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- Case Report -
Clinical pregnancies and livebirths achieved by
intracytoplasmic injection of round headed acrosomeless spermatozoa with
and without oocyte activation in familial globozoospermia:
case report
Enver K. Dirican, Ahmet Isik, Kubilay Vicdan, Eran Sozen, Zekiye Suludere
Department of Embryology, Private Ankara IVF Center, Ankara 06460, Turkey
Abstract
We report the successful outcome of intracytoplasmic sperm injection (ICSI) treatment in two siblings with
familial globozoospermia. After controlled ovarian hyperstimulation and oocyte pick-up, retrieved oocytes were
mechanically activated before ICSI and a fertilization rate of 33.3% was achieved in the first case. The second couple
underwent ICSI without oocyte activation and a 9.1% fertilization rate was obtained. The transfer of two grade I
embryos in the first couple and one grade I embryo in the second couple resulted in clinical pregnancies with healthy
livebirths. It was concluded that the main problem of cases with globozoospermia is a low fertilization rate, and even
though ICSI and oocyte activation can increase this rate it is not necessarily needed to achieve a pregnancy.
(Asian J Androl 2008 Mar; 10: 332_336)
Keywords: intracytoplasmic sperm injection; spermatozoa; acrosome; scanning electron microscopy
Correspondence to: Dr Enver K. Dirican, Department of Embryology, Private Ankara IVF Center, Ankara 06460, Turkey.
Tel: +90-312-472-3334 Fax: +90-312-472-3338
E-mail: kerem@dirican.tr.tc
Received 2006-09-20 Accepted 2006-11-06
DOI: 10.1111/j.1745-7262.2008.00248.x
1 Introduction
Globozoospermia is a severe form of terato-zoospermia characterized by round-headed acrosomeless
spermatozoa [1]. Analysis of the incidence of cases with globozoospermia demonstrated a history of consanguinity and a
familial occurrence. Familial cases of globozoospermia suggest that this pathology has genetic origins, but the mode
of inheritance remains unexplained, probably showing X-linked, sex-restricted dominant, or autosomal recessive
modes of inheritance [2]. Some genes responsible for globozoospermia in mice were identified [3].
Two types of globozoospermia have been described in previous studies. Type I is characterized by a complete
lack of acrosome and acrosomal enzymes, whereas type II has some acrosomal covering with a conical nucleus,
which may be surrounded by large droplets of cytoplasmic material indicating degenerative changes. Some reports
described high levels of sperm aneuploidy and abnormalities of chromatin packaging in globozoospermia [4, 5]. In
addition, the failure of spermatid differentiation causes abnormalities of the acrosome, nucleus, manchette, postacrosomal
sheath, posterior ring and the ectoplasmic specializations surrounding the spermatid [6]. It has been known that
round-headed spermatozoa not only lack acrosomes but also have deformed tails [7] and disorganized mid-pieces [8].
The round-headed spermatozoa cannot penetrate the zona pellucida of an oocyte and therefore cannot provide a
healthy fertilization, leading to male infertility. Therefore, globozoospermia was previously thought to be a sterilizing
pathology of the human male. After recent advances in
assisted reproductive technologies, particularly the
intracytoplasmic sperm injection (ICSI), few successful
fertilization or pregnancies have been reported [9_11].
However, even using ICSI treatment, the fertilization rates
were still poor and this was attributed to the inability of
the round-headed sperm to activate an oocyte mainly
because of premature chromatin condensation [12].
There are also reports about the effects of centrosomal
dysfunction of the round-headed spermatozoa on poor
fertilization rates after ICSI treatment [13].
In this report, we presented the successful outcome
of ICSI in two siblings with familial complete
globozoospermia detected by light and electron microscopy.
2 Case report
Two brothers were admitted to our center after 15
and 8 years of primary infertility. The ages of males
were 35 and 31 and their wives were 37 and 26 years
old, respectively. Neither of the brothers reported any
exposure to potentially toxic chemicals, radiation or other
potential environmental xenobiotics. Both brothers had
normal karyotypes (46, XY) in their peripheral blood
samples. Their hormonal profiles were within normal
range. A basic semen analysis revealed 81.6 ×
106 total sperm count with a 21% total and 5% progressive
motility in the first case and 210 ×
106 total sperm count with a 36% total and 13% progressive motility in the second
case. Strict morphological evaluation after SperMac
(Conception Technologies, San Diego, CA, USA)
staining method for human spermatozoa showed 100%
round-headed spermatozoa in both cases (Figure 1).
Ultrastructural characteristics of the round-headed
spermatozoa were evaluated by scanning electron microscope (JEOL JSM-6060LV; JEOL Technics Ltd.,
Akishima-Shi, Tokyo, Japan). Sperm samples were fixed
in 2.5% glutaraldehyde in 0.1 mol/L sodium cacodylate
buffer, washed in phosphate buffer saline, dehydrated in
alcohol gradients, prepared for microscopy after critical
point drying, and evaluated at 15 kV acceleration.
Detailed evaluation of the cells under SEM pointed out some
severe midpiece and tail abnormalities (Figure 2).
