Human Reproduction Update, Vol.21, No.3 pp. 285–296, 2015
Advanced Access publication on January 21, 2015 doi:10.1093/humupd/dmv001
Artificial gametes: a systematic review
of biological progress towards clinical
application
Saskia Hendriks 1, Eline A.F. Dancet 1,2, Ans M.M. van Pelt 1,
Geert Hamer 1, and Sjoerd Repping 1,*
1
Center for Reproductive Medicine, Women’s and Children’s Hospital, Academic Medical Center, University of Amsterdam, Amsterdam,
The Netherlands 2Leuven University Fertility Centre, Leuven University Hospital, Leuven, Belgium
*Correspondence address. Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail: [email protected]
Submitted on October 29, 2013; resubmitted on December 12, 2014; accepted on December 29, 2014
table of contents
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Introduction
Methods
Search strategy
Article selection
Meta-synthesis
Results
Search strategy
Meta-synthesis
Artificial sperm from a male
Artificial oocytes from a female
Artificial oocytes from a male
Artificial sperm from a female
Discussion
Conclusion
background: Recent progress in the formation of artificial gametes, i.e. gametes generated by manipulation of their progenitors or of
somatic cells, has led to scientific and societal discussion about their use in medically assisted reproduction (MAR). Artificial gametes could
potentially help infertile men and women but also post-menopausal women and gay couples conceive genetically related children. This systematic
review aimed to provide insight in the progress of biological research towards clinical application of artificial gametes.
methods: The electronic database ‘Medline/Pubmed’ was systematically searched with medical subject heading (MesH) terms, and reference
lists of eligible studies were hand searched. Studies in English between January 1970 and December 2013 were selected based on meeting a priori
defined starting- and end-points of gamete development, including gamete formation, fertilization and the birth of offspring. For each biologically
plausible method to form artificial gametes, data were extracted on the potential to generate artificial gametes that might be used to achieve
fertilization and to result in the birth of offspring in animals and humans.
results: The systematic search yielded 2424 articles, and 70 studies were included after screening. In animals, artificial sperm and artificial
oocytes generated from germline stem cells (GSCs), embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have resulted in
the birth of viable offspring. Also in animals, artificial sperm and artificial oocytes have been generated from somatic cells directly, i.e. without
documentation of intermediate stages of stem- or germ cell development or (epi)genetic status. Finally, although the subsequent embryos
showed hampered development, haploidization by transplantation of a somatic cell nucleus into an enucleated donor oocyte has led to fertilized
artificial oocytes. In humans, artificial sperm has been generated from ESCs and iPSCs. Artificial human oocytes have been generated from GSCs,
ESCs and somatic cells (without documentation of intermediate stages of stem- or germ cell development). Fertilization of a human artificial
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Hendriks et al.
oocyte after haploidization by transplantation of a somatic cell nucleus into an enucleated donor oocyte was also reported. Normal developmental
potential, epigenetic and genetic stability and birth of children has not been reported following the use of human artificial gametes. In animals,
artificial oocytes from a male have been created and fertilized and artificial sperm from a female has been fertilized and has resulted in the
birth of viable offspring. In humans, artificial sperm has been generated from female iPSCs. To date, no study has reported the birth of human
offspring from artificial gametes.
conclusion: Our systematic review of the literature indicated that in animals live births have already been achieved using artificial gametes of
varying (cell type) sources. Although experimental biological research is progressing steadily towards future clinical application, data on functionality, safety and efficiency of (human) artificial gametes are still preliminary. Although defining artificial gametes by start- and end-points limited the
number of included studies, the search resulted in a clear overview of the subject. Clinical use of artificial gametes would expand the treatment
possibilities of MAR and would have implications for society. Before potential clinical use, the societal and ethical implications of artificial gametes
should be reflected on.
Key words: artificial gametes / gametogenesis / assisted reproductive technologies
Introduction
Estimates of the 12-month prevalence rate of infertility range from 3 to
17% in more developed nations, where more than half of the couples
confronted with infertility seek medical help (Boivin et al., 2007). Reproductive medicine is currently able to help only 70% of the couples to
deliver a child within 5 years after visiting a fertility clinic while relying
on either patients’ own or donor gametes (Pinborg et al., 2008).
Donor gametes are used in the case of failed homologue treatment
and are the only current treatment option for patients without functional
gametes, which includes men with non-obstructive azoospermia (0.63%
of the general population; Jarow et al., 1989) and women with premature
ovarian insufficiency (0.9% of the general population; Coulam et al.,
1986). Moreover, gay couples are relying on donor gametes. Unfortunately, the current use of donor gametes does not lead to genetic parenthood, which is valued by most couples, although its objective importance
is currently debated (Golomblok et al., 2006; Mertes and Pennings,
2008).
