Relationship between first polar body morphology before intracytoplasmic sperm
injection and fertilization rate, cleavage rate, and embryo quality
Paula Andrea Navarro⁎, Maria Medeiros de Araújo, Carlos Medeiros de Araújo, Marcelo Rocha,
Rosana dos Reis, Wellington Martins
Department of Obstetrics and Gynecology, Ribeirão Preto Medical School, São Paulo University, Ribeirão Preto, São Paulo, Brazil
a b s t r a c t a r t i c l ei n f o
Received 31 July 2008
Received in revised form 3 November 2008
Accepted 10 November 2008
Assisted reproduction outcome
First polar body
Intracytoplasmic sperm injection
Objectives: To evaluate the influence of the morphology of the first polar body (PB) on intracytoplasmic
sperm injection (ICSI) outcomes. Methods: The morphology of the first PB was assessed in 3177 metaphase II
oocytes and classified as: intact and normal size, fragmented, or enlarged size. The rates of fertilization,
cleavage, and embryo quality were evaluated on day 2. Results: The rates of fertilization, cleavage, and
formation of good quality embryos resulting from the insemination of oocytes with an enlarged first PB
(20.7%,18.7%, and 5.0%, respectively) were significantly lower than those for oocytes with an intact first PB of
normal size (70.8%, 62.5%, and 19%, respectively) or a fragmented first PB (69.7%, 60.5%, and 17.1%,
respectively). Rates did not differ significantly between oocytes with an intact first PB of normal size and
oocytes with a fragmented first PB (PN0.05). Conclusions: The presence of an enlarged PB is related to poorer
rates of fertilization, cleavage, and top quality embryos. However, identification of first PB fragmentation does
not seem to interfere with ICSI outcomes.
© 2008 InternationalFederationofGynecologyandObstetrics.PublishedbyElsevierIrelandLtd.Allrightsreserved.
The introduction of intracytoplasmic sperm injection (ICSI) as an
assisted reproduction technique has provided substantial information
about oocyte morphology since the method requires oocyte denudation
by eliminating the cumulus oophorus cells. This permits better observa-
tion before and during the fertilization process, with the possibility of
correlating the parameters of oocyte morphology with embryo quality
and viability [1,2]. The removal of cumulus cells before ICSI permits the
assessment of many morphological parameters of the oocyte such as
oocyte shape, cytoplasm color and granulation, regularity and thickness
of the zona pellucida, the size of the perivitelline space, the presence of
vacuoles, the presence or absence of the germinative vesicle and of the
and the use of objective criteria able to predict oocyte quality would
permit improved results of assisted reproduction techniques byassisting
in oocyte and embryo selection, as well as minimizing the ethical prob-
lems related to embryo freezing because fewer oocytes would be
inseminated and therefore fewer embryos of inadequate quality would
be produced [4,5]. However, the influence of the morphological charac-
on embryo quality is still a matter of controversy [6–8].
ICSI is performed when the oocytes are classified as mature, i.e.,
when they reach metaphase II (MII), which is characterized by the
observation of the first PB under the microscope . The absence of
the first PB, in turn, may indicate that the oocyte is still immature or
that it is already post mature and inadequate for insemination in
either case. The morphology of the first PB was initially thought to
indicate the postovulatory age of the oocyte . However, some
studies have shown that the transfer of embryos selected on the basis
of first PB morphology resulted in a higher fertilization rate and in
embryos of better quality, with higher implantation and pregnancy
rates [6,10–13]. On the other hand, these data are controversial, with
contradictory results reported in the literature [14–16].
The establishment of simple criteria for use with light microscopy to
help embryologists choose the embryo(s) for transfer is fundamental for
improving pregnancy rate and reducing the incidence of multiple preg-
criteria that could predict oocyte quality—one of the most important
determinants of embryo quality —and considering the controversies
the rates of success of assisted reproduction techniques, the aim of the
present study was to assess the effect of first PB morphology on
fertilization and cleavage rates, and on embryo quality.
2. Materials and methods
The study consisted of a retrospective longitudinal analysis of 3177
metaphase II oocytes obtained from 582 consecutive ICSI cycles of
International Journal of Gynecology and Obstetrics 104 (2009) 226–229
⁎ Corresponding author. Department of Obstetrics and Gynecology, Ribeirão Preto
Medical School, São Paulo University, Av. Bandeirantes, 3900, Ribeirão Preto, São Paulo,
14049-900 Brazil. Tel.: +55 16 3602 2821; fax: +55 16 3633 0946.
E-mail address: email@example.com (P.A. Navarro).
0020-7292/$ – see front matter © 2008 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.
