, MBL associated serine protease.
molecules. The central activating components C3 and C5 are
expressed by the embryos themselves already at the oocyte
stage, and the transcripts remain after fertilization. In addition,
proteases and cascade-components normally responsible for
activation of C3, such as factor B and factor D (alternative
pathway) and C1s and C2 (classical/lectin pathways) are also
found. The oocytes and embryos themselves thus have transcripts
for proteins that would allow cleavage and activation of both C3
and C5.
Transcripts from a number of surface receptors, commonly
found to mediate activation of phagocytes and other immune
cells, were also identified in the embryos. These include the
C5a-receptor 1 (C5aR1), the complement receptor 2 (CR2),
and integrin beta chain-2 (ITGB2/CD18—only alternative
splice-form found), which combines with CD11b or CD11c,
forming the complement receptors CR3 and CR4, respectively.
Transcripts of receptors for C1q (linked to clearance of apoptotic
material and tissue-remodeling), such as calreticulin and C1q
globular domain-binding protein (C1QBP, also known as
gC1qR), were found at all investigated stages. Finally, transcripts
from adiponectin receptors 1 and 2 (ADIPOR1/2), which are
linked to cellular homeostasis and metabolism are also present
in the early embryos.
No statistically significant increase in mRNA transcripts
were identified from oocyte to the fertilized stages. The timing
of active transcription is thus likely to be during oogenesis
(28). Transcripts correlating to known alternative splice-forms
(observed for ADIPOR1 and 2, C1QBP, C3, C4BPB, CD55,
CD59, CR2, and ITGB2) may either reflect alternative protein
functions, or partial breakdown of transcripts. Changes over
time in the ratio between 3′UTR/degraded reads and 5′-UTR-
proximal reads, may indicate an active downregulation of genes.
By comparing the 3′/5′-ratios at oocyte vs. 4-cell stage, we
observed an increased ratio for ADIPOR2, C1QBP, C5, and
CD55, suggesting their gradually increasing degradation. In
contrast, a decreased ratio was observed for C3, C5aR1, and
MASP2, suggesting their active transcription (data not shown).
In addition to the above-mentioned verified non-degraded
transcripts, mRNA transcripts from several other complement
genes were also identified in at least one of the four investigated
stages. The lack of TFEs at the 5′UTR proximal region of
these reads may reflect partial breakdown of maternal transcripts,
existence of promiscuous (zygotic) RNAs or novel isoforms (29).
Further validation is needed to determine if these transcripts
produce functional protein at this developmental stage. These
genes are highlighted in the Supplementary Table 1. A number
of additional complement-related genes were investigated;
however, no expression was detected. These are listed in
Supplementary Table 2.
Complement Activation Targets Human
Embryos
Following the observation of embryonic gene expression
of complement molecules, we sought to investigate if human
embryos were targeted by complement activation. We considered
that the complement components on the embryonic surface
could originate from a combination of maternal and embryonic
complement, and initially investigated deposition of the
complement targeting molecules C1q, C3b, the inactivated
C3d as well as C5 (Figure 2). Negative controls are shown in
Supplementary Figure 1.
Strong specific staining of C1q is visible on the cell-surface
of the four-cell stage embryos (Figure 2A). No staining was
observed at the cellular junctions. C1q deposition on a surface
may lead to initiation of classical pathway activation. We
therefore proceeded to investigate the embryonic deposition of
C3 activation products. Staining for C3b/iC3b (recognized by the
anti-C3c antibody, Figure 2B) shows a clear deposition on the
cellular membranes. A disperse staining is also observed on the
surface of the zona pellucida (ZP). The presence of C3b/iC3b
on the cell membranes shows that complement activation targets
human embryos. C3d is the final breakdown product of C3-
inactivation and remains covalently bound to a surface long
after activation has taken place. We therefore next analyzed the
presence of C3d on the embryos (Figure 2C). We observe clear
staining for C3d on the membranes of the embryonic cells. This
shows that a large part of C3b on the embryonic surface has
become degraded to C3d, indicating efficient control. In contrast
to the C3b/iC3b-staining, we observed more staining for C3d
on the ZP. However, variation between embryos was observed.
