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The many problems of somatic cell nuclear transfer in reproductive
cloning of mammals
Katarzyna Malin
a
, Olga Witkowska-Piłaszewicz
a
,
*
, Krzysztof Papis
b
,
c
a
Department of Large Animals Diseases and Clinic, Institute of Veterinary Medicine, Warsaw University of Life Sciences, Nowoursynowska 166, 02-787,
Warsaw, Poland
b
Center for Translational Medicine, Warsaw University of Life Sciences, Nowoursynowska 166, 02-787, Warsaw, Poland
c
Fertility Clinic nOvum, Warsaw, Poland
article info
Article history:
Received 27 March 2022
Received in revised form
20 June 2022
Accepted 20 June 2022
Available online 30 June 2022
Keywords:
Cloning
SCNT
Oocytes
Reproduction
Enucleation
abstract
In 1996, when Dolly the sheep was born, a new, utopian era was expected to begin. Science fiction and
popular culture instantly threatened the public with shortly upcoming human clones, portraying it as a
very easy and instant procedure. Practice has proven otherwise; it exposed how little is known about the
early development of mammals and epigenetic reprogramming. Unfortunately, somatic cell nuclear
transfer success rate in mammals has not changed much since its very beginning. It is not uncommon
that hundreds of oocytes need to be reconstructed to obtain a single live birth. In this review we provide
a brief summary of the progress and problems of the field; beginning with selection of the donor cells
and their susceptibility to different methods of epigenetic reprogramming; methods of the later gene
activation, placental abnormalities, and their possible causes; to health issues that such offspring is prone
to.
©2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
Even though cloning became a matter of public discussion only
in the late twentieth century, the first attempts were undertaken
ca. 1892; when Driesch separated blastomeres of a sea urchin
embryo and demonstrated that two separate organisms were
formed [1]. After 60 years Robert Briggs and Thomas J. King per-
formed the first nucleus transfer, from blastula cells into enucleated
frog eggs [2], but cloning mammals hadn't been made possible for
next 45 years. In 1996, over a hundred years of research resulted in
the first successful attempt to clone a mammal from an adult so-
matic cell [3]. Not only the science world saw it as a breakthrough;
popular media sought to portray the apparently and shortly up-
coming human cloning as the biggest threat to society, resulting in
titles such as “Greetings from Frankenstein: Is the Cloned Human
on the Way?”and continued to portray scientists as the insane [4].
In reality, reproductive cloning is performed by a transfer of a so-
matic cell nucleus into an enucleated oocyte and results in a single
cell, analogous to a fertilized oocyte, usually referred to as a
reconstructed oocyte. Despite the fears of uncontrolled and easy
cloning, practice shows that quarter a century later little is under-
stood in the matter and many moreyears of innovative research are
required for cloning to be as successful as in vitro fertilization (IVF)
or intracytoplasmic sperm injection (ICSI).
This year Dolly the sheep would have been 26 years old. While
many more species were reported to be cloned, overall success rate
from the creation to a viable and healthy offspring remains at a
similar, low level. In 2002, Y. Tsunoda and Y. Kato have published a
commentary, summarizing the live birth ratio as low as 0.5%e18%
in sheep, cattle, pigs and mice [5]. A review from 2007 estimated it
as low as 1e5% [6]. A nation-wide survey in Japan, that covered 9
years of Somatic Cell Nuclear Transfer (SCNT) efforts (1998e2007)
showed no improvement in development and survival until 6
months of cloned calves (on average 4.3% success rate, with 7.5% in
1998 and 2.8% in 2007) [7]. Similar statement was made as of 2020
[8]. Nonetheless, today reproductive cloning serves as a tool of
creation of champion polo horses, farm animals of exceptional
genetic makeup and as a way of bringing back beloved pet
companions.
Dolly was the only viable infant of 277 couplets created with
mammary epithelium cells [3]. In 1998, the ‘Honolulu technique’
was developed [9], resulting in the first successful murine cloning.
*Corresponding author.
E-mail address: olga_witkowska_pilaszewicz@sggw.edu.pl (O. Witkowska-
Piłaszewicz).
