Curr Obstet Gynecol Rep (2013) 2:226–231
DOI 10.1007/s13669-013-0059-2
FEMALE INFERTILITY AND ASSISTED REPRODUCTIVE MEDICINE (Y ZHAO, SECTION EDITOR)
Biology of Polyspermy in IVF and its Clinical Indication
Ping Xia
Published online: 6 September 2013
# Springer Science+Business Media New York 2013
Abstract Normal fertilization involves interaction of one
sperm and one egg. When the first sperm enters the egg, the
egg develops blocks on the zona pellucida and egg plasma
membrane (oolemma) to prevent additional sperm from entering
the egg. If more than one sperm enters the oocyte cytoplasm
(ooplasm), the egg becomes polyspermic. Polyspermy has not
been well studied in humans. As development of assisted repro-
ductive technologies and embryonic stem cell studies, it is
important to understand more about the interaction of sperm
and oocytes and accessory sperm inside the early embryos.
Extensive studies and reviews have summarized the polyspermy
block in mice and large animals. This review focuses on new
discoveries, oocyte quality, multiploidy, ability of embryo’s self-
correction, and the clinical relevance. Studying polyspermy
from different angles in humans, such as oocyte quality and
patient endocrine environment, will allow us to gain more
knowledge about early embryo development in humans. The
ultimate goal of these studies is to avoid genetic abnormalities,
such as mosaicism, from happening in patients.
Keywords Polyspermy . Oocytes . In vitro fertilization .
Embryos . Triploid . Mosaicism . Oocyte cytoplasm .
Fertilization . Perivitelline space . Cortical granules
Introduction
Extensive studies have been done in animals regarding poly-
spermy. However, some areas are unknown in human
polyspermy due to the ethical issues concerning studying human
fertilization. Application of in vitro fertilization (IVF) allows us
High-Complexity Clinical Laboratory Director, certified by the American
Board of Bioanalysis (ABB).
P. Xia (*)
The IVF Center, Department of Women’s Health and Obstetrics,
Hong Kong Sanatorium & Hospital, 6th Floor, LiShuPui Block,
2 Village Road, Happy Valley, Hong Kong
e-mail: pingxia@hksh.com
to observe fertilization during the interval of the procedure.
Routinely, fertilization status is checked 16-20 hours after insem-
ination in vitro. Approximately 7 % of fertilized human eggs are
polyspermic, which is defined by appearance of three pronuclei,
so-called triploid [19].
Theoretically, the triple pronuclei routinely observed in the
IVF laboratories could be a result of two occurrences: 1) triple
nuclei derive from two sperm and oocyte nucleus; 2) triple
nuclei derive from one sperm, oocyte nucleus, and failure of
second polar body extrusion. Therefore, the clinically observed
polyspermic eggs are not always truly polyspermic. Regard-
less, occurrence of multipronuclear formation in the fertilized
oocytes reflects poor oocyte quality. With that being said, do
embryos have the ability to self-correct during early develop-
ment? In this review, the following areas will be discussed.
& Polyspermy block
& Polyspermy and oocyte quality
& Perivitelline space (PVS)/cortical granules (CG)
& Multiploidy - failure of the second polar body extrusion
& Multiploidy - ability of embryo’s self-correction
& Multiploidy and mosaicism
Polyspermy Block
Polyspermic block in mammalian eggs has been well reviewed
[7, 24, 39, 47]. Commonly accepted theories are a temporary
fast block followed by permanent block, which is mediated by
exocytosis of cortical granules to prevent additional sperm
penetration. However, Mio et al. recently [29] described dy-
namic changes of how human polyspermy occurs during real-
time development by using time-lapse cinematography. Their
discoveries challenged traditional concepts that two types of
polyspermy block were thought to exist in marine animals and
mammals, including human-“oolemma block” and “zona reac-
tion.” The authors reported that once the leading sperm pene-
trated the zona pellucida (ZP) and attached to the oocyte
membrane, any following sperm within the ZP immediately
Curr Obstet Gynecol Rep (2013) 2:226–231
would stop penetrating within 10 seconds. Then, the oocyte
membrane block happens.