Following deposition of complement C3b and generation of the
C5 convertase, the cascade leads to the activation of C5 on the
target surface. We therefore investigated the presence of C5 on
the embryonic surface, using a polyclonal antibody recognizing
both cleaved and non-cleaved forms of C5. Here we observed
a very strong staining on the surface of the ZP, but not on the
surface of the cleavage stage embryo blastomeres (Figure 2D).
Despite our RNA-seq data showing cellular expression of both
C3 and C5 (Figure 1), the antibodies used here did not detect any
intracellular signal.
Embryonic Defense Against Complement
Attack
Complement may target “foreign” as well as “self” surfaces.
Therefore, the presence of membrane-bound and soluble
regulators is essential for preventing damage to our own tissue
structures. After identifying specific complement activation on
the surface of the developing embryos, and successful cleavage
Frontiers in Immunology | www.frontiersin.org 4September 2019 | Volume 10 | Article 2234
Reichhardt et al. Complement in Human Embryos
FIGURE 2 | Complement targets developing embryos. Human cleavage stage in vitro fertilization (IVF) embryos were thawed in Vitrolife G-TL serum-free media. The
embryos were then incubated with specific anti-complement antibodies and analyzed by confocal microscopy. Analysis of embryonic binding of complement
activation products revealed binding of the classical pathway initiator C1q (A), and deposition of cascade activation components C3c/C3b/iC3b (B), and C3d (C).
Finally, activation of the terminal pathway is evidenced by deposition of C5 (D).(A) Left panel: Single plane, overlay of C1q (green) and DAPI (blue). Middle panels top
to bottom: DAPI, C1q, and BF. Right panels: Magnification of overlay (orange insert), and below the cross-sectional distribution of fluorescence intensity. This shows
C1q is specifically found on the cell surface. (B–D) Left panels: 3D rendering, overlay of protein stain (green), DAPI (blue), and F-actin (magenta). Right panels top to
bottom: DAPI, protein stain, and brightfield (BF). Scale bars: 50 µm, insert: 10 µm. For each staining, n=3 to 4 +1 to 3 (2PN +3PN embryos).
of C3b to C3d, we investigated the expression of the membrane-
associated inhibitors CD46, CD55, and CD59 (Figure 3 and
Supplementary Videos 1, 2).
The expression of the three membrane regulators showed
varying intensity, as expected from the gene expression data
(Figure 1). In the investigated cleavage stage embryos, we
observed strong staining for CD55 and CD59, but not for CD46.
Interestingly, CD55 and to a lesser degree CD59, displayed a
specific non-uniform localization. Both molecules are observed
on the blastomere membranes. However, a stronger signal is
observed specifically at the cellular junctions. While the majority
of signal for CD55 is seen at the cell-cell interfaces, CD59 is found
more abundantly dispersed throughout the entire cell surface.
This specific pattern of CD59 expression seems to be consistent
through all the early developmental stages (zygote to 8-cell stage,
see Supplementary Figure 2). As expected, no specific signal was
observed for any of these molecules in the ZP.
In addition to the presence of membrane bound regulators,
the ability to recruit soluble complement regulators is crucial for
protection of viable cells against autologous complement attack
(30). We therefore examined, whether embryos have bound the
fluid phase complement regulators C4bp and factor H (Figure 4).
C4bp and factor H are recruited to human surfaces
immediately following complement activation, i.e., after
deposition of C4b and C3b, respectively. Our data show a clear
deposition of both C4bp and factor H on the cell membranes of
the cleavage stage embryo blastomeres. In addition to the cellular
localization of factor H, a strong staining was also observed in the
ZP. This was not observed for C4bp. The binding of factor H but
not of C4bp to the surface of the ZP suggests that a major part
of complement activation on the ZP protein matrix (Figure 2) is
driven by alternative pathway activation, which does not involve
C4 cleavage. Therefore, only factor H, and not C4bp would be
recruited to this surface.