A potential future treatment alternative to using donor gametes is the
use of so-called ‘artificial gametes’, i.e. gametes generated by manipulation of their progenitors or of somatic cells, which would allow children
who are genetically related to both their parents to be conceived. The
possibility of full genetic parenthood could also apply to gay couples, if
reprogramming to the other sex germ line becomes an option, and to
women of post-menopausal age.
Although the potential use of artificial gametes in medically assisted reproduction (MAR) has led to scientific and societal discussion, a systematic overview of the biological progress towards clinical application is
lacking. This systematic review aimed to provide insight into the biological
progress on all biological plausible routes to create artificial gametes in
animals and humans, without evaluating quality, quantity and safety of
the different approaches, which are currently unknown.
Methods
Search strategy
The electronic database MEDLINE was systematically searched with the
search engine PubMed using the following medical subject heading terms
(MeSH-terms): ‘cell differentiation’, ‘cells, cultured’, ‘cell culture techniques’,
‘cytoplasm/transplantation’, ‘diploidy’, ‘DNA, mitochondrial’, ‘embryonic
stem cells’, ‘embryo transfer’, ‘female’, ‘fertilization’, ‘genotype’, ‘germ cells’,
‘germ cells/cytology’, ‘haploidy’, ‘human reproduction, assisted’, ‘meiosis’,
‘microinjections’, ‘micromanipulaton’, ‘nuclear transfer techniques’, ‘oocytes’,
‘oocytes/cytology’, ‘oocytes/physiology’, ‘oocytes/transplantation’, ‘oogenesis’, ‘oogonia/cytology’, ‘pluripotent stem cells’, ‘pregnancy’, ‘pregnancy
outcome’, ‘reproductive techniques, assisted/ethics’, ‘reproductive techniques/ethics’, ‘spermatogenesis’, ‘spermatogonia/cytology’, ‘spermatozoa/cytology’, ‘stem cells/cytology’ and ‘stem cells/metabolism’. The
reference lists of the eligible articles were subsequently hand searched (i.e.
snowball strategy).
Article selection
Articles reporting on artificial gametes, published in English between January
1970 and December 2013, were considered for inclusion by screening their
titles, abstracts and if necessary full-text reports.
Only studies reporting on originally collected biological data were considered, while reviews, opinion studies and other non-original work were
excluded. Furthermore, studies needed to report on specific biological starting and end-points, which were defined a priori.
As starting points, i.e. cells that in a clinical application would be derived
from the patient, adult GSCs and differentiated somatic cells were defined.
Adult GSCs with the reported capacity of migration to the stem cell niche in
vivo after (xeno)transplantation (male GSCs) or proof of survival in vivo after
(xeno)transplantation (female GSCs) were included as starting points. For
the purpose of this review, we exclude the use of primordial germ cells,
which, although they too are GSCs, cannot be retrieved from an adult
commissioning parent and are thus not clinically relevant as starting point
of treatment.
Differentiated somatic cells could be a starting point for artificial gamete
formation in three ways.
First, artificial gametes could be formed from differentiated somatic cells
via embryonic stem cells (ESCs). For clinical application, this would require
somatic cell nuclear transfer of a patient nucleus into a donor ESC (Tachibana
et al., 2013a, b); in this way, the ESCs contain the commissioning parent’s
genetic material. For the purpose of this review, however, ESCs as starting
point of preclinical research were accepted for inclusion.
Second, artificial gametes could be formed from differentiated somatic
cells via induced pluripotent stem cells (iPSCs). Articles that have iPSCs as
starting point or use somatic cell reprogramming by ectopic expression
were also included.
Third, artificial gametes could be formed from differentiated somatic cells
by ‘direct’ differentiation into gametes without the occurrence or documentation of our defined intermediate stages of stem- and germ cell development
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Artificial gametes: progress to clinical application
[i.e. ESCs, iPSCs and germline stem cells (GSC)]; in this review referred to as
‘without documented intermediate stages’.
The following end-points were defined: artificial gamete formation, fertilization and birth of offspring.
For males, the formation of artificial gametes was defined as: the presence
of haploid male sperm cells, as proven by DNA content analyses or similar
techniques and/or the presence of post-meiotic markers. This does not
include sperm morphology, motility and fertilization potential without the
use of ICSI. For females, artificial gamete formation was defined as: the presence of metaphase II secondary oocytes proven by DNA content analyses,
meiotic markers and/or polar body extrusion. This does not include epigenetic normality and developmental potential, for example, to, or past, the
blastocyst stage. These conditions were chosen for the review as they describe gametes which could be used for MAR, yet they are not necessarily
genetically and epigenetically equal to normal gametes with respect to
quality and developmental potential. They will therefore all be referred to
as artificial sperm or artificial oocytes.
Fertilization was defined as the presence of a zygote with two pronuclei
or a cleavage-stage embryo in which donor-derived DNA is detected.
Birth of offspring was defined as the birth of one or more viable offspring,
in which donor-derived DNA is detected.
