REPRODUCTION

RESEARCH

Zinc as a non-hormonal contraceptive: an alternative to the

copper intrauterine device (IUD)
K N Shankie-Williams , L A Lindsay, C R Murphy and S N Dowland
School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, Sydney, New South
Wales, Australia
Correspondence should be addressed to K N Shankie-Williams; Email: kirst​en.sh​ankie​-will​iams@​sydne​y.edu​.au

Abstract
In brief: The many side effects of current contraceptives leave a large proportion of women without adequate protection. This study
shows that zinc, a highly physiologically compatible metal, provides effective long-acting reversible contraception in rats,
without requiring the use of hormones.
Abstract: Long-acting and reversible contraceptives (LARC) are the most widely used form of female contraception worldwide;
however, they have significant side effects that often result in early removal. Most LARCs are hormonal, but the use of
exogenous hormones is not suitable for all women and causes side effects in many others. The copper IUD (CuIUD) is the
only non-hormonal LARC, but a large proportion of users suffer severe side effects. This study proposes the use of zinc as a
suitable alternative to the CuIUD. A rat intrauterine device (IUD) model was established to test the efficacy of a zinc IUD
(ZnIUD) against a CuIUD. The IUD was surgically implanted into one uterine horn while the other remained untreated. Both
the ZnIUD and CuIUD resulted in zero implantation sites which were significantly fewer compared to non-treated horns.
Histological assessment revealed damage and inflammation in the endometrium of CuIUD-treated horns but only minor
epithelial changes in ZnIUD-treated horns. This suggests ZnIUDs may not share the side effect profile of the CuIUD. To test
the long-term efficacy of the ZnIUD, rats had a ZnIUD surgically implanted into both horns and cohoused with males for 3
months. These rats mated regularly but did not get pregnant, confirming long-term effectiveness. Reversibility of the ZnIUD
was also established, as removal of the ZnIUD after 3 months resulted in no significant difference in the number of
implantation sites between treated and untreated horns. This study demonstrated the contraceptive efficacy of zinc and its
potential as a LARC.
Reproduction (2022) 164 135–142

Introduction that when inserted into the uterus provides the user
up to 12 years of protection against pregnancy. This
Intrauterine devices (IUDs) are the most commonly long-term contraceptive comes with many practical
used form of reversible contraception worldwide benefits distinguishing it from alternatives, such as the
(d’Arcangues 2007). Almost all long-acting reversible contraceptive pill, including low cost, no potential for
contraceptives (LARCs) contain hormones as their patient error and no exogenous hormone induced side
contraceptive agent, and many women are either effects. These characteristics provide the foundation for a
precluded from using these by a medical condition low risk and financially accessible contraceptive option
or wish to avoid the many physical and physiological for women around the world. However, the CuIUD has
side effects of exogenous hormones. Depression, mood its own unique side effect profile causing more painful
swings, irritability, anxiety, weight gain, migraines and periods for 38% of women (Hubacher et al. 2009).
decreased libido are just some of the commonly reported This excessive pain and bleeding, both menstrual and
side effects experienced by hormonal contraceptive intermenstrual, has led to persistent rates of early removal
users in numerous studies over the last few decades of around 25%. For this reason, it is not recommended
(Stephenson et al. 1994, Robinson et al. 2004, Beksinska for women with moderate to heavy periods (Hubacher
et al. 2010, Zettermark et al. 2018, Guen et al. 2021). et al. 2009). One in 10 women worldwide have an
These can lead to discontinuation or inconsistent use, unmet need for contraception, and side effects are a
both increasing the chance of unplanned pregnancies. primary reason women discontinue use, resulting in 121
Only one non-hormonal option currently exists, the million unintended pregnancies each year (Sedgh et al.
copper IUD (CuIUD), which is a medicated model 2016, Bearak et al. 2020, UNDESA 2020).