Contents lists available at ScienceDirect
Theriogenology
journal homepage: www.theriojournal.com
https://doi.org/10.1016/j.theriogenology.2022.06.030
0093-691X/©2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Theriogenology 189 (2022) 246e254
There, in mice, the cumulus cells showed to be the most suitable
nuclei donors and the only ones able to produce offspring. Out of 4
experiment series, the highest birth rate was reported to reach
2.8%. The perhaps unexpected difficulties caused a 5 year gap be-
tween the first mouse and rat cloned [10]. In rats, specifically, oo-
cytes activate spontaneously within an hour after being removed
from the oviducts. The original protocol, allowing for the first
successful rat nuclear transfer followed by live births, involved
collecting the oocytes in the presence a proteasomal inhibitor
(MG132). It reversibly inhibited the oocyte from entering anaphase
II, thus preventing precocious activation. Starting with 129 recon-
structed and transferred embryos, three pups were delivered alive
and two of them survived (which marks 1,5%) and later also pro-
duced offspring [11]. It is highly desired to create rat clones pop-
ulations, as they make a useful model for medical research. As new
lines of inbred animals are created and other become excinct, to
achieve a 98.6% homozygous, isogenic population, over twenty
consecutive generations of brother x sister or parent x offspring
crossing must occur [12,13]. This process could perhaps be replaced
by somatic cell-cloned animals.
The first cloned pet animal, “CC”, simultaneously the first ever
cloned cat, was particularly famous for a different coat color than
the nuclear donor, as it is influenced by developmental factors,
other than genetic [14]. She was also the first clone to reproduct,
with three out of four kittens viable. The offspring and the mother
herself were reported to live in good health, and CC died in 2020 at
18 years old [15]. Many difficulties had to be overcome to clone the
first dog as well, and the specimen was born in 2005.1095 embryos
were transferred to 123 bitches and three pregnancies were
confirmed, with “Snuppy”being the only viable offspring [16].
Later, in 2011, Jung et al. reported their effort to clone pekinese
dogs, where the reconstructed oocytes were activated using 4
different techniques (needle and chamber fusion methods, both
divided in groups using low and high voltage). 180 embryos were
transferred, and out of 3 fetuses only one pup was born alive (0.5%)
[17]. Despite inaccesibility to canine oocytes, relatively rare ovula-
tion and very low in vitro maturation efficacy of canine oocytes [18],
a resounding success was achieved and canine alongside feline
cloning is commercially available as of 2022 at a cost of ca. $50 000
and $35 000, respectively [19]. Recently an attempt to clone Ma-
caque Monkeys was reported [20]. With an optimized protocol, out
of 301 reconstructed embryos, four live births were obtained and
only two of them survived. This makes 1,3% birth rate, which is also
rather similar to the results from 20 years prior.
The first ever cloned calves were obtained from only ten embryo
transfers, and of eight live births four survived [21]. The authors
speculated that no abnormalities were observed post-mortem, and
the demise was due to environmental causes, including pneumonia
following a heatstroke, drawing in superfluous amniotic fluid, and
one as a consequence of dystocia. Despite high success ratio of the
initial study, a 2001 report on cloning efficiency, which included 13
citations of adult SCNT in cattle, showed that most of them didn't
achieve 2% live births compared to the number of reconstructed
embryos [22]. In 2007, 49.1% of 160 laboratories worldwide were
reported to clone cattle [23]. As this practice became widely
available among breeders and due to the consumers' concerns,
Food and Drug Administration (FDA) offers Consumer Health In-
formation. It addresses, among others, safety of consumption of
meat derived from cloned specimens and the fact that the cloned
animals are meant for breeding, and not direct consumption, as the
cost of obtaining such animals is also very high [24]. SCNT remains
the main method, when it comes to obtaining gene edited livestock
[25].
Prometea, the first equine clone, was born in 2003. She was the
only viable foal obtained from 841 reconstructed zygotes [26]. In
2008, a review of SCNT progress in horses was released [27]. Au-
thors cited five studies (all of them published from 20 03 to 2007) in
which a total of 3577 equine oocyte reconstructions were per-
formed and only 15 foals were born. Highest success rate was
achieved when donor cells were treated with roscovitine, which is
thought to synchronize the treated donor cells in G0/G1. Several
protocols of activation were additionally applied in these groups,
including ionomycin and/or sperm extract [28]. Commercial equine
cloning is also performed (ex. Viagen, Sinogene), but international
studbooks registering racing thoroughbreds refuse to recognize
clones [29]. Argentine Polo Pony clones are accepted in competi-
tions. It is claimed that Crestview, a commercial laboratory that
offers such service, cloned over 200 horses before 2016 [30] with a
30e40% pregnancy rate and 10% of the pregnancies end up with a
live birth rate [31].