Therefore, for example, studies that reported on spermatogenesis until
the stage of the first meiotic division were excluded, as they did not achieve
one of the three predefined end-points.
donor oocyte (Route 9), in which the somatic cell nucleus of the commissioning mother is injected into an enucleated (donor) oocyte, which by division
induces haploidization of the somatic nucleus.
Third, for each potential route of artificial gamete generation, clinical applications are specified, as shown in Table I. For the treatment of heterosexual
couples (or infertile singles relying on material from a healthy donor instead of
a partner), eight routes are biologically plausible to result in artificial sperm
from a male, whereas nine routes are biologically plausible to result in artificial
oocytes from a female. The routes for which GSCs are the starting point obviously cannot be used for patients whose gonads do not contain GSCs.
For the treatment of gay couples, five routes might theoretically result in
artificial oocytes from males, and six routes may result in artificial sperm
from females.
Data were extracted for each of the nine plausible routes of artificial gamete
formation based on the achievement of the farthest of the following end-points:
artificial gamete formation, fertilization and birth of offspring. This review
reports only on the farthest end-point reached, because of clinical relevance
and because not all studies documented the achievement of intermediate endpoints (e.g. a study reporting birth of offspring might not have investigated or
documented fertilization). Achievements from animal and human research
were differentiated and the type of animals examined was specified.
Results
Search strategy
Meta-synthesis
Data were extracted using standardized data extraction sheets. Given the
nature of the collected data, meta-synthesis, rather than meta-analyses
was performed. The meta-synthesis was performed by two reviewers, independently, who discussed until consensus was met. In this way, the chance of
a human error in classification or interpretation was reduced.
To structure the abundance of biological data, three strategies were used.
First, data were organized by the sex of the animal or commissioning parent
whose genetic material was used (male or female) and by the type of artificial
gamete that was formed (sperm or oocyte). More specifically, besides artificial generation of sperm from a male and of oocytes from a female, theoretically, it could be possible to create artificial sperm from a female and artificial
oocytes from a male. Clinically, the latter could be relevant for gay couples
wishing to have a child that is genetically related to both of them, instead of
having to rely on donor gametes.
Second, nine biologically plausible routes of artificial gamete generation
were defined based on insight in the literature and clinically available cell
types, as described in Figs 1 and 2. Of the nine routes, two routes (Routes
1 and 2; the nature of Route 2 is exemplified in Figs 1 and 2) start from
adult GSCs of the animal or commissioning parent whose genetic material
will be used. Adult GSCs are spermatogonial stem cells (SSCs) or oogonial
stem cells (OSCs), which would have to be retrieved from, respectively,
the testis or ovary of the animal or commissioning parent whose genetic material will be used. The other seven routes (Routes 3 – 9) start from a somatic
cell of the animal or commissioning parent.
Four routes starting from a somatic cell (Routes 3– 6) require either ESCs
(Routes 3 and 4) or iPSCs (Routes 5 and 6; the nature of Route 5 is exemplified in Figs 1 and 2) as transitional cell types. To generate transitional ESCs, the
nucleus of an ESC of a donor embryo has to be replaced by the nucleus of a
somatic cell from the animal or commissioning parent (Tachibana et al.,
2013a, b), while iPSCs result directly from reprogramming somatic cells
from the animal or commissioning parent (Takahashi et al., 2007a, b).
In the remaining three routes starting from somatic cells, artificial gametes
are formed without a documented intermediate stage: somatic cell transformation into gametes without transitional cell types (Routes 7 and 8) and
haploidization by transplantation of a somatic cell nucleus into an enucleated
The systematic search yielded 2424 articles (Fig. 3). Based on eligibility,
46 studies were included. Hand searches of the reference lists of these
studies, resulted in the inclusion of 24 additional studies. Thus, in total,
70 studies were included.
Meta-synthesis
Table I reports on the most advanced end-points achieved in animals
and humans of all nine biologically plausible routes for the formation of
artificial sperm.
Artificial sperm from a male
Of the eight biologically plausible routes leading to the formation of artificial sperm from a male (Fig. 1), seven were reported in literature as
achieving at least one of the specified end-points in animals and/or
humans (Table I). In animal research, for one route (Route 5), the formation of artificial sperm was achieved as the most advanced end-point,
while for six routes (Routes 1, 2, 3, 4, 6 and 8), one or more studies
reported the birth of viable offspring. In humans, only two of the biologically plausible routes to artificial gamete formation (Routes 3 and 5) actually led to the generation of artificial sperm. In humans, no studies
reported on fertilization or the birth of offspring.
In vitro differentiation of SSCs
In vitro culture and differentiation of SSCs resulted in artificial sperm in
cattle (Izadyar et al., 2002). Birth of offspring was achieved in mice (Sato
et al., 2011).
In vitro proliferation of SSCs followed by (auto)transplantation
In vivo haploidization after in vitro proliferation of SSCs resulted in the
formation of artificial sperm in mice (Nagano et al., 2000; Lee et al.,
2009) and rats (Hamra et al., 2005). Fertilization was reported in hamsters (Kanatsu-Shinohara et al., 2008). Birth of offspring was reported
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Hendriks et al.