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136 K N Shankie-Williams and others

Side effects experienced by users of the CuIUD are a impact of IUD removal in early pregnancy rather than
direct result of the copper content of the device. Animal demonstrating reversibility of these contraceptives as
models have shown that copper cytotoxicity causes claimed. In addition, authors acknowledged that surgical
necrosis, erosion, stromal hypertrophy and metaplasia of trauma to the uterus during pregnancy was expected to
epithelial cells in mucosal surfaces in direct contact with contribute to the reported findings of decreased fertility
copper, resulting in polyps (Zipper et al. 1969, Chvapil in the IUD-treated horns.
et al. 1978). Exposure to metallic copper also resulted Although these earlier studies provided initial
in an increase in lysosomes and swollen, vesiculated evidence supportive of zinc as a contraceptive agent, true
mitochondria in vaginal epithelial cells and infiltration efficacy and reversibility of a ZnIUD have not yet been
of leukocytes into the underlying mucosa (Chvapil established. Therefore, the present study has developed
et al. 1978). This is consistent with the morphological a rat model to examine the contraceptive efficacy of a
changes observed in human endometrial biopsies taken ZnIUD with consideration of its potential mechanism of
from women using the CuIUD (Misra et al. 1989). action. This article also examines the potential for zinc
Lymphocytic, monocytic and leukocytic infiltrations to provide continued contraception over an extended
have been found in the uterine epithelium and stroma period of time and assesses the return to fertility after
of CuIUD users, and a loss of epithelial integrity was such long-term use of a ZnIUD, to determine the true
observed (González-Angulo & Aznar-Ramos 1976). reversibility of such a contraceptive. The impact of a
During early experiments exploring non-hormonal ZnIUD on the endometrium is also compared with that of
IUD materials, a range of metals were trialled for a CuIUD. This work aims to evaluate the potential utility
antifertility effects and tested in the rabbit uterus of a ZnIUD as a non-hormonal LARC, in comparison
by placing an IUD near the cervix (Zipper et al. with the only current option, the CuIUD.
1969). Copper and zinc resulted in the highest anti-
fertility rates of all the metals studied. Although only
one implantation site was found with the zinc IUD
Methods
(ZnIUD), further investigation of the use of zinc was Ethics approval
largely abandoned. However, we suggest that the
All procedures were approved by The University of Sydney
slightly lower efficacy of zinc in this study may have
Animal Ethics Committee.
resulted from methodological limitations. Later studies
have shown that the two-cell embryo has a unique
sensitivity to Zn2+, impeding further development Animals and mating
(Andrews et al. 1991); therefore, we hypothesise that
In total, 23 nulliparous female Wistar rats were housed in
this may be responsible for the contraceptive effect of plastic cages under a 12h light:12h darkness cycle at 21°C. All
a ZnIUD. As such, the placement of the experimental rats were between 10–12 weeks old at the start of the protocol

suitable for copper, but suboptimal for zinc − being

IUDs near the cervix of rabbits (Zipper et al. 1969) was and weighed 280–300 g. Rats were fed and watered ad libitum.
The oestrous cycle of the rats was tracked by vaginal smearing
located distant from the site at which we now expect and analysis of the cellular composition (Long & Evans 1922).
it to act. Mating was also confirmed by smearing, the presence of
There has only been one study that previously sperm indicating day 1 post-coitum (pc). Pseudopregnancy
considered the reversibility of a ZnIUD (Medel et al. was confirmed by the cellular composition of daily vaginal
1972). This model involved rats and rabbits being smears (Long & Evans 1922).
mated 2–3 days after insertion of IUDs, which were
then removed on day 2 after mating, with pregnancy
outcomes evaluated on day 10 (Medel et al. 1972). This Treatment groups
work showed pregnancy was able to proceed in these Rats were randomly allocated to three experimental groups (Fig.
animals; however, this study design only evaluates the 1). In the Efficacy Group, rats had either a zinc (n = 5), copper

Figure 1 Flowchart of experimental treatment

groups. The Efficacy Group had either a zinc
(n = 5), copper (n = 6) or nylon IUD (n = 5)
inserted into one uterine horn. The Long-term
Group (n = 3) rats had ZnIUDs inserted into
both uterine horns for 3 months. The
Reversibility Group (n = 4) had a ZnIUD
inserted into one uterine horn which was
removed 3 months later. Animals were mated
and tissue was collected on day 9 pc.