Despite extreme difficulties, SCNT is often seen as a hope to
restore extinct species or help preserve the endangered. In popular
media, Mammoth “de-extinction”is brought up every few years as
some small progress is made in the matter [32]. Pyrean Ibex was
successfully cloned, using tissue from the last known specimen, but
the infant died shortly after birth due to left lung atelectasis. An
additional lobe was also found occupying majority of its thoracic
cavity, with no connection to the tracheobronchial tree [33].
Similarly, the first cloned gaur died within the first 48 h after birth
[34]. Other endangered species, including Grey Wolves [35], Black
Footed Ferret [36], Przewalski's Horse [37], were successfully
cloned. To the authors' best knowledge, as of 2022, SCNT is not
considered a major strategy in preserving any endangered species.
In Table 1, we summarized selected publications, focused on
species and corresponding success rate which illustrate two and a
half decades of advances in the field. They are extremely valuable,
especially in the cases where the species are endangered by
extinction.
2. Protocol issues
The procedure of cloning by SCNT begins with the nucleus
donor cell and the reciepient ooplast. A brief summary of the
process is illustrated in Fig. 1. While the process can be presented
generally, it needs to be recognized that in practice, there is high
species-specificity involved.
It is common for the rather older literature to suggest that
achieving G0 phase is necessary for the nucleus donor cell before it
is fused together with the ooplast [38], which can be obtained
through serum starvation. More modern evidence shows that non-
starved G1 cells serve the same purpose with success [39,40] and in
some cases, are equally successful to G0 cells [41]. From this
perspective cumulus cells might provide a great facillitation, as 80%
of them prove to be arrested in G0/G1 stage naturally and are very
suitable as nucleus donors due to the fact that no in vitro culture is
required [42,43]. On the other side of the fusion, to obtain the
ooplast, the oocyte's meiotic spindle complex is removed, requiring
a proper visualization technique. For example, bovine oocytes can
be treated with UV and fluorochrome bisbenzimide (Hoechst
33342) staining, to which they are relatively resistant [44]. Primate
embryos only show comparable blastocyst formation rate (15e16%)
when Oosight imaging system is introduced, which does not
involve the staining. A speculation was raised by the authors, that
Hoechst 33342 might activate primate oocytes prematurely or
cause degradation of maturation factors, which could be later
linked to epigenetic reprogramming failure [45]. Later, the positive
impact of replacing staining with Oosight imaging system in bovine
embryos was also demonstrated [46]. In mice specifically, the
spindle complex can be made visible using Nomarski or Hoffman
optics, also without staining, in 37
C[47].
K. Malin, O. Witkowska-Piłaszewicz and K. Papis Theriogenology 189 (2022) 246e254
247
There is an ongoing debate, whether oocytes stripped of Zona
Pellucida are better candidates for this process, as SCNT using Zona
Pellucida-Free (ZF) oocytes tends to be simpler and quicker [48]. It
seems to have no affect on bovine [49,50], equine [26,51] and
murine embryo development [52]. However, it does not apply to
any species given - in cats, removing zona pellucida later leads to
complete impairment and failure of such embryos' implantation
[53]. ZF embryos require individual, longer lasting culture, i.e., until
the blastocyst develops. Additionally, treating the cells with
phytohemagglutinin further increases the chances of both cells'
fusion [54]. Zona-enclosed embryos, which are harder to obtain,
can be transferred at cleavage [54]. Regarding the difficulties with
ZF oocytes in cats, specific gene expression abnormalities were
identified during the in vitro culture of such: SOX2 and NANOG -
considered as markers of pluripotency expression - were under-
expressed and BAX, apoptosis marker, proved overexpressed [53].
To authors’best knowledge, there are no successful cases reported
of ZF feline cloning.
The metaphase II stage arrested, and enucleated oocytes
become the recipients of the donor nucleus, whether it is injected
or the cells are fused [55]. Until their activation, the development is
managed by factors which were already present in the recipient
oocyte, such as Maturation/Meiosis/Mitosis-Promoting Factor
(MPF) [56]. Physiologically, sperm cells introduce Phospholipase C
Zeta 1 (PLCZ1, absent in somatic cells), which, through calcium
oscillation and MPF breakdown, induces the meiotic arrest release
and further development [57]. Alike fertilized oocytes, the recon-
structed oocytes need to be activated to enter the division cycle.
Table 1
Summary of the species and corresponding success rate.
Year Species Results Other comments
1997 [3] Sheep Dolly the sheep was born; the only viable offspring of 277
attempts
The first mammal cloned from adult somatic cells
1998 [9] Mouse Four experiments, highest birth rate 2.8% First mice cloned
2003 [10] Rat 3 pups born of 129 reconstructed embryos First rats cloned, later reproduced naturally
2002 [14] Cat One viable offspring of 82 transferred embryos, total 188
reconstructed oocytes
The first cat cloned, the first pet cloned, later produced healthy offspring.