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patients under 40 years at the Assisted Reproduction Laboratory of
the University Hospital, Faculty of Medicine of Ribeirão Preto, Uni-
versity of São Paulo, from July 2003 to July 2005. Only severe male
factor infertility (sperm count b100 000/mL, sperm morphology b4%
according to Kruger strict criteria, and/or azoospermic patients) was
excluded from the study.
The menstrual period was programmed with the use of combined
oral contraceptives. For pituitary suppression, 10 IU/day subcuta-
neously of GnRH agonist leuprolide acetate (Lupron, Abbott Labora-
tories, Abbott Park, USA; or Reliser, Serono, Sao Paulo, Brazil), or
400 µg/dayof nafarelin (Synarel; Pharmacia, Brazil) by the nasal route,
was administered starting 10 days before the programmed menstrua-
tion. Transvaginal ultrasound (TVUS) was performed from the 1st to
the 3rd dayof the menstrual cycle toconfirm ovarian suppression, and
controlled ovarian stimulation was then started using recombinant
follicle-stimulating hormone (Puregon, Organon, Brazil; or Gonal-F,
Serono, Brazil) at a dose of 200–400 IU/day during the first 5 days and
adjusted according to ovarian response starting on the 6th day of
stimulation. When 2 or more follicles ≥18 mm in mean diameter were
present, 10 000 IU of urinary hCG (Profasi; Serono, Brazil) was admi-
nistered intramuscularly, or 250 µg of recombinant hCG (Ovidrel;
performed 34–36 hours after hCG administration. Follicles were
aspirated with a negative pressure of 110 mm Hg with transvaginal
ultrasound guidance. The cumulus–oocyte complexes (COC) were
separated from the follicular fluid under a stereomicroscope (Nikon
SMZ645) and washed in human tubal fluid (HTF; Irvine Scientific,
Santa Ana, CA, USA) culture medium modified with HEPES (Irvine
Scientific) supplemented with 10% synthetic serum substitute (SSS;
Irvine Scientific) before being transferred to culture plates previously
incubated at 37 °C in the presence of 5% CO2in air, containing HTF
culture medium supplemented with 10% SSS. After 2–5 hours of incu-
bation in this medium, cumulus cells were removed by COC exposure
by mechanical removal with glass pipettes.
Semen samples were obtained soon after oocyte retrieval and
manipulated and analyzed for ICSI according to previous recommen-
of HTF-HEPES supplemented with 10% SSS on Falcon plates and
observed under the inverted microscope (400×magnification) with
Hoffman optical contrast. The morphology of the first PB was assessed
sizewith an ovoid shape and a smooth surface; (2) fragmented first PB
with an irregular shape and a rugose surface; and (3) first PB of en-
larged size much larger than those of the previous two groups and
without fragmentation (Fig.1). A single observer (MCPMA) character-
ized the first PB according to the criteria of Ebner et al. .
ICSI was performed as previously described , 36–41 hours after
hCG administration. After ICSI, the oocytes were incubated separately
in 25-µL microdroplets of HTF plus 10% SSS, at 37 °C in the presence of
visualization of 2 pronuclei and 2 PBs being considered to represent
fertilization. Oocytes presenting 3 or more pronuclei were classified as
abnormal and were discarded. Cleavagewas verified at about 24 hours
after fertilization by the observation of cell division. Embryo quality
was assessed according to variables such as percent cytoplasm frag-
mentation, symmetry, and number of blastomeres. On the second day
after ICSI, embryos with 4 cells, symmetrical blastomeres, and no
fragmentation were considered to be of good quality.
For statistical analysis, the rates of fertilization, cleavage, and for-
with an intact first PB of normal size were compared with a fragmented
Prism 4.0 software (GraphPad Software Inc., San Diego, CA, USA). The
level of significance was set at 5% (Pb0.05).
Atotalof 3177metaphase IIoocytesobtainedfrom582consecutive
cycles for ICSI were analyzed and divided into 3 categories according
to first PB morphology: (1) intact first PB of normal size (n=1366);
(2) fragmented first PB (n=1448); and (3) enlarged first PB (n=363)
Of the 1366 oocytes with a normal first PB that were injected, 967
were normally fertilized (70.8%), 854 cleaved (62.5%), and 260
Fig.1. Morphology of the first polar body (PB): (A) intact polar body of normal size; (B) fragmentedpolar body; (C) enlarged polar body. Images obtained by inverted light microscopy
at 400×magnification. Scale bar=20 µm.