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 Zinc as a non-hormonal contraceptive 137

(n = 6) or nylon IUD (n = 5) inserted into one uterine horn, over 4.5 h at 40°C before clearing with two 45-min changes
were mated after recovery and tissue was collected on day 9 of xylene and infiltration with paraffin (Thermo Fisher)
pc. In the Long-term Group (n = 3), rats had ZnIUDs inserted over 3 changes of 30 min at 60°C. Finally, specimens were
into both uterine horns, allowed to recover for 1 week and embedded using the Tissue-Tek TEC 5 tissue embedding
then co-housed with a male for 3 months. Rats were regularly console system (Sakura FineTek, Torrance, CA, USA).
smeared to track the oestrous cycle, mating events and number
of days in pseudopregnancy. At the end of the 3-month period,
tissue was collected 9 days after mating. In the Reversibility Sectioning and staining
Group (n = 4), rats had a ZnIUD inserted into one uterine horn
Transverse sections (7 µm) were cut on an HM 325 Rotary
which was removed 3 months later. After recovery, animals
Microtome (Thermo Fisher Scientific), and sections were
were mated and tissue was collected on day 9 pc.
floated on a water bath (40°C) and placed onto gelatin-chrom-
alum-coated glass slides. At least five sections were collected
Surgical insertion and removal of IUDs per block and two blocks were used for each animal. Slides
were then dried overnight in a 40°C oven.
Insertion Sections were stained using a routine haematoxylin and
ZnIUD and CuIUD were made from 1 cm of 99.994% pure eosin protocol (Carleton et al. 1980). Images were captured
zinc and 99.999% pure copper wire (Alfa Aesar, MA, USA), on an Olympus BX53 brightfield microscope (Olympus), using
0.5 mm in diameter and were finished with a small loop an Olympus DP73 camera (Olympus) and Olympus CellSens
at one end. Rats received an intraperitoneal injection of software (version 1.11).
ketamine (75 mg/kg; TROY Laboratories, NSW, Australia) and
xylazine (10 mg/kg; Parrell Laboratories, NSW, Australia). The
IUDs were inserted into the uterine lumen through a small Statistical analysis
incision in the uterine wall, 1 cm from the ovary. All IUDs In the Efficacy Group, changes in the number of implantation
were secured in place with a nylon suture through the IUD sites in IUD-treated horns and contralateral non-treated horns
loop and uterine wall, closing the uterine wall incision. A were analysed using two-way ANOVA, with IUD material
foreign body control was also performed by inserting a 1-cm and uterine horn as factors. Tukey post hoc test for multiple
loop of nylon thread into the uterine horn to test the effect comparisons (reporting multiplicity-adjusted P values)
of the presence of a foreign object in the uterine lumen. Rats was then applied to determine which pairs of means were
were allowed to recover for at least 1 week after surgery. significantly different. Differences were determined to be
statistically significant when P < 0.05. For the data obtained in
Removal the Reversibility Group, a paired t-test was used to determine
if a statistical difference existed between the number of
Removal surgery for animals in the Reversibility Group
implantation sites present in a horn that had a ZnIUD removed
occurred during dioestrus, at which time the uterus is small,
of ≤0.05 was considered statistically significant. All statistical
after 3 months and a contralateral non-treated horn. A P value
not oedematous and lacks prominent vasculature, allowing
easier removal and minimising tissue damage. Similar to
analysis was performed using GraphPad Prism Software
insertion surgery, rats received an intraperitoneal injection
(version 7.02; GraphPad Software, Inc), and bar graphs were
of ketamine (75 mg/kg) and xylazine (10 mg/kg), the uterine
generated where values represent the mean + s.e.m.
horn was exposed and the IUD was carefully removed. Rats
recovered for at least a week.
Results
Tissue collection Copper and zinc IUDs prevent pregnancy
Rats were anaesthetised with 20 mg/kg sodium The contraceptive efficacy of copper and zinc was assessed
pentobarbitone (TROY Laboratories, NSW, Australia) and by counting the number of implantation sites in the IUD-
dissected under deep anaesthesia before euthanasia. Both
treated and untreated horns on day 9 pc in rats receiving
uterine horns were excised, pinned onto dental wax and
different IUD materials (Fig. 2). In rats receiving a nylon
photographed. The implantation sites were counted and
IUD, implantation sites were evident in both the IUD horn
compared to the control horn to determine contraceptive
efficacy (Liechty et al. 2015). Uteri were cut into and untreated, control horn with no significant difference
approximately 5 mm blocks from the region of the horn in the number of implantation sites in control vs treated
containing the IUD or inter-implantation sites in the control horns (P = 0.1059) (Fig. 2). In rats with either a CuIUD
(Christofferson & Nilsson 1988) and immediately fixed in or ZnIUD, there were no implantation sites in uterine
10% neutral buffered formalin (Fronine, NSW, Australia). horns containing an IUD. Implantation sites were evident
The tissue was then washed in distilled H2O and dehydrated in the untreated uterine horns, resulting in a statistically
in 30, 50 and 70% ethanol for 30 min each. Samples were significant difference in the number of implantation sites
then placed in the Excelsior ES tissue processor (Thermo in treated compared to untreated horns in both CuIUD
Scientific) to further dehydrate tissue in graded ethanol baths (P < 0.0001) and ZnIUD (P < 0.0001) animals (Fig. 2).