DISCUSSION
Complement is a very potent mediator of inflammation, and
untimely activation on self-surfaces contributes to a great
number of pathologies, such as atypical hemolytic uremic
syndrome, paroxysmal nocturnal hemoglobinuria, pregnancy
disorders and kidney diseases (13,31,32). The canonical
understanding of the complement system includes an ability of
the alternative pathway to auto-activate—this is what makes the
expression and recruitment of regulators essential for human
health. An understanding of the precise targeting of complement
activation in various physiological settings is therefore of
great importance. Animal models have been a great tool in
Frontiers in Immunology | www.frontiersin.org 5September 2019 | Volume 10 | Article 2234
Reichhardt et al. Complement in Human Embryos
FIGURE 3 | Embryonic expression of surface-tethered complement inhibitors. Human cleavage stage IVF embryos were thawed in Vitrolife G-TL serum-free media.
The embryos were incubated with anti-complement antibodies and analyzed by confocal microscopy. The analysis revealed a clear staining for both CD55 and CD59,
particularly at cellular junctions. In contrast, no positive signal was observed for CD46. (A) CD46 (B) CD55 (C) CD59. Left panels: Single plane, overlay of protein stain
(green) and DAPI (blue). Second column panels top to bottom: DAPI, protein, and BF. Third column panels: Magnification of overlay (orange insert), and below the
cross-sectional distribution of fluorescence intensity. Right panels (B,C): 3D rendering, overlay of protein stain and DAPI. Scale bars: 50 µm, insert: 10 µm. For each
staining, n=3+7 (2PN +3PN embryos).
understanding these processes. However, it is well-established
that variation exist between the human complement system
and that of other species, such as mice (33–35). The continued
investigation of human complement function is therefore crucial.
The current study is the first full analysis of the expression
and localization of complement activation molecules and their
regulators in human pre-implantation embryos (Figure 5). We
show that complement targets the embryonic surfaces and
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Reichhardt et al. Complement in Human Embryos
FIGURE 4 | Embryonically bound soluble complement regulators. Human
cleavage stage IVF embryos were thawed in Vitrolife G-TL serum-free media.
The embryos were incubated with anti-complement antibodies and analyzed
by confocal microscopy. Embryonic binding of the soluble complement
regulators C4bp and factor H are displayed. (A) C4bp is recruited to the
embryonic surface and show strong staining on the cell membrane. No
binding is observed to the ZP. (B) Factor H stains both the blastomere surface
as well as the ZP. Left panels: single planes, overlay of protein stain (green) and
DAPI (blue). Right panels top to bottom: DAPI (blue), protein stain (green), and
BF. Scale bars: 50 µm. For each staining, n=3+5 (2PN +3PN embryos).
observe deposition of complement activators, such as C1q, C3,
and C5. To balance this activation, we also show expression
of surface inhibitors (CD46, CD55, and CD59), as well as the
deposition of soluble complement regulators, such as C4bp and
factor H. This is in line with previous work showing the presence
of CD55 and CD59, and possibly CD46, on human embryos
(11,12). Interestingly, the pattern of CD55 and CD59 expression
at cellular junctions suggests that these molecules, in addition to
complement regulation, may be directly involved with cellular
interactions, such as signaling or adherence. While being GPI-
anchored to cell membrane rafts or caveolae CD55 and CD59
would be in a position to transmit robust activating signals
to cells.