Distribution of the rates of fertilization, cleavage, and goodqualityembryos accordingto
first polar body morphology
Parameters First polar body morphology
Inseminated mature oocytes
No. of fertilized oocytes
Fertilization rate, %g
Cleavage rate, %h
Good quality embryos
Proportion of good quality embryos, %
a,c,eSame superscript letters on the same line indicate the absence of significant
a,b,c,d,e,fDifferent superscript letters on the same line indicate a significant difference
(Pb0.05 using Fisher exact test).
gFertilization rate: number of fertilized oocytes×100 divided by the number of oocytes
hCleavage rate: number of cleaved embryos×100 divided by the number of fertilized
iProportion of good quality embryos: number of embryos with 4 symmetrical cells
without fragmentation on the second day after ICSI×100 divided by the total number of
P.A. Navarro et al. / International Journal of Gynecology and Obstetrics 104 (2009) 226–229
progressed to good quality embryos (19%). Of the 1448 mature oocytes
with a fragmented first PB that were injected, 1008 were normally
fertilized (69.7%), 876 cleaved (60.5%), and 248 progressed to good
PB that were injected, 75 were normally fertilized (20.7%), 68 cleaved
(18.7%), and 18 progressed to good quality embryos (5%) (Table 1).
The rates of fertilization, cleavage, and formation of good quality
embryos resulting from the insemination of oocytes with an enlarged
first PB were significantly lower than those for oocytes with an intact
first PB of normal size or with a fragmented first PB. These rates did
not differ significantly between oocytes with an intact first PB of
normal size and oocytes with a fragmented first PB (Table 1).
is important to identify and use noninvasive and harmless parameters
quality and of the subsequent success of the procedure performed. The
removal of cumulus cells before ICSI provides for better oocyte
observation before and during the fertilization process, permitting a
correlation of oocyte morphology parameters, among them the
presence and morphology of the first PB, with embryo quality and
viability. However, data are controversial about the influence of first PB
morphology on ICSI outcomes, a fact that motivated the present study.
The oocytes retrieved after ovarian stimulation with gonadotropins
may present different degrees of nuclear maturation and of first PB
nuclearmaturation.ICSI is performed when theoocytesare classifiedas
observation of the first PB under the microscope . The absence of the
already post mature, both stages being inadequate for insemination.
Oocyte maturation from prophase I to metaphase II starts with the
break of the germinative vesicle associated with a reduced flow of
meiosis inhibitors released from the granulosa cells to the oocyte .
At this point the oocytes should have reached the ideal level for
nuclear and cytoplasmic maturation necessary to guarantee adequate
conditions of fertilization and subsequent embryo development. The
literature emphasizes that synchrony between oocyte nuclear and
cytoplasmic maturity is a fundamental aspect for the good develop-
ment and viability of the embryo . There are controversial reports
indicating that the presence of an intact first PB and of a normal
perivitelline space may suggest a better synchrony between the
cytoplasmic and nuclear maturation of the oocyte and consequently a
better oocyte quality . These data were supported by encouraging
results [10,11] suggesting an important prognostic role of first PB
morphology regarding embryo quality.
We detected lower rates of fertilization, cleavage, and formation of
good quality embryos resulting from oocytes with an enlarged first PB
compared with oocytes with an intact first PB of normal size or a
fragmented first PB. Our results agree with those reported by others
[21,22], suggesting that oocytes with these morphological character-
istics should not be used for ICSI procedures. We believe that the
presence of an enlarged first PB may be associated with disproportional
of normal fertilization and embryo development preimplantation, a
possibility that requires further elucidation.
In the present study, no significant differences were observed
between the rates of fertilization, cleavage, and formation of good
quality embryos resulting from ICSI of oocytes with an intact first PB
of normal size or with a fragmented first PB, suggesting that the
presence of first PB fragmentation does not interfere with these
variables. Our findings are similar to those reported in other studies
that also observed a relationship between first PB fragmentation and
fertilization rates or embryo quality [7,14,15].
There are indications that fragmentation of the first PB may result
from oocyte aging during in vitro culture . Thus, different protocols
of gamete manipulation and the time interval between oocyte re-
trieval, oocyte denudation, and the execution of ICSI may be respon-
siblefor thedifferencesin therates ofsuccessandfor thecontroversial
results reported in the literature about the potential influence of first
PB morphology on fertilization rates, embryo quality, and subsequent
implantation. Similarly, different protocols of ovarian stimulation and
different criteria for evaluation may also contribute to the divergence
The current data suggest that the presence of an enlarged first polar
does not seem to interfere with the variables and therefore does not
justify oocyte and/or embryo selection based on the analysis of this
parameter. Different protocols of ovarian stimulation, details regarding
oocyte and embryo manipulation and culture, and different criteria for
analysis, among other factors, may contribute to the divergent data
reported in the literature, which requires more detailed evaluation in
studies involving pertinent methodology.
 De Santis L, Cino I, Rabellotti E, Calzi F, Persico P, Borini A, et al. Polar body
morphology and spindle imaging as predictors of oocyte quality. Reprod Biomed
 Ebner T, Moser M, Tews G. Is oocyte morphology prognostic of embryo
developmental potential after ICSI? Reprod Biomed Online 2006;12(4):507–12.