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138 K N Shankie-Williams and others

Figure 3 Uterine histology of horns containing metal IUDs (Cu2+ and

Zn2+) on day 9 pc. (A) Haematoxylin and eosin showing inflammation in
the underlying stroma and epithelial damage in the CuIUD-treated horn.
(B) Metaplasia (arrows) of the uterine epithelial cells is present in the
Figure 2 Contraceptive efficacy of metal IUDs (Cu2+ + Zn2+) and a control
CuIUD-treated horn. (C) The ZnIUD-treated horn exhibits normal overall
nylon IUD on day 9 pc. (A) Implantation sites (arrow heads) were visible
uterine morphology. (D) Mild metaplasia of the uterine epithelial cells is
in both IUD and control horns of the nylon treated rats, as well as the
present in the ZnIUD-treated horn. L, lumen; S, stroma; E, epithelium
region containing the IUD (bracket). (B) CuIUD-treated horns did not
contain implantation sites while the non-treated control horn contained
multiple. (C) ZnIUD-treated horns did not contain implantation sites underlying stroma, glands and myometrium appeared
while the non-treated control horn contained multiple. (D) There was no normal. The untreated uterine horn did not show signs
significant difference in the number of implantation sites between of damage, inflammation or metaplasia. Uterine horns
treated and control uterine horns in rats receiving a nylon IUD (n = 5). containing a nylon IUD exhibited no abnormalities
No implantation sites were seen in the CuIUD-treated (n = 6) and and displayed normal uterine morphology (Fig. 4A
ZnIUD-treated (n = 5) uterine horns in any animals, while the respective
and B). The glands and myometrium appeared normal.
control horns contained implantation sites. (**** P < 0.0001).
The untreated horn demonstrated normal histological
appearance with no signs of damage, inflammation or
Short-term exposure to metal IUDs impacts metaplasia (Fig. 4C and D).
endometrial histology
Endometrial histology was assessed after exposure to Zinc IUD provides long-term contraception
each of the IUD materials. The endometrium of the
copper-treated uterine horn exhibited infiltration of The long-term contraceptive efficacy of a ZnIUD was
inflammatory cells into the stroma, including dense assessed by inserting IUDs in both uterine horns and
clusters of leukocytes, including neutrophils (Tigner et al. co-housing rats with males for 3 months. Daily vaginal
2021) and sites of epithelial damage in the region of the smears showed that the rats regularly mated but did not
IUD (Fig. 3A). Along areas of intact epithelium, the simple get pregnant. Cervical stimulation during mating causes
columnar epithelial cells had undergone metaplasia with rescue of the corpus luteum in rats resulting in rats spending
a resulting saw-tooth appearance (Fig. 3B). The underlying an average of 10 days in a pseudopregnant state before
stroma, glands and myometrium of the uterus appeared returning to oestrus and mating again (Smith et al. 1975).
normal. The untreated uterine horn demonstrated normal Despite rats mating at least 8 times each, there were no
histological appearance of the endometrium with no pregnancies during the 3-month treatment period (Table 1).
epithelial damage, inflammation or metaplasia. In the
ZnIUD-treated uterine horns, the epithelium remained The contraceptive effect of the zinc IUD is reversible
intact, with no regions of epithelial damage or exposure The reversibility of the method was investigated by
of the stroma and no dense clusters of leukocytes inserting a ZnIUD into one uterine horn, removing it 3
(Fig. 3C). The epithelium showed mild metaplasia months later and then examining implantation success.
resulting in an uneven apical border (Fig. 3D). The No significant difference in the number of implantation
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 Zinc as a non-hormonal contraceptive 139