After giving detailed information on outcome, both
couples decided to undergo ICSI treatment. The
gynecological and infertility work-up of the female partners
revealed no abnormality. A microdose-flare protocol was
preferred in the first case because of the age and basal
ultrasonographic evaluation of the ovaries and long
protocol in the second case was carried out. In the first
case, 450 IU recombinant FSH (rFSH)/day (Puregon; Organon, Istanbul, Turkey) s.c. and 80 mg leuprolide
acetate/day (Lucrin; Abbot, Istanbul, Turkey) b.i.d. s.c.
were applied. In the second case, 225 IU rFSH with
0.5 mg leuprolide acetate/day s.c was used. Final
oocyte maturation and ovulation stimulation was provided
by 10 000 IU of HCG (Pregnyl; Organon, Istanbul,
Turkey). Thirty-five hours later, oocytes were retrieved
by transvaginal follicle aspiration under ultrasound
guidance with a total intravenous anesthesia. Six and 11
metaphase II oocytes were obtained respectively.
After denudation by hyaluronidase treatment, ICSI
procedure was performed by using Olympus IX 71 Inverted
Research Microscope (Olympus Corporation,
Shibuya-ku, Tokyo, Japan) equipped with Narishige ON-2
Microinjector (Narishige CO. LTD., Setagaya-ku, Tokyo,
Japan).
In both cases, ejaculated samples were allowed to be
liquefied, washed in bicarbonate buffered commercial
sperm wash media (Sperm Preparation Medium, Medicult,
Denmark), and layered on 95% discontinuous density
gradient medium carefully (Suprasperm, Medicult, Denmark). After 20 minutes of centrifugation at 300 ×
g, pellet was aspirated and washed twice in sperm wash
media, resuspended to the desired volume and placed in
the sperm droplet of the ICSI dish. ICSI was performed
after aggressive sperm immobilization.
In the first case, 6 mature oocytes were injected
after mechanical oocyte activation using a previously
described technique [19]. Briefly, plasma membranes of
the oocytes were broken using a microneedle to
maintain a Ca2+ influx and after a short period, the ICSI was
carried out. Two oocytes were fertilized (33.3%) and
cleaved to two 4-cell grade I embryos without any fragmentation and granulation with even blastomeres
on day 2. These embryos were transferred on day 2
after mechanically assisted hatching had been performed. The second case had 11 mature oocytes
and these oocytes were injected without oocyte activation. Only one oocyte was fertilized (9.1%) and
one 4-cell grade I embryo without any fragmentation
or granulation with even blastomeres transferred after
mechanical assisted hatching on day 2. Luteal phases
were supported by vaginal suppositories of micronized
progesterone 600 mg/day t.i.d. (Progestan, Kocak, Turkey). A singleton clinical pregnancy was obtained
in the first attempt in both couples. Pregnancies had
uncomplicated clinical courses and resulted in delivery
of two healthy female infants at 38 weeks and 38.5
weeks respectively. Table 1 shows the patient and cycle
characteristics.
3 Discussion
Teratozoospermia is a common phenotype with
several deformations ranging from cellular to subcellular levels
in the head and tail of spermatozoa [15]. This condition
is frequently associated with infertility and usually ICSI
is used as the treatment of choice. However, severe
sperm morphological abnormalities may result in poor
fertilization rates and embryo quality even with ICSI
application [16]. Many studies reported various low
fertilization rates with complete globozoospermic samples and
total fertilization failures were observed in some cases
[9]. Previous studies showed that the majority of the
oocytes injected by round-headed acrosomeless
spermatozoa remain intact and cannot complete second meiotic
division, probably because of the inability of the
round-headed sperm to activate an oocyte as a result of
premature chromatin condensation [12,17].
Globozoospermia, which is a pathology of acrosome,
probably shows X-linked, sex-restricted dominant, or
autosomal recessive modes of inheritance [2], but so far,
no responsible genes have been identified. Recently,
murine studies revealed some hereditary information on
severe teratozoospermia and globozoospermia [3]. Since
these patients display normal haploid, sex chromosome
and aneuploidy status, ICSI can be conceivably offered
as a treatment for their infertility [18]. There have been
several reports of healthy offsprings resulting from ICSI
applications with round-headed spermatozoa [9_11]. Our
cases were counseled under the light of the published
literature and were informed about the elevation of
aneuploidy rates [4] and possible inheritance of
globozoospermia leading to infertility in the male offspring.
Both cases decided to undergo ICSI treatment.
In familial globozoospermia, pregnancies and one
livebirth were achieved previously without oocyte
activation in repeated ICSI treatment cycles [11]. One of
our cases underwent oocyte activation procedure together
with ICSI and achieved a livebirth without repeated ICSI
treatment cycles in familial globozoospermia.
It was previously reported that oocyte activation prior
to ICSI does not result in better fertilization rates [19].
On the contrary, it was suggested that assisted oocyte
activation enables normal fertilization and pregnancy in
cases with sperm and oocyte related fertilization failure
caused by the impairment of the oocyte activation [14].
Mechanical oocyte activation in our study yielded a
better fertilization rate and two good-quality 4-cell embryos
were obtained in this case. The second case without
oocyte activation had only one fertilized oocyte, although
more oocytes were injected. Oocyte activation in this
case was not performed because of the availability of
more than 10 mature oocytes at that time but a low
fertilization rate was achieved probably because of
abnormal chromatin condensation [12] or centrosomal
dysfunction [13].
In conclusion, this report supported the genetic
origin of globozoospermia with a familial occurrence in two
brothers. Although the fertilization rate was still low
after ICSI, current ICSI procedures may overcome the
infertility associated with globozoospermia and result in
normal healthy livebirth with and without assisted
oocyte activation. In our cases, oocyte activation before
ICSI resulted in a better fertilization rate, and provided
more fertilized oocytes.
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