She died at the age of 18 due to kidney failure [130]
2005 [16] Dog Three pregnancies obtained from 1095 embryos
transferred; one viable offspring
Snuppy the dog was later cloned as well [131]
2011 [17] Dog One viable offspring of 180 transferred embryos Four different activation techniques were tested in this experiment
2007 [35] Wolf 251 reconstructed embryos, 2 live births Endangered species; Grey Wolf somatic cells nuclei were transferred to
canine oocytes
2003 [26] Horse Only one viable foal obtained from 841 embryos First cloned horse
2016 [109] Horse 16 blastocysts resulted in 11 pregnancies, 3 foals delivered
viable
All of the placentas showed abnormalities
2020 [127] Rabbit 61 blastocysts formed from 165 reconstructed oocytes This result was achieved using electrofusion and melatonin used as a
protective agent against electrofusion damage
2020 [36] Black footed
ferret
1 live birth Donor cells cryopreserved over 30 years prior to the procedure.
2021 [128] Dromedary
Camel
1033 reconstructed oocytes, 28 births, 19 calves survived No significant differences in effectiveness regarding the breed of the oocyte
donor
2020 [37] Przewalski's
horse
One foal born to a domestic horse dame Cells cryopreserved in 1980 were used in this procedure
2019 [100] Crab eating
macaque
5 live births out of 325 embryos transferred Involved (CRISPR)/Cas9 editing
Fig. 1. A general illustration of the SCNT protocol.
K. Malin, O. Witkowska-Piłaszewicz and K. Papis Theriogenology 189 (2022) 246e254
248
This process must be recreated artificially and so far multiple
activation methods have been investigated.
The protocol which resulted in Dolly's birth consisted of elec-
trical pulses between 34th and 36th hour after the ovulation was
induced in donor ewes [3]. In the aforementioned Honolulu tech-
nique, best results were achieved when the oocytes were activated
one to 3 h after the nucleus transfer, in calcium-free, 10 mM Sr
2þ
and 5
m
gml
1
cytochalasin B environment [9]. Other reports
describe PLCZ cRNA injection, which results in repetitive
fertilization-like Ca
þþ
rises in bovine reconstructed oocytes [58].
This method was tested alongside with two more common pro-
tocols involving ionomycin/6-dimethylaminopurine (DMAP) and
ionomycin/cycloheximide (CHX). In this study, PLCZ showed lower,
but comparable efficiency (27.7% embryos developed to blastocyst
stage vs. 35.9% for ionomycin/CHX). Another study describes a
comparison of three activation methods in porcine embryos:
electrical fusion/activation, electrical fusion/activation combined
with MG132, and electrical fusion in low Ca
2þþ
followed by
chemical activation with thimerosal/dithiothreitol. The second
treatment was the most effective, but only 2 piglets (1.9% of em-
bryos transferred) were delivered [59]. It was also proposed to
activate the reconstructed oocyte by reducing zinc ions concen-
tration within the cell. As a result, even in the absence of cellular
Ca
2þ
changes, the oocyte can be activated, when Zn
2þ
is depleted
with heavy metal chelators in the intercellular space [60].
In 2019 another study was published, which focused on creating
a faster but equally reliable porcine protocol. Four experiments took
place and were compared to a control group, which was treated
with the routine protocol for the laboratory (ionomycin exposure
for 5 min followed by SrCl
2
and CHX for 4 h, total 245min). It was
concluded that similar percentage of embryos which developed to
the blastocyst stage was achieved with all methods, including a
20 min procedure, where oocytes were exposed to ionomycin for
5 min plus 15 min of TPEN (N,N,N0,N0-Tetrakis(2-pyridylmethyl)
ethylenediamine) treatment [61]. Unfortunately, time-saving pro-
cedures might not be as beneficial after all, as a 2019 meta-analysis
reveals that delaying the activation by over an hour significantly
increases the blastocyst formation rate [62].
3. Epigenetics
It has been clear for decades that the biggest challenge
regarding the cloned embryo development lays in its epigenetic
reprogramming; physically, among many, resulting in hardships
regarding the implantation, placenta development and function,
but also abnormal offspring syndrome [63], later obesity, immu-
nodeficiency, respiratory defects and early death [64]. It has been
concluded that this epigenetics issue is vastly responsible for the
low birth rate [65,66]. It is critical for the cell nucleus to return to
totipotency through undifferentiation. If incomplete before
genome activation, the failed patterns will be copied and expressed,
resulting in fetal abnormalities and death [67,68]. Dysregulations at
the time of genome activation have been studied and multiple
aberrations have been proven. As many as 259 differently expressed
mRNAs have been reported in a study only at the one cell stage of an
SCNT embryo, compared to in vivo fertilized embryos, making ca.