Figure 1 Roadmap to the eight biologically plausible routes of artificial sperm formation in men. (1) In vitro differentiation of GSCs. (2) In vitro proliferation
of GSCs followed by autotransplantation. (3) In vitro differentiation of ESCs. (4) In vitro differentiation of ESCs followed by autotransplantation. (5) In vitro
differentiation of iPSCs. (6) In vitro differentiation of iPSCs followed by autotransplantation. (7) In vitro somatic cell transformation into gametes without
documented transitional cell types. (8) In vivo somatic cell transformation into gametes without documented transitional cell types.
in mice (Kanatsu-Shinohara et al., 2003, 2005a, b, 2006, 2010, 2011;
Takahashi and Yamanaks, 2006; Kubota et al., 2004, 2009; Kita et al.,
2007; Nagano et al., 2001; Ohta et al., 2009; Shiura et al., 2013), rats
(Hamra et al., 2002; Ryu et al., 2005; Wu et al., 2009) and zebrafish
(Kawasaki et al., 2012).
In vitro differentiation of ESCs followed by autotransplantation
In mouse studies, in vitro differentiation of ESCs followed by autotransplantation resulted in the formation of artificial sperm (Toyooka et al.,
2003) and the birth of offspring (Chuma et al., 2004; Ohinata et al.,
2009; Hayashi et al., 2011; Nakaki et al., 2013).
In vitro differentiation of ESCs
In mice, this route resulted in fertilization (Geijsen et al., 2003; Kerkis
et al., 2007; Yu et al., 2009). In one report, the birth of offspring was
reported (Nayernia et al., 2006). Human artificial sperm, expressing
post-meiotic markers and/or haploidization attested by RNA expression analyses and DNA content analysis, respectively, has been generated from ESCs (Aflatoonian et al., 2009; Kee et al., 2009; Panula et al.,
2011; West et al., 2011; Easley et al., 2012).
In vitro differentiation of iPSCs
Artificial sperm cells were created by in vitro iPSC differentiation in mice
(Yang et al., 2012). Human artificial sperm was also created by in vitro
differentiation of iPSCs (Eguizabal et al., 2011; Panula et al., 2011;
Easley et al., 2012), some of which revealed incomplete imprinting
(Eguizabal et al., 2011).
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Artificial gametes: progress to clinical application
Figure 2 Roadmap to the nine biologically plausible routes of artificial oocyte formation in women. (1) In vitro differentiation of GSCs. (2) In vitro proliferation of GSCs followed by autotransplantation. (3) In vitro differentiation of ESCs. (4) In vitro differentiation of ESCs followed by autotransplantation.
(5) In vitro differentiation of iPSCs. (6) In vitro differentiation of iPSCs followed by autotransplantation. (7) In vitro somatic cell transformation into
gametes without documented transitional cell types. (8) In vivo somatic cell transformation into gametes without documented transitional cell types. (9)
Haploidization by transplantation of a somatic cell nucleus into an enucleated donor oocyte (DO).
In vitro differentiation of iPSCs followed by autotransplantation
Differentiating iPSCs in vitro followed by autotransplantation resulted
in the formation of artificial sperm in mice (Zhu et al., 2012). Mouse
offspring were born from iPSCs that were differentiated into primordial
germ cell-like cells (PGCLCs) in vitro and subsequently formed artificial
sperm in vivo after transplantation into the seminiferous tubules
(Hayashi et al., 2011).
In vitro somatic cell transformation into sperm without documented
intermediate cell types
No studies achieving artificial sperm formation, fertilization or birth of
offspring via this route were identified.
In vivo somatic cell transformation into sperm without documented
intermediate cell types
Injection of mesenchymal stem cells into rats, previously sterilized
by busulfan treatment, led to fertility and live births (Cakici et al., 2013).
Artificial oocytes from a female
Of the nine biologically plausible routes to artificial oocyte formation
from a female (Fig. 2), seven achieved at least one of the specified endpoints in animals and/or humans (Table I). In animal research, as the
most advanced end-point, formation of animal artificial oocytes was
achieved via three routes (Routes 1, 7 and 8), fertilization via one
route (Route 9) and the birth of offspring was achieved via three
290
Hendriks et al.
Table I Studies demonstrating possible routes to create artificial gametes.
Route creating artificial gamete
Most advanced outcomes reacheda
...............................................................................................................................
Animal model
......................................................................
Gamete
Fertilization
Offspring
Human
......................................................
Gamete
Fertilization
Offspring
.............................................................................................................................................................................................