Single-cell RNA sequencing revealed that mRNA from a
number of complement components are found at various stages
of early development, i.e., in oocytes, zygotes, as well as at
the 4-cell stage and 8-cell stage blastomeres. Our analysis
identified validated 5′UTR-proximal TFEs (a reliable indicator
for protein translation), or alternative splice-forms of transcripts
from the genes ADIPOR1, ADIPOR2, C1QBP, C1S, C2, C3,
C4BPB, C5, C5AR1, CALR, CD46, CD55, CD59, CFB, CFD,
CFI, CR2, ITGB2, and MASP2. The CD-transcripts representing
alternative splice-forms may have unknown functional relevance
at the embryonic stage. The role of these TFEs require further
validation. Comparing oocyte to the 4-cell stage, we observed
increased degradation of ADIPOR2, C1QBP, C5, and CD55, and
decreased degradation of C3, C5aR1, and MASP2 (measured by
the 3′/5′ratio). Though no statistically significant increase in
transcription was detected, a decreased degradation supports a
role in helping the embryo on its way to the uterus. In contrast,
increased degradation suggests a primary function at the oocyte
stage. In addition to the 5′UTR-proximal reads, we identified
partially degraded transcripts or potentially novel isoforms from
additional complement genes. These include genes encoding
important molecules such as ADIPOQ, C1q-C, C1R, C7, CDH13,
CR1, and CFP. While our method cannot distinguish between
explicit embryonic expression or earlier maternal expression
of these partially degraded mRNAs, the presence of these
gene-transcripts suggest expression during oogenesis. Proteins
translated during oogenesis may be important at this stage only,
but are also likely to remain in the early fertilized embryo.
Combined with the validated 5′-UTR and splice-form reads, we
thus demonstrate that human oocytes and pre-implantation stage
embryos produce a very wide range of complement molecules.
Immunological Targeting of Human
Embryos
By confocal microscopy we observed targeting of healthy human
embryos by complement. Auto-activation is the default of the
alternative pathway of complement activation, thus making the
expression and recruitment of regulators essential for human
health. While the potent inflammatory role of complement has
mainly been studied in the context of serum, it is well-established
that complement components are found in mucosal secretions,
e.g., from cervix, uterus, and fallopian tubes (22,38–40). The
presence of complement in the uterine compartment, alongside
the data presented here, indicates that human embryos are
targeted by complement in a physiological setting. This makes the
observed expression of surface inhibitors and the recruitment of
soluble regulators essential for the survival of the embryo already
prior to implantation. Numerous links between complement and
pregnancy complications have been described in the literature,
all related to implantation, placentation or later development (5–
7,9,13). Our data provide a novel, much earlier, mechanism,
whereby a faulty or insufficient complement regulation may
predispose to pregnancy disorders and miscarriage.
The expression of CD55 and CD59 throughout the
investigated period of embryogenesis, and CD46 at certain
stages, may be important for embryo survival. However,
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Reichhardt et al. Complement in Human Embryos
FIGURE 5 | Functional overview of the embryonic complement system. Indicated are the canonical functional roles of membrane-expressed complement regulators
(squares), soluble complement components (circles), and their cleaved activated membrane-deposited forms (demi-circles). Finally, embryonically expressed
complement receptors are shown (pentagons). All molecules depicted were found to be expressed or bound by the embryos in this study (exceptions: C4, mannose
binding lectin (MBL), ficolins (FCNs), and some MAC-components). The classical and the lectin pathways are initiated by target-binding of pattern recognition
molecules such as C1q, or MBL and FCNs, respectively. Utilizing their associated proteases C1r/s or MASPs, they activate C4 and C2, which subsequently activate
C3. Alternative pathway activation of C3 occurs when factor D cleaves C3-associated factor B, which generates a novel C3-cleaving enzyme; C3bBb. Cleavage of C3
by either pathway leads to generation of soluble C3a and surface-deposited C3b, which amplifies alternative pathway C3 activation, and subsequently activates C5 to
C5a and C5b. Finally, C5b initiates the assembly of the pore-forming MAC (1,36). To avoid excessive immunological targeting of self, human cells express or recruit
inhibitors of complement activation. Membrane regulators may function; (1) by disrupting the enzymes cleaving C3 and C5 (CR1, CD55), (2) as co-factors for factor
I-mediated degradation of C3b and C4b (CR1, CD46), or (3) by inhibiting MAC-formation directly (CD59). Soluble regulators such as factor H and C4bp inhibit
activation by mechanisms 1 and 2, while clusterin work through mechanism 3. Inactivation of C3b, leads to generation of iC3b, C3dg, and finally C3d. While C3b and
iC3b function as opsonins for increased phagocytosis by antigen presenting cells (through CR3 and CR4), C3d has important biological functions as an important
internal adjuvant aiding antigen uptake by dendritic cells and inducing efficient antibody responses in B cells (through CR2) (1,36,37). While CR2, CR3 and CR4
expression is mainly described on immune cells, our study found embryonic expression of these receptors, along with the signaling receptors for C1q; calreticulin
(CALR) and C1qbp.