More recently, Zumoffen et al. [51] reported that a protein
called lactoferrin, detected in human oviductal secretion, is
involved in gamete interaction, which may be associated with
polyspermy block. In vivo, the oviductal modification of ZP
resistance to proteolytic digestion has been demonstrated to
influence fertilization and this prefertilization mechanism is
considered to contribute to the control of polyspermy. ZP
resistance to proteolysis, induced by oviductal fluid, was ob-
served in mouse, rat, hamster, rabbit, sheep, goat, pig, and cow,
but not in human [30]. In the circumstances of IVF, the oocytes
are aspirated directly from the ovaries. They are not exposed to
the oviductal proteins. Obviously, proteolytic digestion is not a
prerequisite for human oocytes to be fertilized normally. How-
ever, the singletons derived from IVF/ICSI have poorer peri-
natal outcomes than those naturally born ([35], systematic
review and meta-analysis). In pigs, zona hardening is regulated
by the oviductal proteins. Such interaction is associated with
frequencies of monospermy and polyspermy [31]. Despite
differences of ZP resistance among species and human [30],
the oviductal contribution to egg quality and occurrence of
polyspermy cannot be ignored. Further studies are needed to
address the difference of oocytes/sperm exposed with or with-
out oviductal environment. Perhaps the short-term exposure to
the oviductal proteins/fluid would enable the oocytes to reach
full cytoplasmic maturation, which could reduce the occur-
rence of polyspermy.
Polyspermy and Oocyte Quality
Although polyspermy was considered abnormal fertilization,
in some species, physiological polyspermy does occur ([23•]
review). Dale and DeFelice [7] opened a debate about whether
mammalian oocytes actively repel supernumerary sperm.
Considering the fact that the ratio of sperm:oocyte at final
destiny in nature is so low, these authors indicated that
polyspermy preventing mechanisms may not be necessary.
Aged eggs appear to have reduced ability to prevent
polyspermy at the level of the oolemma, i.e., to establish a
membrane block to polyspermy [8]. However, women aged
45 years or older had the same fertilization rate via IVF and
ICSI [5••]. These authors suggested that zona hardening does
not appear to be a consequence of reproductive aging, which
would challenge the application of assisted hatching and also
the theory of zona hardening in the prevention of polyspermy.
The experiment using pig as a model also suggested that the
ZP and oolemma are not competent factors for the prevention
of polyspermy [43].
Mammalian oocyte maturation includes two independent
processes-nuclear maturation and cytoplasmic maturation [11].
In the controlled ovarian stimulation, the oocytes are retrieved
227
from follicles in different sizes. Although most oocytes com-
pleted first meiosis (metaphase II stage), with released first polar
body, their cytoplasmic maturation varies. More recently, it was
reported that mouse oocyte growth and differentiation are ge-
netically dissociable from the chromosomal events of the first
meiosis [10••], which further proved that completion of oocyte
nuclear maturation reaching metaphase II does not mean the
oocytes are ready to accept the sperm. The oocyte cytoplasm
could be at different status: immature, mature, or overmature.
Even with natural conditions, the egg is ovulated with different
status of cytoplasmic maturation. Most likely, polyspermy
happens to those eggs with immature cytoplasm or overmature
cytoplasm (aging). Therefore, theoretically, for the clinical
application of conventional IVF, the insemination time should
be based on their status of cytoplasmic maturity, meaning
that oocytes from the same patient should be inseminated at
different times.
Progesterone levels are closely related to the oocyte matu-
ration status, especially the ratio of estrogen/progesterone.
Increased level of progesterone decreases ZP hardening,
resulting in a high rate of polyspermy in pigs [30, 31]. In
humans, serum premature progesterone rise on the day of
human chorionic gonadotropin (hCG) administration has been
reported to be correlated negatively with live birth rate [20, 34].
These authors concluded the progesterone premature rise did
not affect pregnancy rates when transferring the embryos in
frozen cycles, indicating endometrium damage occurrence.
However, the results are controversial. Check et al. [6] pub-
lished results of a retrospective cohort analysis that egg donor
progesterone level (>3.4 ng/ml) may have lower pregnancy rate
in recipients. Studies regarding premature progesterone rise and
its association with polyspermy may shed light on improving
egg quality by using different ovarian stimulation protocols.
Application of in vitro maturation (IVM) revolutionized
the field of assisted reproductive technologies. Many publica-
tions have reported successful outcomes using different ap-
proaches of IVM. Midkine was reported to facilitate mamma-
lian oocyte cytoplasmic maturation [22]. To date, it is still
unknown whether IVM oocytes have lower polyspermic rate
because of application of ICSI in most cases [12, 38]. How-
ever, Walls et al. [46] demonstrated IVM oocytes (150 oocytes
of 8 cycles) achieved the same fertilization rate and clinical
pregnancy rate comparing two insemination approaches: con-
ventional IVF and ICSI. As application of conventional IVF to
in vitro matured oocytes, more information about polyspermic
occurrence of IVM would allow us to understand the relation-
ship of oocyte quality and polyspermy.
Perivitelline Space and Cortical Granules
Perivitelline space is the space between oolemma and zona
pellucida of the oocyte. This space does not develop in oocyte
228
at germinal vesicle stage until oocyte starts meiotic maturation.