1.6% of all detected transcripts (137 of higher, 122 of lower
expression) [69].
As mentioned, no epigenetic manipulations were applied in the
murine protocol [9]. In case of Dolly, the cell cultures were serum-
starved only with a suggestion, that such quiescence could possibly
help the reprogramming [3]. Both cases show that the oocyte itself
has the ability to reprogram the transferred nucleus. To increase the
birth rate pharmaceutical intervention can be applied. Among
many, DNA methylation, histone acetylation and methylation were
studied as potential molecular targets with a variety of results.
Trichostatin A (TSA) is a drug of Histone Deacetylase Inhibitor
(HDACi) class [70]. Its action, through interfering with enzymatic
removal of acetyl groups from histones, assures the DNA accessi-
bility for the transcription factors. In case of somatic cell nucleus
remodeling, the addition of TSA is expected to support unfolding of
the nucleosome so it can resemble the totipotent pattern. In 2006,
two groups, independently, have established the most appropriate
protocol for murine SCNT using TSA and fivefold increase in the
embryo's development was reported [71,72]. Another report
delivered data of enhanced blastocyst development rate in monkey
(4%e18%) [73]. Embryos reconstructed using adult bone marrow
Mesenchymal Stem Cells (MSC)and treated with TSA prove the
success rate to grow exponentially, with 65,2% of electrofused
embryos reaching the blastocyst stage, of higher cytological and
molecular quality compared to 45,5% in those untreated [74 ].
Nonetheless, questions were raised about TSA's toxicity and tera-
togenicity [75]. In a recent meta-analysis of its effectiveness on pig
SCNT blastocysts yield, an important conclusion was drawn, that
TSA improves the embryo development when the nuclear donor
cell is of fetal and not adult source. Furthermore, even though the
embryonic development is usually increased, births are reduced
[76]. In rabbits, a study was performed where out of six recipients
two became pregnant with reconstructed embryos treated with
TSA [77]. Out of eleven pups, seven were born alive but all died
within an hour to 19 h. The authors recommend further investi-
gation on long-term effects of such treatment, but don't associate
the mortality with it as early deaths are common in cloned animals.
Another substance of the same class, Scriptaid, has been shown to
provide similar therapeutic effects [78] with less toxicity [79] but a
different study showed improved blastocyst formation in bovine
embryos only when treated with TSA and not Scriptaid [80]. A 2022
meta-analysis on Scriptaid's potency to increase the number of
blastocyst cells or the cleavage rate in porcine embryos derived
from SCNT concluded that only the former might be true [81].
Other epigenetic treatment candidates researched include
Chetomin, a fungal secondary metabolite (which supposably in-
hibits the trimethylation on histone 3 lysine 9 (H3K9 me3)) and
caffeine (as a protein phosphatase inhibitor) [82]. In the cited article
authors did not observe any improvement in bovine and equine
embryo development compared to TSA, even after a 48 h exposure
to Chetomin. Caffeine, on the other hand, was shown to improve
the developmental capability of porcine embryos [83] and primate
embryos [84]. Another H3K9 methyltransferase inhibitor, Chaeto-
cin, was studied but opposite to expectations, it impaired the ovine
embryo development [85]. More recently, a different, promising
approach on epigenetics was proposed: Mutliple Barriers Removal
[86]. The following was proposed: Histone 3 lysine 9 trimethyla-
tion, the first barrier, can be removed by injecting a demethylase
(Kdm4d) mRNA into reconstructed embryos 5 h post-activation.