attempts of stem-cell transplantation show the importance of
soluble regulators for cell survival as well (30). Our data reveal
presence of clusterin and factor I mRNA, and deposition of
C4bp and factor H on the cleavage stage embryos. The inhibitory
effects of these molecules were substantiated by our staining for
C3-degradation products. Importantly, only the non-degraded
C3b will lead to continuation of the complement cascade and
formation of the C5 activating convertase. No staining was
observed for C5 on the blastomere membranes, thus showing
that the kinetics of the complement regulation favor degradation
of C3b deposited on the cell surfaces. However, on the ZP, the
lack of membrane regulators may favor a different outcome. The
presence of factor H, but not of C4bp, on the surface of the ZP
suggests that the majority of complement activation against the
ZP protein matrix is a result of alternative pathway activation,
which does not involve C4 cleavage. A strong C5 deposition
was observed on the ZP, indicating that this layer absorbs the
most intense complement attack. Though our staining for C3d
revealed that a lot of the C3b deposited has become degraded,
the kinetics of C3b-degradation vs. C5-convertase formation
has still favored C5 activation. Given the stronger deposition
of complement activation products, a function of the ZP may
be to divert complement from the cell membranes, and act as a
protective layer for the developing embryo also in this respect.
The role of factor H as the main inhibitor of complement
activation against the ZP, highlights the potentially detrimental
impact of factor H deficiencies on reproduction. Still, while we
here observe potentially harmful pro-inflammatory stimuli in
response the pre-implantation embryo, the impact on pregnancy
outcome may be less clear. Local inflammatory processes
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Reichhardt et al. Complement in Human Embryos
are crucial for decidualization of the endometrium following
implantation and in establishing immune tolerance toward the
developing conceptus (41). While immunological opsonization
and clearance of the conceptus would be catastrophic, the
activation of complement may therefore also contribute to
priming the immunological landscape following implantation.
Cellular Complement Activation and
Signaling
From the perspective of the developing embryo, the expression
of complement regulators and binding of inhibitors is useful
for protection against the clearance function of complement.
However, our data show that complement activation components
are also expressed. This suggests other functional roles of
complement, e.g., in cellular signaling or in metabolism as
has been suggested for a wide range of cells, including stem
cells (2,27,42). With the local expression of C1s, C2, C3,
C5, factor B, and factor D, the oocyte and potentially later
embryonic cells themselves produce the molecules necessary
for initiating complement. Activation of cell-derived C5 and
subsequent autocrine binding to its receptors C5aR1 and C5aR2,
has been shown to initiate a number of cellular signaling events
(2,42). As we also identify expression of C5aR1, it is possible that
complement is utilized for cellular signaling events, as has been
suggested for human stem cells (14,43,44). It has previously been
shown in the human oviductal epithelium, that the combined
expression of molecules such as factor B and factor I, together
with C3 is enough to produce an active C3-convertase and
generate C3-cleavage products such as iC3b (45). The study by
Tse et al. revealed an embryotrophic effect of iC3b on mouse
embryos. Our data show that expression and/or deposition of
the molecular machinery mediating the embryotrophic effects in
mice, also exists in humans.
C1q and Tissue-Remodeling
While the gene expression data suggest that C1q and C1r may
be expressed in oocytes, they appear to be degraded later.
However, the staining data show a clear binding of C1q to
the blastomere membranes. The origin of C1q at this stage
may thus either represent remnants from ovarian complement
deposition, or protein produced by the oocyte surviving the
half-life of its corresponding mRNA. The strongly expressed
C1qbp may function as an essential regulator of complement
activation at this stage (46). Mouse studies have indicated C1q
as an important signaling molecule in stem cell differentiation,
and later during development as a crucial molecule for tissue
organization (24,47). Furthermore, human studies have related
altered C1q-mediated clearance of debris and apoptotic material
from the placenta in pre-eclampsia (9). Calreticulin, of which
we found mRNA transcripts at all investigated stages, is thought
to act as a receptor for C1q and collectins to mediate clearance
of apoptotic materials. The deposition of C1q observed in the
current study and the high levels of mRNA from complement-
modulatory proteins in the early embryo suggest that C1q may
have similar functions already at this developmental stage.