Glycoproteins secreted by cumulus cells are involved in making
the space more obvious [42]. In mice, polyspermy occurs at a
higher rate in the oocytes with smaller PVS treated with
Tunicamycin, an inhibitor of glycoprotein synthesis [25]. In
the physiological condition, the size of PVS is not a reason for
causing polyspermy. It is a result of oocyte maturity, especially
for patients treated with the controlled ovarian stimulation.
Ovarian apoptosis happens at every stage of follicular develop-
ment. The oocytes retrieved from these follicles could come
from normal-size follicles but already at atretic stages. The
oocytes from the atretic follicles could be at immature or
overmature, thus resulting oocytes with bigger or small PVS.
The human oocyte grading [49] based on the size of PVS and
status of polar body (fragmented vs. nonfragmented) specified
the best synchronized oocyte morphology-normal PVS and
nonfragmented first polar body. The results of this publication
were from patients who underwent intracytoplasmic sperm
injection (ICSI) treatment involving a single sperm injection.
To date, no studies have been performed concerning whether
this group of oocytes (with normal PVS and intact first polar
body) have lower polyspermic rate using conventional IVF.
More criteria are needed regarding the association of oocyte
morphology and polyspermy. In another words, what type of
morphological features of oocytes has higher incidence of
polyspermy in humans?
Cortical granules (CG) in mammalian oocytes have been
well studied over the past decades (reviewed by [27]). The
content of CGs is released into the PVS immediately after
fertilization (exocytosis) for the establishment of polyspermy
block. Distribution of CGs in immature oocytes and in vitro
matured oocytes varies under different culture conditions
comparing with the CG distribution in vivo matured oocytes
in mice [28]. The total number of GCs in unfertilized oocytes
was increased during 3- to 6-hour culture in vitro. Increasing
number of GCs also was associated with fertilization and
embryo development [37]. These authors indicated that pro-
liferation of GCs could be used as criteria for nuclear matura-
tion. However, if CGs developed from Golgi complexes dur-
ing oocyte development [15, 27], their total number could be
more related to the cytoplasmic maturation rather than nuclear
maturation. Using a mouse model, Liu et al. [28] also sug-
gested that CGs were associated with oocyte cytoplasmic
maturation. Future studies are needed regarding the mecha-
nisms of CGs maturation and oocyte cytoplasmic maturation,
thus the polyspermy rate would be reduced via improving
cytoplasmic maturity.
Moreover, cortical granules can be released before fertili-
zation, after fertilization and even up to the first cleavage stage
[27]. Observing more than 3,000 embryos cultured in time-
lapse EmbryoScope, we found that CGs do not exist in PVS of
the oocyte at germinal vesicle stage but exist in the PVS of the
oocytes at metaphase II stage. They do not seem to have much
Curr Obstet Gynecol Rep (2013) 2:226–231
change morphologically until 2-cell stage. Then, after 4-cell
stage, the total number of granules in PVS is reduced and
disappeared by Day 3 of the culture (unpublished data).
Apparently, the cortical granules may continue their contri-
bution to the early development during cleavage stage,
perhaps up to blastocyst stage to facilitate embryo hatching.
Mouse ovastacin, a cortical granule protease, cleaves zona
pellucida protein 2 [4]. Swine CGs contain hydrolytic
enzymes, proteases, and peroxidases [1]. Hopefully, future
studies would prove the concept of contribution of CGs to
blastocyst hatching, which would shed light on understanding
the controversial results about application of the assisted
hatching in human IVF field.
Multiploidy - Failure of the Second Polar Body Extrusion
Mammalian oocytes undergo two rounds of meiotic divisions.
The first round results oocytes at Metaphase II stage with the
first polar body. The second meiotic division happens after
fertilization by releasing the second polar body (PB2). The polar
body extruding processes are driven by the spindle migration
which is controlled by cytoplasmic actin filaments [2, 26].
Failure of the second polar body extrusion is related to the
oocyte polarization during which a series of signaling cascade
is associated with the emission of PB2. These signal pathways
are not well studied in humans. In mice, it was recently
reported that Cdc42 activation (Cdc42-GTP) is related to the
mammalian oocyte polarization [9], together with down-
stream of cytoplasmic Ran-GTP gradient and upstream of
N-WASP activation at the oocyte cortex. These authors also
reported that Cdc42-GTP accumulates, in a polarized fashion,
in the cortex overlying the meiotic spindle during both meiosis
I and II in mice.