Figure 5 Number of implantation sites in uterine horns after a

3-month treatment and subsequent removal of a ZnIUD (n = 4). There
was no significant change in implantation rate between treated and
untreated horns after IUD removal from the treated horns.
Figure 4 Uterine histology of horn containing nylon IUD and
contralateral non-treated horn on day 9 pc. (A) Haematoxylin and 100% contraceptive efficacy, completely preventing
eosin showing no abnormalities present in the nylon IUD-treated
horn. (D) The contralateral non-treated horn exhibits normal uterine
pregnancy in the IUD-treated uterine horns. This study
morphology. L, lumen; S, stroma; E, epithelium also demonstrated for the first time that a ZnIUD can
provide 100% contraceptive efficacy, equal to that of the
sites in the ZnIUD-treated uterine horn compared to CuIUD. Comparison with the nylon IUD demonstrated
the untreated horn was found (P = 0.86) (Fig. 5). The that the contraceptive effect of both the ZnIUD and
average number of implantation sites in ZnIUD-treated
uterine horns was 5.75 (±1.4) compared to 6 (±0.4)
implantation sites in control horns.

Long-term exposure to the zinc IUD does not impact

endometrial histology
Histological analysis of the endometrium after 3 months
exposure to a ZnIUD revealed no pathologies such
as metaplasia or inflammation in the ZnIUD-treated uterus
(Fig. 6A and B). The untreated horn did not show signs of
damage, inflammation or metaplasia (Fig. 6C and D).

Discussion
Both copper and zinc demonstrate 100%
contraceptive efficacy in the rat IUD model
In agreement with previous animal studies, the
present study determined that the CuIUD provided

Table 1 Contraceptive efficacy of the ZnIUD over a 3-month period.

Mating Mating Mating pair

pair 1 pair 2 3
Figure 6 Uterine histology of horn containing a ZnIUD for 3 months
No. of matings 8 8 9 and the non-treated control horn after removal and mating. (A and B)
No. of pregnancies 0 0 0 Haematoxylin and eosin showing no abnormalities of the uterine
Average no. of days in 11 11 9 epithelial cells and underlying stroma in the ZnIUD-treated horn,
pseudopregnancy closely resembling that of the non-treated control horn (C and D). L,
Average no. of days in oestrus 1 1 1
lumen; S, stroma; E, epithelium

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140 K N Shankie-Williams and others