In conclusion, we here provide evidence for complement
targeting of early human embryos, along with a substantial
expression and/or recruitment of complement inhibitors. These
findings suggest that a lack of appropriate inhibition of the
activation cascades, and the generation of C3 and C5 cleavage
products on the pre-implantation embryonic tissue may be
detrimental to the developing embryo and the inflammatory state
of the maternal endometrium. However, the functional relevance
of complement-deposition on pre-implantation embryos is very
likely to extend beyond mis-directed immunological clearance.
Our finding of early cellular expression of complement-activating
molecules supports emerging roles of complement in basic
cellular processes, such as metabolism and differentiation. This is
the first study to identify the extent of complement involvement
in the pre-implantation developmental stage in a bona fide
human model. The data presented here thus highlight the
importance of further studies into the role of complement,
both in relation to fertility and pregnancy complications, as
well as in relation to basic cellular processes during early
human development.
MATERIALS AND METHODS
Ethical Considerations
Oocytes and embryos utilized for the single-cell RNA sequencing
were collected in Switzerland and Sweden. All analyses were
performed in Sweden. The full protocol was approved by the
ethical committees in Switzerland (authorization CE2161 of the
Ticino ethical committee, Switzerland) and in Sweden (Dnr
2010/937–31/4 of the Regional Ethics Board in Stockholm).
Embryos used for confocal imaging were donated by patients
at the Helsinki Women’s clinic Fertility unit, Finland, after
informed consent. The study was approved by the local ethics
committee (124/13/03/03/2015 and DNr 308/13/03/03/2015).
All cells and embryos were generated for the sole purpose
of IVF. Following standard procedures embryos generated for
IVF treatment, but not immediately transferred to the uterus,
were cryopreserved. Upon termination of the freezing contract,
embryos were either discarded or donated for research purposes.
Abnormally fertilized triploid (3PN) embryos were donated at
fresh cycles and used either fresh or after vitrification and
warming at later time point.
Reanalysis of Single-Cell RNA Sequencing
Data on Human Oocytes and Blastomeres
in the Pre-implantation Embryos
Initial transcriptomics data were generated earlier (26). Embryos
utilized for single cell-RNA sequencing were collected and
cultured as described. Individual blastomeres were obtained by
laser-assisted biopsy, the ZP was removed and cells were placed
in lysis buffer in individual wells of a 96-well plate. For single-
cell RNA sequencing STRT was applied (48). Three individual
libraries were prepared from the total number of single cells;
oocytes: 19 cells, zygotes: 29 cells, 4-cell stage blastomeres: 30
cells and 8-cell stage blastomeres: 21 cells, as described (26).
Expression levels were correlated to eight synthetic spike-in
RNAs (ArrayControl RNA spikes Ambion, cat. no. AM1780)
(49). Following amplification, the synthesized cDNA was
Frontiers in Immunology | www.frontiersin.org 9September 2019 | Volume 10 | Article 2234
Reichhardt et al. Complement in Human Embryos
sequenced on the Illumina platform, filtered, demultiplexed and
trimmed as described (26). Estimation of the ratio of transcripts
per cell was done by comparing total reads to total spike-in RNA
associated reads (all with sample-specific barcodes). Following
pre-processing, the reads were aligned to human UCSC genome
hg19, ArrayControl RNA spikes and human ribosomal DNA
complete repeat unit (GenBank: U13369) by TopHat version
2.0.6 (45) and annotated by genomic features. The aligned STRT
reads were assembled by sample types using Cufflinks (45) and
counted as TFEs, as described (26).