The second polar body contains one set of segregated
chromatids. If not released, it would form a nucleus-like
structure, which looks like the third pronucleus in the fertilized
egg. Clinically, it also is called triploid. Such triploids also
occur in the event of application of intracytoplasmic sperm
injection (ICSI), which only involves injection of a single
sperm. From our observation by time-lapse EmbryoScope,
77 of 3,213 (2.39 %) eggs injected were triploid. These
embryos do become 8-cell embryo and even form blasto-
cysts. Under such circumstances, triploid embryos generated
by ICSI are more prone to generating genetically abnormal
babies. A severe case was reported that a triploid infant, 69,
XXX karyotype, resulted from nondisjunction at maternal
second meiosis [3••, 18]. However, a small-scale study that
included 32 triploid embryos by ICSI and 18 by IVF has shown
that 50 % of these embryos could become normal blastocysts as
detected by fluorescence in situ hybridization (FISH) and pa-
rental inheritance analysis by PCR [14••]. These authors sug-
gested the ability of the self-correction depends on the parental
Curr Obstet Gynecol Rep (2013) 2:226–231
origin of the extra pronucleus, which indicated that the triploid
embryos via ICSI have higher chance of becoming normal
embryos, whereas the embryos derived by IVF could be at a
risk of genetic abnormalities. Regardless, the triploid embryos
derived from ICSI should be discarded in the IVF laboratories.
Multiploidy - Ability of Embryo’s Self-Correction
As mentioned earlier, triploid embryos via ICSI could become
normal embryos, but to date it is still unknown whether human
embryos derived from polyspermic fertilization possess the
ability of self-correction.
Studies in pigs have confirmed that the polyspermic eggs
were able to develop to term, resulting piglets with normal
chromosome numbers [17••]. The same group also reported
that the polyspermic eggs developed to the blastocyst stage at
a developmental rate similar to that of normal 2PN eggs [16].
In mice, the embryo at 2-cell stage is able to engulf sperm
present in the perivitelline space [44]. Ultrastructural studies
showed that approximately 50 % of blastomeres at the 3- and
4-cell stage contain accessory sperm heads and additional
sperm head was seen inside the lysosomal body [50].
Lysosomal proteolysis is triggered by posttranslational pro-
tein modification by ubiquitination in mammalian oocytes. The
deubiquitinating enzymes (DUBs) could reverse ubiquitination
process. Two members of Ubiquitin C-terminal hydrolase family
of DUBs, UCHL1 and UCHL3, are associated with murine
oocyte cortex (UCHL1) and meiotic spindle (UCHL3). Their
mRNAs are highly expressed in murine oocytes at germinal
vesicle stage and metaphase II stage [33]. Antibodies against
UCHL3 into mature metaphase II oocytes blocked fertilization
by reducing sperm penetration of the zona pellucida and sperm
incorporation into the ooplasm [32]. Sutovsky suggested that
polyspermy can be ameliorated by modulating sperm-associated
deubiquitinating enzymes ([41•], review). Perhaps the balancing
of ubiquitination and deubiquitination processes controls the
proteolysis process in oocytes and sperm, which may be asso-
ciated with removing extra sperm inside the oocyte cytoplasm
and blastomeres of embryos at the cleavage stages. Future
studies are needed in these areas.
Multiploidy and Mosaicism
Multiploidy resulting from polyspermy or retention of sec-
ondary polar body, or in rare cases binuclear sperm, is the
major cause of embryo mosaicism. Patients with diploid/
triploid chromosome mosaicism present significant clinical
abnormalities, including similar genetic syndromes to aberrant
genomic imprinting [36], dysmorphic formation [45], cutane-
ous pigmentary dysplasia [48], and hydatidiform mosaic mole
[40]. More recently, Huisman et al. [21] reported that patients
229
have high rate of somatic mosaicism (10/44; 23 %) for an
NIPBL (encodes Nipped-B-like protein) gene mutation,
which results in Cornelia de Lange syndrome.
Clinically, many mosaicism cases were underdiagnosed or
neglected. The patients could have normal karyotype with
abnormal clinical phenotype [3••]. The amniocentesis is not
sufficient to make diagnosis of diploidy-triploid mosaicism
[13]. Analyzing cleaved embryos by preimplantation genetic
diagnosis (PGD) also has the risk of missing the mosaicism.
The application of trophectoderm PGD at blastocyst stage also
would be subjected to the underdiagnosis of mosaicism due to
the fact that inner cell mass eventually differentiates into the
human body. Therefore, genetic prenatal testing for the babies
via application of assisted reproductive technologies should
be provided routinely to all patients.
Conclusions
Collectively, polyspermy is not a normal phenomenon. To
avoid it from happening requires improving the quality of
the oocytes. If additional sperm inside the oocyte did not affect
the embryonic genome, the embryos may have some ability of
removing the additional sperm. Under such conditions, if the
quality of oocyte is not favorable, the polyspermic embryos
would not survive, not because of the polyspermy but because
of poor oocyte quality. Therefore, improving oocyte quality
by variety of ovarian stimulation protocols in the ART field is
a key issue.
Compliance with Ethics Guidelines
Conflict of Interest Ping Xia declares no conflict of interest.
Human and Animal Rights and Informed Consent This article does
not contain any studies with human or animal subjects performed by any
of the authors.
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