Similarly can be done with Kdm5b mRNA injection into the
enucleated oocyte. Combining both increased the rate of murine
cloned embryos’development into live animals to 11% [87]. Also
knock-out of H3K9me3 transferases in the donor cells prior to SCNT
brought improvement of almost 50% in blastocyst formation
compared to 6,7% in control group [88]. Authors point out reme-
thylation to be another barrier, which could be overcome by
injecting siRNAs of methyltransferases Dnmt3a and Dnmt3b into
the enucleated oocytes. Finally, the same authors reported that
92.3% of enucleated oocytes that underwent Kdm4bþ5b mRNA and
siDnmt3aþ3b co-injection reached the blastocyst stage. So far no
data is available whether any viable offspring was ever obtained
using this method. Unfortunately, promising embryonic develop-
ment during the in vitro culture might have nothing to do with
survival until birth and later viability of such newborn. In
K. Malin, O. Witkowska-Piłaszewicz and K. Papis Theriogenology 189 (2022) 246e254
249
epigenetics, it is perfectly illustrated when donor cells of different
origin are used: equine adult skin fibroblasts show similar blasto-
cyst development rate, and even birth rates to equine MSC, but foals
created with MSC are not burdened with malformations or diseases
and present higher viability [89]. This might be due to the fact that
MSC show lower activity of Histone Deacetylases and DNA meth-
yltransferases (DNMTs) [74], which is possibly the reason for the
susceptibility to epigenetic reprogramming. Using MSC as the nu-
cleus donors also seems to improve the success rate of the pro-
cedure in equines ein a study where MSC (along with adult
fibroblasts) were compared as donor cells against a control group of
Artificial Insemination (AI) embryos. A total of 594 MSC embryo-
transfers resulted in 37 pregnancies and 20 viable foals (3,4% suc-
cess rate, compared to 1,9% for adult fibroblasts and 71,6% for
embryo transfer) [89]. Good developmental perspectives have been
shown in porcine [90e92], caprine [93] and bovine embryos [94]
cloned with MSC.
4. Abnormal placental development
Although the placenta developed de novo many times
throughout the evolutionary history [95], it is usually associated
with Placentalia, a subdivision of the class Mammalia. In the live
bearing, the placenta allows for the embryo to implant in the
uterine wall; this process, if imperfect, may result in multiple pa-
thologies. Generally, they are inevitable in SCNT pregnancies. Due
to this reason, the majority of them are lost during the first
trimester [96] or peri-implantation [97]. In bovine embryos, a
reduced number of placentomes, hydroallantois and general
enlargement are observed [98,99]. In 2006 genetic aberrations in
the developing placenta have been proven. Authors confirmed that
certain genes responsible for the trophoblast proliferation, differ-
entiation and function were expressed differently to the control
group (embryos derived from AI and IVF, leaning towards high
proliferation genes expression and low or absent function-
associated ones both pre- and post-implantation. At day 17 the
SCNT-derived trophoblast showed no signs of interferon-tau pro-
duction, which is the pregnancy recognition signal in cattle [100].
Another study identified 123 differentially expressed circRNAs,
which were found related to 60 host genes and 32 miRNAs [101].
Especially the miRNA abnormality is to be linked to the problematic
epigenetics of SCNT. In the cited study, transcriptomes of the mRNA,
lncRNA and miRNA of placental cotyledon tissue at day 180 after
gestation were sequenced and 362 mRNAs, 1272 lncRNAs and nine
miRNAs were found to differ in expression [102]. Thus, the prob-
lematic placental development is most likely emergent and not
fundamental.
Placentas and fetal membranes of SCNT animals have also been
examined by ultrasound imaging and histologically which revealed
even more abnormalities [103]. Amniotic membranes, pla-
centomes, umbilical cords, and fetal fluid were studied by ultra-
sound and compared to an AI control group. It disclosed focal
edema, larger placentomes and increased umbilical cord diameter
with hyper-echodensity areas and spikes around it. Histological
findings included degenerated inflammatory cells, chorioallantoic
membrane edema, and decreased epithelial thickness. Clinical ob-
servations prior to the study fall into place with it, as placentome
hypertrophy, enlarged umbilical vessels and placental edema were
reported [104,105].
In mice, placental hyperplasia and expanded spongiotropho-
blast layer develop [106] with poor proliferation rates of the
trophoblast cells followed by rapid growth in later stages [107].
Recently, causes of this enlargement were studied [108]. In natural
conception in mice, genes Sfmbt2, Gab1, and Slc38a4 are paternally
expressed, but in SCNT placentas, they are expressed biallelically. At
first, a maternal knockout of previously recognized enlargement-
related genes (Sfmbt2, Gab1, Slc38a4) was performed with no
improvement in placental quality but correcting the expression of
clustered miRNAs within the Sfmbt2 gene the physiological
placental phenotype was restored. The pups grew into normal,
fertile adults. Although after miRNA knockout the birth rate
increased more than twice (6.7% vs 3% in the wild SCNT clones) the
numbers were considered statistically insignificant. Therefore,
even though the abnormal placental development can at least be
partially prevented, more underlying issues are probably to be
identified.