Complement Gene-Expression Analysis
A list of genes relevant to complement function was generated
based on an exhaustive analysis of functional studies in the
field (1,2,27). The method of TFE-based quantification
is implemented as open-source software (https://github.com/
shka/STRTprep). This method was used to identify expressed
complement genes. In brief, the TFEs were defined by STRT
RNAseq reads, which correspond to the 5′-end of polyA-tailed
RNAs. Therefore, TFEs not at the 5′-UTR or the proximal
upstream were excluded from the investigation of known
protein-coding genes, as mRNAs suggested by these TFEs were
less likely to have a methionine for translation of functional
protein. Furthermore, TFE-based quantitation provides an
advantage over normal sequencing approaches particularly in
studies of pre-implantation development, where the gene-based
quantitation methods also sum promiscuous (zygotic) RNAs and
degraded (maternal) RNAs, which are less likely to translate
into proteins (29). Therefore, the tagging of RNAseq reads to
the 5′UTR was a positive selection criterion for expression. The
integrity of the mRNA reads not directly tagged to the 5′-UTR in
human GRCh37/hg19 build was assessed by the Zenbu Genome
browser, the UCSC Genome Browser and the GENSCAN online
tools (50,51). This led to the inclusion of a number of reads
tagged to the CDs of coding transcripts. Specifically, sequences
tagged a few codons downstream of the 5′end were included.
Also, sequences corresponding to alternative splicing events and
sequences that aligned with expressed spliced human sequence
tag reads within the target gene were included in the analysis.
Statistical Analysis for Differential
Expression
The R package pvclust (52) was applied to exclude outlier
samples. Subsequently, differential expression levels were tested
by SAMstrt (53), a version of SAMseq modified for spike-in-
based normalization.
Collection and Culturing of Cleavage-Stage
Human Embryos for Confocal Imaging
Cleavage stage embryos utilized for confocal microscopy were
cultured to 4- or 8-cell stage embryos in a sequential culture
system (G-IVF/G-1PLUS, Vitrolife) at 37 ◦C and 5% CO2,
5% O2. Embryos were frozen and thawed using Vitrolife
FreezeKit Cleave and ThawKit Cleave, respectively (Vitrolife
Sweden AB, SE-421 32 Västra Frölunda, Sweden). After thawing,
embryos were cultured in Vitrolife G-TL medium until processed
for immunostaining.
Confocal Microscopy
Embryos were fixed with 4% paraformaldehyde in phosphate-
buffered saline (PBS) solution for 15 min at room temperature,
washed with PBS and permeabilized using 0.5% Triton R
X-100
(Fisher Scientific, Geel, Belgium) in PBS for 15 min, and blocked
for unspecific staining using Ultra Vision Protein Block (Thermo
Scientific, MI) for 8–10 min, all at room temperature. Primary
antibodies, mouse anti-CD46 (GeneTex Inc., Irvine, California,
US; 1:100), mouse anti-CD55 (IBGRL Research Products, Bristol,
UK; 1:100), mouse anti-CD59 (IBGRL Research Products,
Bristol, UK; 1:100), goat anti-factor H (Calbiochem, La
Jolla, California; 1:100), mouse anti-C4bp (Quidel, San Diego,
California; 1:100), rabbit anti-C1q (DAKO Denmark A/S,
Glostrup, Denmark; 1:100), goat anti-C5 (Cappel, Organon
Teknika Corp., West Chester, Pennsylvania; 1:300), rabbit anti-
C3d (DAKO Denmark A/S, Glostrup, Denmark; 1:100), rabbit
anti-C3c (DAKO Denmark A/S, Glostrup, Denmark; 1:100)
were diluted in PBS +0.1% Tween20 (Fisher Scientific, Geel,
Belgium) and incubated over night at +4◦C. After three washes
in PBS +0.1% Tween20, embryos were incubated 2 h at room
temperature while rocking in secondary antibody donkey anti-
mouse AlexaFluor R
594, donkey anti-goat AlexaFluor R
488, or
donkey anti-rabbit AlexaFluor R
488 (all from Thermo Fisher
Scientific; 1:500) diluted in PBS +0.1% Tween20. F-actin
was stained with AlexaFluor R
647 Phalloidin (Thermo Fisher
Scientific; 1:100) and nuclei were stained with DAPI (Thermo
Fisher Scientific; 1:500). Images of the embryos were acquired
using an inverted TCS SP8 MP CARS confocal microscope (Leica
Microsystems, Mannheim, Germany) and Leica HC PL APO
CS2 40x/1.10NA water and Leica HC PL APO CS2 63x/1.20NA
water objectives.