Artificial sperm from male
(1) In vitro differentiation of germline stem cells
(GSCs)
1
—
2
—
—
—
(2) In vitro proliferation of GSCs followed by
autotransplantation
3, 4, 5
6
7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19,
20, 21, 22, 23
—
—
—
(3) In vitro differentiation of embryonic stem
cells (ESCs)
—
24, 25, 26
27
28, 29, 30,
31, 32
—
—
(4) In vitro differentiation of ESCs followed by
autotransplantation
33
—
34, 35, 36, 37
(5) In vitro differentiation of induced pluripotent
stem cell (iPSCs)
38
—
—
29, 39, 31
—
—
(6) In vitro differentiation of iPSCs followed by
autotransplantation
40
—
35
—
—
—
(7) In vitro somatic cell transformation into
sperm without documented transitional cell
types
—
—
—
—
—
—
(8) In vivo somatic cell transformation into
sperm without documented transitional cell
types
—
—
41
—
—
—
(1) In vitro differentiation of GSCs
42
—
—
42
—
—
(2) In vitro proliferation of GSCs followed by
autotransplantation
—
42
43
42
—
—
(3) In vitro differentiation of ESCs
26, 44, 45, 46
—
47, 48
28
—
—
(4) In vitro differentiation of ESCs followed by
autotransplantation
—
—
—
—
—
—
(5) In vitro differentiation of iPSCs
—
—
47, 48
—
—
—
(6) In vitro differentiation of iPSCs followed by
autotransplantation
—
—
—
—
—
—
(7) In vitro somatic cell transformation into
oocytes without documented transitional cell
types
49, 50
—
—
51, 52
—
—
(8) In vivo somatic cell transformation into
oocytes without documented transitional cell
types
53, 54
—
—
—
—
—
(9) Haploidization by transplantation
of a somatic cell nucleus into an
enucleated donor oocyte
55, 56, 57, 58,
59, 60, 61
62
—
58, 63
59, 64, 65
—
Artificial oocytes from a male
44
24
—
—
—
—
Artificial sperm from a female
—
66, 67
68, 69, 70
39
—
—
—
Artificial oocyte from female
ESC, embryonic stem cell; iPSC, induced pluripotent stem cell; GSC, germline stem cell; —, refers to no publication reporting on the respective outcome as a furthest end-point.
a
Numbers (Supplementary data) indicate the appropriate reference with the respective outcome as furthest end-point.
routes (Routes 2, 3 and 5). In humans, four routes (Routes 1, 2, 3 and 7)
achieved the formation of artificial oocytes as farthest end-point and one
route (Route 9) resulted in fertilization.
In vitro differentiation of OSCs
Both mouse and human female germ stem cells have undergone meiosis
in vitro, resulting in artificial oocytes (White et al., 2012).
In vitro proliferation of OSCs followed by (auto)transplantation
After proliferation in vitro, mouse OSCs were transplanted to ovaries of
recipient mice and the resulting artificial oocytes could be fertilized
(White et al., 2012) and, in another study, contributed to the birth of
offspring (Zou et al., 2009). Human OSCs were proliferated in vitro
and transplantated to human ovary tissue xenografted into mice, resulting in the formation of artificial oocytes (White et al., 2012).
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Artificial gametes: progress to clinical application
Figure 3 Flowchart of the study selection process.
In vitro differentiation of ESCs
In vitro differentiation of iPSCs
Starting with ESCs, in vitro differentiation led to the formation of artificial
oocytes in mice but did not result in viable embryos or offspring so far
(Hübner et al., 2003; Qing et al., 2007; Salvador et al., 2008; Yu et al.,
2009). Offspring were born in mice by differentiating iPSCs into
PGCLCs that formed aggregates in culture that, after being grafted
under the ovary bursa, formed mature artificial oocytes under in vivo-like
conditions (Hayashi et al., 2012; Hayashi and Saitou, 2013). In humans,
follicle-like structures were formed, but no oocytes with zona pellucida
were identified (Aflatoonian et al., 2009).
Offspring were born in mice by differentiating iPSCs into PGCLCs that
formed aggregates in culture that, after being grafted under the ovary
bursa, formed mature artificial oocytes under in vivo-like conditions
(Hayashi et al., 2012; Hayashi and Saitou, 2013).
In vitro differentiation of iPSCs followed by autotransplantation
No studies achieving artificial oocyte formation, fertilization or birth of
offspring via this route were identified.
In vitro differentiation of ESCs followed by testicular transplantation
In vitro somatic cell transformation into oocytes without documented
intermediate cell types
No studies achieving artificial oocyte formation, fertilization or birth of
offspring via this route were identified.
Somatic cells were shown to develop into artificial oocytes starting
from rat pancreatic stem cells (Danner et al., 2006) and porcine skin
292
stem cells (Dyce et al., 2006) with uncharacterized genomic integrity and
epigenetic status. In humans, amniotic stem cells differentiated in vitro
into artificial oocytes with unknown ploidy status and a diameter of
50– 60 mm (Cheng et al., 2012) and a hepatic cell line differentiated in
vitro into artificial oocytes, of which some spontaneously activated and
developed germ cell/embryonic tumors in vivo (Ma et al., 2013).