In equines the placenta faces developmental issues as well. A
detailed study of equine SCNT pregnancies was published in 2016
[109]. Nuclei obtained from fibroblasts of a 29-years old mare were
transferred. Sixteen blastocysts underwent an embryo transfer,
resulting in eleven pregnancies, during which the placentas were
monitored for abnormalities. Three foals were delivered viable.
Pathologies were found in all assessed placentas; even in the three
successful pregnancies. In these, placental edema, engorged allan-
toic vessels and a large umbilical vessel were found. Two mares
were diagnosed with bacterial placentitis and treated with
trimethoprim sulfamethoxazole, pentoxifylline and flunixin
meglumine. In the five mares that experienced abortion the pla-
centas were found heavier than normal, up to 35% of the fetal
weight. All of them showed some form of placentitis. Villous hy-
poplasia, atrophy or necrosis, vasculopathy of allantoic and um-
bilical vessels, hemorrhages and placental mineralization were
reported as well. Additionally, four of five umbilical cords were
longer than regular and edema, hemorrhage and mural thickening
of the umbilical vessels were observed. There were four cases of
large allantoic vesicles. Severe placental separation was diagnosed
in some as well. Two more mares were found to have had bacterial
placentitis only after the abortion, despite no ultrasonographic
findings. The viable foals experienced hypoxic-ischemic encepha-
lopathy, pneumonia, omphalophlebitis and omphaloarteritis. Two
foals had umbilical hernias blood clots in the urinary bladders.
Incomplete calcification of carpal bones and multiple rib fractures
were found in one foal. Two foals were discharged on day 12, the
third one suffered hemothorax due to a fractured rib causing
puncture of the pericardium and was euthanized. These findings
are mostly consistent with previous research [110 ,111], but this
particular study was the first one to report placentitis in SCNT
pregnancies.
5. Other findings
Even though the majority of cloned animals live in good health,
it is estimated that nearly one in three dies within the first 6
months of life [112 ]. Large/Abnormal Offspring Syndrome (LOS),
respiratory failure, abnormal kidney development, cardiovascular
and liver pathologies are often reported [22,112,113]. LOS is widely
associated with cloned and IVF offspring. By definition, it results in
a phenotype consisting of excessive overall fetus size, muscular
deformities, abnormal and asynchronous organ growth combined
with placental dysfunctions. Its occurrence is unpredictable [114],
but so far has not been observed in horses [27], when overgrowth is
expected, parturition induction can be performed a week before
the due date [115 ]. Abnormal fetal development is linked to obesity
later in life in animals [116] and adult murine clones struggle with
increased body mass [117].
Il-Hwa Hong et al. [118 ] reported necropsy findings in 12 still-
born/deceased SCNT canine neonates. Significant increase in mus-
cle mass and macroglossia were commonly observed. The tongue
size caused airway obstruction in many cases. Moreover, anterior
abdominal wall defects, edema and atelectasis of the lung tissue,
K. Malin, O. Witkowska-Piłaszewicz and K. Papis Theriogenology 189 (2022) 246e254
250
increased heart and liver size were present in almost all of the dogs
and one case of failed cerebrum medulla development was found.
Interestingly, this particular neonate presented normal muscle
mass and tongue size. These abnormalities raised questions on
myostatin expression in such specimens, as myostatin-deficient
animals share such phenotype. The team found reduced expres-
sion in myostatin in their tongues and muscle and provided some
interesting discussion. They explain that myostatin is one of the
TGF-
b
family members, which is regulated by IGFs during cell cycle
and its differentiation. Another study showed variable, abnormally
high IGF2R, IGF1R mRNA expression in adult SCNT produced goats
[119], otherwise healthy. IGF2 mRNA expression also varies in
bovine clones [120].
Aging in animals cloned from adult somatic cells is researched
due to justified concerns of the cloned offspring to be at the bio-
logical age of the nucleus donor at birth. Among the most con-
cerning are probably the epigenetic dysregulations, problematic in
these specimen as discussed above. Telomere length also rose
questions, as they were expected to be shorter, proportionally to
the age of the donor of the nucleus. Dolly's telomeres were
confirmed to be 20% shorter to the control group [121], and it seems
to be a tendency in cloned sheep, according to a 2017 review [122].
Surprisingly, it also reported telomere length in cloned cattle to be
appropriate for their age or even elongated, but overall, in other
species it was often found reduced compared to natural offspring of
the same age. Recent study in Dromedary Camels showed that
telomere lengths in clones was adequate to the control group [123].
Interestingly, TSA was proved to facilitate telomere elongation in
cloned pigs [124].