 Ebner T, Moser M, Sommergruber M, Gaiswinkler U, Shebl O, Jesacher K, et al.
Occurrence and developmental consequences of vacuoles throughout preimplan-
tation development. Fertil Steril 2005;83(6):1635–40.
 Kahraman S, Yakin K, Donmez E, Samli H, Bahce M, Cengiz G, et al. Relationship
between granular cytoplasm of oocytes and pregnancy outcome following intra-
cytoplasmic sperm injection. Hum Reprod 2000;15(11):2390–3.
 Meriano JS, Alexis J, Visram-Zaver S, Cruz M, Casper RF. Tracking of oocyte
dysmorphisms for ICSI patients may prove relevant to the outcome in subsequent
patient cycles. Hum Reprod 2001;16(10):2118–23.
 Ebner T, Moser M, Sommergruber M, Yaman C, Pfleger U, Tews G. First polar body
morphology and blastocyst formation rate in ICSI patients. Hum Reprod 2002;17(9):
 Ciotti PM, Notarangelo L, Morselli-Labate AM, Felletti V, Porcu E, Venturoli S. First
polar body morphology before ICSI is not related to embryo quality or pregnancy
rate. Hum Reprod 2004;19(10):2334–9.
 Eichenlaub-Ritter U, Schmiady H, Kentenich H, Soewarto D. Recurrent failure in
polar body formation and premature chromosome condensation in oocytes from a
human patient: indicators of asynchrony in nuclear and cytoplasmic maturation.
Hum Reprod 1995;10(9):2343–9.
 Rienzi L, UbaldiFM, Iacobelli M,Minasi MG, RomanoS,Ferrero S,etal.Significance of
metaphase II human oocyte morphology on ICSI outcome. Fertil Steril 2008;90(5):
 Ebner T, Moser M, Yaman C, Feichtinger O, Hartl J, Tews G. Elective transfer of
embryos selected on the basis of first polar body morphology is associated with
increased rates of implantation and pregnancy. Fertil Steril 1999;72(4):599–603.
 Ebner T, Yaman C, Moser M, Sommergruber M, Feichtinger O, Tews G. Prognostic
value of first polar body morphology on fertilization rate and embryo quality in
intracytoplasmic sperm injection. Hum Reprod 2000;15(2):427–30.
 Xia P. Intracytoplasmic sperm injection: correlation of oocyte grade based on polar
body, perivitelline space and cytoplasmic inclusions with fertilization rate and
embryo quality. Hum Reprod 1997;12(8):1750–5.
 Ubaldi F, Rienzi L. Morphological selection of gametes. Palcenta 2008;29:115–20
 Verlinsky Y, Lerner S, Illkevitch N, Kuznetsov V, Kuznetsov I, Cieslak J, et al. Is there
any predictive value of first polar body morphology for embryo genotype or
developmental potential? Reprod Biomed Online 2003;7(3):336–41.
 De Sutter P, Dozortsev D, Qian C, Dhont M. Oocyte morphology does not correlate
with fertilization rate and embryo quality after intracytoplasmic sperm injection.
Hum Reprod 1996;11(3):595–7.
 Balaban B, Urman B, Sertac A, Alatas C, Aksoy S, Mercan R. Oocyte morphology
does not affect fertilization rate, embryo quality and implantation rate after
intracytoplasmic sperm injection. Hum Reprod 1998;13(12):3431–3.
 Balaban B, Urman B. Effect of oocyte morphology on embryo development and
implantation. Reprod Biomed Online 2006;12(5):608–15.
 Sharlip ID, Jarow JP, Belker AM, Lipshultz LI, Sigman M, Thomas AJ, et al. Best
practice policies for male infertility. Fertil Steril 2002;77(5):873–82.
 Van Steirteghem AC, Nagy Z, Joris H, Liu J, Staessen C, Smitz J, et al. High
fertilization and implantation rates after intracytoplasmic sperm injection. Hum
P.A. Navarro et al. / International Journal of Gynecology and Obstetrics 104 (2009) 226–229
 Pickering SJ, Johnson MH, Braude PR, Houliston E. Cytoskeletal organization in Download full-text
fresh, aged and spontaneously activated human oocytes. Hum Reprod 1988;3(8):
 Martini E, Flaherty SP, Swann NJ, Payne D, Matthews CD. Analysis of unfertilized
oocytes subjected to intracytoplasmic sperm injection using two rounds of
fluorescence in-situ hybridization and probes to five chromosomes. Hum Reprod
 Fancsovits P, Tothne ZG, Murber A, Takacs FZ, Papp Z, Urbancsek J. Correlation
between first polar body morphology and further embryo development. Acta Biol
P.A. Navarro et al. / International Journal of Gynecology and Obstetrics 104 (2009) 226–229