In vivo somatic cell transformation into oocytes without documented
intermediate cell types
Mouse bone marrow transplants were shown to lead to the formation of
artificial oocytes containing the genetic material of the donor. These
oocytes, however, failed to lead to offspring (Johnson et al., 2005;
Lee et al., 2007). No studies described the achievement of the specified
end-points using human cells.
Haploidization by transplantation of a somatic cell nucleus into
an enucleated donor oocyte
Artificial oocytes were formed by injecting somatic cell nuclei into
enucleated (donor) oocytes. Although usually with low efficiency and
unknown imprinting patterns, this induced haploidization of the
somatic nucleus in mice (Fulka et al., 2002; Nagy et al., 2002; Palermo
et al., 2002a, b; Tateno et al., 2003; Chang et al., 2003) and rabbits
(Zhang et al., 2005). Fertilization achieved after haploidization was infrequent and the development of embryos into 2-cell and blastocyst
stages was reported without further knowledge about their developmental potential (Heindryckx et al., 2004). Haploidization by transplantation of a somatic cell nucleus into an enucleated donor oocyte also
resulted in human artificial oocytes (Palermo et al., 2002a; Galat et al.,
2005) that could be fertilized by ICSI (Tesarik et al., 2001; Palermo
et al., 2002b; Takeuchi et al., 2005).
Artificial oocytes from a male
Of the nine biologically plausible routes that could theoretically lead to
the formation of artificial oocytes from a male (Fig. 1), one (Route 3)
was reported in literature as achieving at least one of the specified endpoints in animals and/or humans (Table I). Artificial mouse oocytes were
derived from male ESCs in vitro (Hübner et al., 2003). Male mouse ESCs
differentiated in vitro into both artificial sperm and artificial oocytes.
While being cultured in the same Petri dish, these artificial oocytes
were fertilized (parthenogenetically) by the artificial sperm cells from
the same ESC donor (Kerkis et al., 2007). No studies described the
achievement of the specified end-points using human cells.
Artificial sperm from a female
Of the nine biologically plausible routes leading to the formation of artificial sperm from a female (Fig. 2), three were reported in literature as
achieving at least one of the specified end-points in animals and/or
humans (Table I). The injection of a mouse cumulus cell into a mouse
oocyte resulted in fertilization and embryos with chromosomal abnormalities (Lacham-Kaplan et al., 2001; Route 8; Chen et al., 2004). A variation to method nine (i.e. haploidization by transplantation of somatic cell
nucleus into an enucleated donor oocyte), namely serial nuclear transfer of
an oocyte nucleus (of Mother 1) into enucleated donor oocytes, could
generate an ‘imprint-free’ oocyte from genetically manipulated females,
which once injected into a final (nucleated) oocyte (of Mother 2), resulted
in the birth of viable mice (Kono et al., 2004; Kawahara et al., 2007;
Hendriks et al.
Kawahara and Kono, 2010). In the latter case, the offspring had a different
phenotype (longevity) from the donor (Kawahara and Kono, 2010).
Haploid artificial sperm was formed by differentiating female human
iPSCs, although the authors specifically refer to their incorrect or
unknown epigenetic status (Route 5; Eguizabal et al., 2011).
Discussion
This systematic review on the biological progress on artificial gametes
towards clinical use is relevant to all MAR professionals. Previous
reviews on artificial gametes (Roelen, 2011; Yao et al., 2011; West
et al., 2013) were narrative rather than systematic and did not include
all studies identified by our extensive search.
Our overview of biological progress documents important advances in
the formation of artificial gametes in animals and in humans. The ultimate
proof that artificial gametes were generated is the birth of normal healthy
offspring. The birth of animal offspring has been achieved using several
methods, and although long-term safety has not been unambiguously
proven, animal models thus provide a proof of principle that artificial
gametes can be generated. The birth of animal offspring has been
proven for both the generation of artificial sperm from a male and the
generation of artificial oocytes from a female starting from both ESC
and iPSCs. Additionally, animal offspring have been born using artificial
sperm from females starting from somatic cells (via iPSCs and without
documented intermediate stages) and oocytes.
To date, no study has reported the birth of human offspring from artificial gametes. The creation of human sperm from males was reported for
two of the eight biologically plausible routes, but fertilization was not
reported. The creation of human oocytes from females was reported
for four of the nine biologically plausible routes and one route even
resulted in fertilization. Human and/or animal research more often
focused on creating artificial sperm (n ¼ 41) than on creating artificial
oocytes (n ¼ 27). Research less often focused on creating artificial
gametes from the opposite sex of the animal or commissioning parent
than on creating artificial gametes from the same sex (i.e. 8/70 versus
66/70, respectively; of note, three studies reported on both). In
humans, artificial sperm from a female was created, but fertilization
was not reported. The shortage of research on creating artificial
gametes from the opposite sex might, amongst others, be due to the following factors: (i) technical challenges (e.g. it is more challenging to
develop oocytes from males than spermatozoa from males; Hinxton
group, 2008) that are imposed by the biological nature of male and
female cells (e.g. sex-specific differences in the meiotic processes
(Hunt and Hassold, 2002); (ii) less presumed demand from gay
couples than from infertile heterosexual couples and (iii) less societal
acceptance (Mertes and Pennings, 2010).