The aforementioned abnormal placental development might be
linked to cardiovascular issues in the early life, according to
Batchelder et al. [125]. The study suggests that anomalous placental
membranes, causing abnormal circulatory pressure, interfere with
the fetal heart's development. This thesis was supported by echo-
cardiographic findings (initially decreased aortic diameters and
increased myocardial thicknesses) in seven cloned calves, which
showed that during the first 30 ex utero life, they made adjustments
and by the end of the study the differences to the control group
resolved. Other authors also agree that although SCNT animals vary
from non-SCNT control offspring in the first two months of life, it is
no longer the case after this period is over [112]. In another report,
seven of ten cloned piglets were similarly evaluated between 40
and 65 day of life [126]. Decrease in fraction ejection, mitrial
insufficiency and decreased cardiac output was observed in some.
Authors also included necropsy findings in five piglets who died
during the study and in four of them, cardiovascular deviations
were observed, including an enlarged heart with dilated, thin-
walled ventricles with myocardial necrosis, myocardial degenera-
tion and excess pericardial fluid and congestive heart failure. The
surviving five piglets showed no symptoms of cardiac disease.
General examination, hormonal and hematologic assessments
have been performed in cattle cloned from somatic cells [127].
Fetuses obtained before term showed variable organ sizes and
deformities, including abnormally large kidney, autolysis of both
kidneys in a stillborn and large fatty liver. Neonates had higher
average birth weight compared to the AI and IVP control groups.
Rectal temperature of clones was found to be higher until 50 days of
age, including random 24e36 h peaks up to 41
C with no other
clinical signs observed, non-reactive to any treatment applied
(NSAIDs, wet towels and ventilation). T
4
plasma levels were
examined in speculation, whether it could be the reason for the
fever, but the answer was negative. RBC parameters were found to
be consistent within all groups. The neutrophil:lymphocyte ratio,
reflecting cortisol levels at birth, was higher in clones. Despite this,
later ACTH stimulation tests found no significant differences.
Regarding the growth hormone, normal level was found, as well as
in insulin and glucose responses. To the authors’best knowledge,
these macroscopic findings are not found to originate within any
specific developmental deviation.
6. Suggestions for further research
In a short summary, the low efficiency reflects how complicated
SCNT and further embryo development is. Some recommendations
and assumptions can be made with a precaution, that even though
sharing similar problems and results, probably no two studies can
be directly compared. Different methods and approaches are
researched in order to further rectify the whole procedure as such.
A faster and simplified SCNT would limit both oocyte's and donor
cells' exposure to external factors and manipulations and require
shorter training of the staff with similar success rates. Here, an
inevitable conclusion has to be stated - that more successful cloning
will require deeper understanding of oocyte's physiology, not only
as a mammal oocyte, but also the species specificity and unique-
ness. Especially the relationship between nucleus-donor cell type
and introduced pharmaceuticals need further studies; which might
be very complicated due to multiple possibilities and combinations.
Perhaps the future of SCNT lies in the Mutliple Barrier Removal
technique, as it potentially withdraws the need of using any clas-
sical pharmaceutical treatment, but on the other hand, may require
more injections per se. Yet again, blastocyst formation or cleavage
rate, often presented as determinants of a successful study, may not
have anything to do with the embryo's pregnancy survival and the
newborn's viability. While many studies are promising, their actual
value can only be verified by obtaining healthy offspring.
Aside from discussed technical difficulties, ethics of cloning,
widely discussed in the popular media, cannot be forgotten. So far
no successful human reproductive cloning attempt was docu-
mented, not only due of the legal regulations, but also due to lack of
reasonable cause for such; when it comes to infertility, multiple
methods other than SCNT can be used. In animal cloning, doubts
could be raised especially in case of commercial dog cloning. World
famous Snuppy, the first dog cloned by SCNT, out of 1000 embryos
transferred into 123 surrogates, was the only pup surviving until
adulthood [16]. To our knowledge, such businesses do not publicly
share information on the surrogates’living conditions, especially
those, who experience abortion, nor about the dogs that do not get
adopted due to lack of predictability regarding the live births count.
Despite multiple issues, of which some we briefly described,
progress continuously takes place. In the end, SCNT may not be as
easy as portrayed quarter a century ago, but each and every advance
may be a giant leap for the science of medicine.
Author contributions
Conceptualization: K.M., O.W.P., K.P.; Roles/Writing - original
draft: K.M., O.W.P. Writing - review &editing: K.M., O.W.P., K.P.
Funding
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Declaration of competing interest
None.
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