Confocal Image Processing
Confocal images were processed using Fiji (http://fiji.sc) and
Imaris (BitPlane, Oxford Instruments). Depending on the
dataset, preprocessing consisted of applying a sliding-window
averaging in the z dimension, denoising with Rolling Ball and
smoothing by applying either a Gaussian filter or a 3D-Median
filter (kernel 1). When necessary, DAPI signal was isolated from
the background by applying a binary mask (obtained by local
maxima functions) to the raw image to specifically render nuclei
only. Fluorescence intensity profiles were obtained by averaging
lines cropped in the inset (to smooth the noise) and are plotted as
normalized to the maximum peak. 3D renderings were obtained
using Imaris.
DATA AVAILABILITY
The raw data supporting the conclusions of this manuscript will
be made available by the authors, without undue reservation, to
any qualified researcher.
ETHICS STATEMENT
The studies involving human participants were reviewed
and approved by Oocytes and embryos utilized for the
single-cell RNA sequencing were collected in Switzerland
Frontiers in Immunology | www.frontiersin.org 10 September 2019 | Volume 10 | Article 2234
Reichhardt et al. Complement in Human Embryos
and Sweden. All analyses were performed in Sweden. The
full protocol was approved by the ethical committees in
Switzerland (authorization CE2161 of the Ticino ethical
committee, Switzerland) and in Sweden (Dnr 2010/937-31/4
of the Regional Ethics Board in Stockholm). Embryos used
for confocal imaging were donated by patients at the Helsinki
Women’s clinic Fertility unit, Finland, after informed consent.
The study was approved by the local ethics committee
(124/13/03/03/2015 and DNr 308/13/03/03/2015). All cells
and embryos were generated for the sole purpose of IVF.
Following standard procedures embryos generated for IVF
treatment, but not immediately transferred to the uterus, were
cryopreserved. Upon termination of the freezing contract,
embryos were either discarded or donated for research purposes.
Abnormally fertilized triploid (3PN) embryos were donated
at fresh cycles and used either fresh or after vitrification
and warming at later time point. The patients/participants
provided their written informed consent to participate in
this study.
AUTHOR CONTRIBUTIONS
MR, JT, JK, SM, and TT designed the research. MR, KL, and SK
performed research. MR, KL, AL, GR, and SV analyzed data. MR,
KL, AL, and SM wrote the paper.
FUNDING
Funding for the project was provided by the Finnish Cultural
Foundation, the Jenny and Antti Wihuri foundation, the
Academy of Finland, Helsinki University Hospital Funds,
Foundation ARC for cancer research (grant #20171206504),
Knut and Alice Wallenberg Foundation, Swedish Research
Council, Sigrid Jusélius Foundation, and Jane and Aatos Erkko
Foundation. GR was a member of the CNRS ImaBio GdR.
ACKNOWLEDGMENTS
We are grateful to the donors of cells enabling this study.
The computations were performed on resources provided by
SNIC through Uppsala Multidisciplinary Center for Advanced
Computational Science (UPPMAX) under Project b2010037.
Confocal microscopy was carried out at the Bioimaging Unit,
University of Helsinki, Finland.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fimmu.
2019.02234/full#supplementary-material
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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2019 Reichhardt, Lundin, Lokki, Recher, Vuoristo, Katayama,
Tapanainen, Kere, Meri and Tuuri. This is an open-access article distributed
under the terms of the Creative Commons Attribution License (CC BY). The use,
distribution or reproduction in other forums is permitted, provided the original
author(s) and the copyright owner(s) are credited and that the original publication
in this journal is cited, in accordance with accepted academic practice. No use,
distribution or reproduction is permitted which does not comply with these terms.
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