Clearly, the full range of findings, including both the results that have
been reported (e.g. birth of mice offspring) and the results that have
not (yet) been reported (e.g. birth of children), is crucial to understanding
the level of progress of the field. The functionality of human artificial
gametes, the chromosomal and epigenetic stability of animal and
human artificial gametes, and the viability and long-term health of artificial
gamete-derived offspring have not been unambiguously proven. Furthermore, many findings are yet to be validated, by different research groups
repeating the experiments and enhancing the efficiency of the techniques. Accordingly, the findings presented here should be considered
as preliminary data, opening up new avenues for research that may
Artificial gametes: progress to clinical application
eventually lead to clinical applications of artificial gametes. The pace of
scientific progress and, with that, the timeframe for any potential
future clinical application of artificial gametes, is difficult to predict
(Hinxton Group, 2008).
The systematic rather than narrative approach in this review has
resulted in a complete overview of the current literature. The unconventional broad focus of our review, although relevant for MAR health care
professionals, required overcoming several challenges.
First, the search strategy had to be very broad to identify articles
covering different novel techniques, which are generally poorly
indexed. We therefore used the snowball strategy to identify about
one-third of our studies that were not in our initial search but were
mentioned in the initially included studies.
Second, risk of bias in the included studies was not assessed, as no sets
of quality criteria are available for biological proof-of-concept studies or
opinion studies. Initially, we attempted to develop a set of quality criteria
for biological proof-of-concept studies, but this resulted in a very limited
number of quantifiable and reproducible quality criteria, which did not
cover the entire quality of the studies. For example, specifying certain
markers to be used or validation of gamete morphology assessments
were not considered objective enough. Identifying quality criteria for biological proof-of-concept studies remains a challenge. We therefore did
not exclude any of the studies meeting our predefined inclusion criteria
and end-points for means of fairness. This review synthesizes all that has
been reported, including methods that seem controversial at the
moment. More specifically, (i) some groups (Oatley and Hunt, 2012;
Zhang et al., 2012) doubt the presence of OSCs that have been reported
by others (a.o. White et al., 2012); (ii) some groups doubt the biological
plausibility of haploidization by transplantation of a somatic cell nucleus
into an enucleated donor oocyte (Tateno et al., 2003) reported by
others (Tesarik et al., 2001; Palermo et al., 2002b; Takeuchi et al.,
2005) and there is controversy on whether bone marrow cells can contribute to the formation of female germ cells (Eggan et al., 2006. As we
refrain from assessing the quality of the individual studies cited in the
absence of objective bias assessment criteria, we need to consider alternatives. The number of references (ideally from different research
groups) for each end-point mentioned in Table I reflects reproducibility,
which is an important quality criterion in biological studies.
Third, some critical remarks on the three predefined end-points (artificial gamete formation, fertilization, the birth off offspring) chosen for
this review should be made. These end-points do not merely reflect
phases in the process of the use of artificial gametes, but also reflect different levels of proof of functionality, in which the birth of offspring is the
only real proof while ‘artificial gamete formation’ and ‘fertilization’ can be
considered pseudo-proof. However, it should be noted that functionality
does not necessarily mean that artificial gametes are genetically and epigenetically equal to normal gametes. Within our end-point ‘artificial
gamete formation’, several forms of proof were accepted (i.e. DNA
content analyses, presence of markers and, for oocytes, polar body
extrusion). It is important to note that in general, DNA content analyses
are seen as more reliable than the presence of markers, and oocyte polar
body extrusion (a form of morphological evidence) is seen as least reliable. Moreover, recent evidence suggests the production of a polar
body is dissociable from the chromosomal events of meiosis (Dokshin
et al., 2013), stressing the importance of discerning between the different
levels of proof of gamete formation presented by the papers included in
this review, varying between polar body extrusion and the birth of
293
offspring. Furthermore, excluding intermediate end-points (e.g. the formation of primordial germ cells; Tilgner et al., 2008) resulted in the exclusion of interesting studies. Finally, the predefined end-points for artificial
oocyte formation were less strict than those for artificial sperm formation
in accordance with the natural differences between spermatogenesis and
oogenesis, resulting in less robust evidence on artificial oocyte formation.
For example, polar body extrusion was accepted as indication of artificial
oocyte formation, whereas morphological evidence only was not
accepted as indication of artificial sperm formation.