CuIUD is due to the metal content of the IUD, rather and a positive correlation with vascular endothelial
than resulting from the presence of a foreign body in growth factor (VEGF), a potent angiogenic factor (Li
the uterine lumen. Our IUD model demonstrated higher et al. 2016). In addition, higher fibrinolytic activity
contraceptive efficacy than Zipper et al. (1969), showing has been found in areas of the endometrium that have
this model successfully takes into consideration the direct contact to the CuIUD, indicating inhibition of
mechanism of action. homeostasis (Liedholm et al. 1983). The histological
data in the present study support these observations,
further supporting that copper directly exerts negative
Zinc IUD is an effective long-term and reversible
effects on the endometrium.
contraceptive in the rat
These effects are particularly severe soon after
In order to mimic the clinical setting, this study for the insertion, when the rate of dissolution of copper ions
first time showed the long-term contraceptive efficacy into the uterine fluid is highest upon insertion of the
and reversibility of a ZnIUD. The ZnIUD prevented CuIUD and drops exponentially over the duration of
all pregnancies in female rats co-housed with males its use, an effect known as the ‘burst release’ (Alvarez
for a continuous 3-month period with no change in et al. 2012). This high initial Cu corrosion rate irritates
the number of days in oestrus or the average days of the endometrium, enhancing the inflammatory
pseudopregnancy after cervical stimulation (Long & response and deposition of proteins and cell debris
Evans 1922). This indicates that zinc may have long-term and may contribute to the inflammation and damage
efficacy in clinical settings similar to that of the CuIUD. of the endometrium after short-term exposure to the
The present study is also the first to clearly demonstrate CuIUD, seen in this study. This period is when the
that the contraceptive effect of a ZnIUD is reversible. highest removal rates occur, preventing many women
When ZnIUDs were removed after 3 months exposure, from having successful, long-term use of the CuIUD
rats were able to rapidly return to fertility and exhibited (Hubacher et al. 2009).
successful implantation in uterine horns previously The impact of a ZnIUD on uterine histology has
containing an IUD, at rates comparable to untreated not been previously examined. Short-term exposure
uterine horns. This is in contrast to the previous study (approximately 2–3 weeks; Efficacy Group) to the
which found a significant decrease in implantation sites ZnIUD resulted in mild metaplasia in the uterine
when ZnIUDs were removed 2 days after mating, in horn, with epithelial cells appearing irregular in
treated compared to control horns (Medel et al. 1972). height. Unlike the CuIUD-treated horn, the epithelium
As such, this study has shown that the ZnIUD could be remained intact and no areas of inflammation were
an effective LARC and offer an alternative contraceptive observed. Therefore, it is possible that a ZnIUD would
option to women. cause users less pain and bleeding compared to the
CuIUD, due to the absence of localised inflammation
(Julius & Basbaum 2001, Ferenczy 2003). Uterine
Copper IUD impacts endometrial histology more
horns that were subjected to long-term exposure
severely than zinc
to Zn (approximately 16 weeks; Long-Term Group)
The morphological changes and pathologies of the exhibited no pathologies, having the same appearance
CuIUD-treated horn, observed histologically in the as the control horn. This indicates that Zn acts as
present study, reflect those seen in the earlier rabbit and a contraceptive without any long-term impact to
human trial of the CuIUD (Zipper et al. 1969). The CuIUD endometrial integrity. Although there have been
in our rat IUD model caused severe metaplasia in the significantly fewer studies conducted on the interaction
uterine epithelium and epithelial damage. Inflammation of zinc in the uterus compared to those of copper, the
was also present in the CuIUD horn, in alignment present findings, in conjunction with the emerging use
with the stromal hypertrophy documented by Zipper of zinc in biocompatible implants provide a positive
et al. (1969) in copper-treated rabbit uterus (Zipper indication of the potential biocompatibility of a ZnIUD
et al. 1969). An influx of inflammatory cells around (Drelich et al. 2017, Kannan et al. 2017). Zinc is
areas that had lost epithelial integrity were common relatively harmless to humans as it is the second most
amongst the copper-treated horns. This inflammation abundant essential metal in the body and found at a
and endometrial damage may lead to many commonly mean serum concentration of 1000 μg/mL in humans,
reported side effects. Inflammatory mediators activate with a recommended daily intake of 10–15 mg (Plum
and sensitise local nociceptors to prevent further tissue et al. 2010). This is in contrast to copper, which is
damage (Julius & Basbaum 2001), whilst the continuous found at a mean serum concentration of 0.1 μg/mL
release of proteolytic agents from leukocytes suppress and a recommended daily intake of 0.9 mg. Because
endometrial angiogenesis, resulting in pain and excess of this, the body is physiologically adept at transporting
bleeding (Ferenczy 2003). Recent studies have found a and storing larger concentrations of zinc with no risk of
higher concentration of copper in the endometrium of producing reactive oxygen species, a consequence of
women experiencing abnormal bleeding with a CuIUD high concentrations of copper and mediators of acute
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 Zinc as a non-hormonal contraceptive 141

and chronic inflammatory pain (Khattab 2006, Babula Funding

et al. 2012). Zn is now recognised as an excellent
This work was supported by an NWG Macintosh Memorial
candidate for Zn-based orthopaedic implants, being Grant from The Ann Macintosh Foundation of the Discipline of
chosen for its biocompatibility and safety (Kannan Anatomy and Histology and Murphy Laboratory Funds.
et al. 2017, Yang et al. 2020).

Contraceptive effect of a zinc IUD is likely via a Author contribution statement

unique mechanism, acting on the two-cell embryo K N S-W designed the study, carried out experimental work,
The improved contraceptive efficacy of zinc in the analysed data and prepared manuscript. L A L and C R M gave
technical advice as well as experimental design and editing
present study compared to earlier studies (Zipper et al.
of manuscript. S N D designed the study, gave technical and
1969, Chang et al. 1970, Kincl & Rudel 1971) supports
experimental assistance and critical editing of manuscript.
our hypothesis that Zn exerts its contraceptive effect via a
unique effect on the two-cell embryo. Previous research
has shown that the two-cell embryo exhibits specific References
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