Fourth, frameworks had to be developed to structure the metasynthesis of our findings to ensure understandability for professionals
who are not specialists in artificial gametes. This resulted in the need
to use a priori defined start- and end-points and to differentiate
between all biologically plausible routes to create artificial gametes.
However, excluding ‘patient gametes’ as a starting point prevented us
from that addressing some interesting techniques (e.g. oocyte nuclear
transfer, in which the nucleus of a patient’s oocyte is transferred into
an enucleated, younger, donor oocyte; Tachibana et al., 2013a, b). Moreover, excluding intermediate end-points (e.g. the formation of primordial
germ cells; Tilgner et al., 2008) resulted in the exclusion of other interesting studies. Finally, the predefined end-points for artificial oocyte formation were less strict than those for artificial sperm formation, in
accordance with the natural differences between spermatogenesis and
oogenesis, resulting in less robust evidence on artificial oocyte formation.
For example, polar body extrusion was accepted as indication of artificial
oocyte formation, whereas only morphological evidence (i.e. an elongated cell with a ‘tail’) was not accepted for sperm formation.
Fifth, this review was limited to describing effectiveness (gamete
formation, fertilization, the birth of offspring) rather than efficiency
and/or long-term safety, as the included studies focus on effectiveness.
Future preclinical research could contribute to safeguarding the
following three dimensions of quality of healthcare defined by the Institute of Medicine: ‘effectiveness’ (i.e. providing services based on scientific knowledge in order to result in benefit), ‘efficiency’ (i.e. avoiding waste)
and ‘safety’ (i.e. avoiding injuries to patients and their offspring; Corrigan
et al., 2001). Currently, the main preclinical biological research focus is on
the proof of principle (i.e. effectiveness) proven by its final test: the birth
of viable offspring. For some groups of beneficiaries (e.g. gay men requiring artificial oocytes from males), however, more research on effectiveness still needs to be performed than for others (e.g. heterosexual
couples with male infertility requiring artificial sperm from males). In
our opinion, the focus of preclinical biological research should be on
healthy offspring, rather than viable offspring, which is a combined
measure of effectiveness and safety. Only a minority of studies assessed
safety in terms of genetic and epigenetic normality of artificial gametes, or
the epigenetic status and (long-term) health of offspring derived from
them. Some studies (e.g. Zou et al., 2009; Kawahara and Kono, 2010;
Sato et al., 2011) did describe (ab)normal growth, the capability to reproduce and/or the life expectancy of offspring. Obviously, clinical application will require rigorous production of the appropriate safety data, for
which we recommend setting up long-term follow-up of offspring, first
in animals and later in children.
Regarding efficiency of the biologically plausible methods, little has
been reported (e.g. how many attempts were required to end up with
one artificial gamete or offspring). Before clinical application, efficiency
requires extensive research. After all, for clinical purposes, it is crucial
that treatment options fall within humanly reasonable scales relating
294
to, for example, the number of GSCs needed or the number of donor
oocytes required for one pregnancy.
Based on reviewing the biological evidence, we identified the different
routes to generate artificial gametes, and their progress. However, there
is insufficient evidence to recommend focusing on one or more superior
route(s), as all routes are at the forefront of biology (i.e. they challenge
our understanding and technical possibilities).
Conclusion
Although they are currently still in an experimental stage, and the time
frame until possible clinical application is difficult to predict, studies on
artificial gametes seem to be progressing steadily towards possible
future clinical application. The increasing amount of biological studies
will point us towards the safest and most efficient method to create artificial gametes. Deciding to introduce artificial gametes in clinical practice,
however, requires a point of view that goes beyond biologic parameters.
First, artificial gametes could change the field of MAR dramatically by discarding the entire concept of infertility, and potentially allowing new
groups of patients (e.g. heterosexual couples without functional
gametes, post-menopausal women and gay couples) to have genetically
related children. Second, we are unable to acquire informed consent
from the children that will be conceived. Therefore, to prevent premature implementation of artificial gametes, driven by profit and patients’
demands, as with ICSI and PGD (Steele et al., 1999; Leese and Whittall,
2001; Schatten, 2002; Winston and Hardy, 2002; van Steirteghem, 2008;
Harper et al., 2012), all stakeholders should be involved in deciding on the
timing and conditions (including, but not limited to, safety and efficiency)
of any future implementation into clinical practice.
Supplementary data
Supplementary data are available at http://humupd.oxfordjournals.org/.
Authors’ roles
S.H. contributed to the study design, execution, analysis, manuscript
drafting and critical discussion. E.A.F.D. contributed to the study
design, manuscript drafting and critical discussion. A.M.M.P. contributed
to the study design, manuscript drafting and critical discussion. G.H. contributed to the execution, analysis, manuscript drafting and critical discussion. S.R. contributed to the study design, manuscript drafting and
critical discussion
Funding
This study was funded by Academic Medical Center of the University
of Amsterdam.
Conflict of interest
The author(s) report no financial or other conflict of interest relevant to
the subject of this article.
Hendriks et al.
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