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International Journal of Reproductive BioMedicine
Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences of Yazd
ISSN: 1680-6433 EISSN: 2008-2177
Vol. 1, Num. 1, 2003, pp. 1-6
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Iranian Journal of Reproductive Medicine, Vol. 1, No. 1, 2003, pp. 1-6
Is There a Place for Round and Elongated Spermatids Injection
inAssisted Reproduction?
Byron
Asimakopoulos1, Nikos Nikolettos1, Safa Al-Hasani2
1 Laboratory of
Reproductive Physiology - IVF, Faculty of Medicine, Democritus Univ. of Thrace,
Dragana, 68100 Alexandroupolis, Greece - Hellas.2
Department of Obstetrics/Gynecology, Universitat zu Schleswig-Holstein,
Ratzerburger Allee 160, D-23538 Lübeck, Germany.
Corresponding Author: Safa Al-Hasani, Department of Obstetrics/Gynecology,
Universitat zu Schleswig-Holstein, Ratzerburger Allee 160, D-23538 Lübeck, Germany. E-mail: Sf_alhasani@hotmail.com
Code Number: rm03001
Summary
Spermatids are the earliest male germ
cells with one set of haploid chromosomes. After experiments, mainly in
rodents, the spermatid injection was introduced in human assisted reproduction
to the treatment of men with non-obstructive azoospermia. Spermatid injection
is a technique with particular difficulties that may negatively influence the
outcome. The identification, isolation and the assessment of viability,
especially for round spermatids, require intensive work and considerable
experience. Up to date, it appears that the rates of fertilization and
implantation with round spermatid injection are dramatically low and
significantly less compared to the use of elongated spermatid injection. The
extremely low fertilization potency of the round spermatids led to attempts for
their in-vitro culture and maturation. The immaturity of round and elongated
spermatids has raised concerns regarding the potential genetic risk for the
offspring. Under these facts, a reconsideration of the use of spermatids in
assisted human reproduction is necessary.
Introduction
Forty years have passed since the first intracytoplasmic sperm
injection (ISCI). It was 1962 when sea urchin eggs were successfully fertilized
by microinjection of live spermatozoa (Hiramoto, 1962). Thirty years later this
technique was introduced for the treatment of human infertility (Palermo et
al., 1992). Today, ICSI is the state of the art in assisted reproduction
technologies. It gives reliable solutions in cases of severe male factor
infertility there are still cases of azoospermia where ICSI is not possible
even after MESA or TESE. In such cases, spermatid injection is considered as
a
promising alternative.
Spermatids are the youngest male germ cells with a single set
of haploid chromosomes (complete meiosis). Once, they have completed meiosis,
they undergo a complex cellular differentiation and maturation process known
as spermiogenesis. Spermiogenesis starts at puberty and continues throughout
the
reproductive life of males. During spermiogenesis, round spermatids (Sa) have
approximately 7μm size, which will transform into mature
spermatozoa. One
of the most important changes that takes place in this process is nuclear DNA
packaging. DNA condensation is associated with biochemical alterations such as
the replacement of lysine-rich histones, first by transition proteins and later
by arginine-rich protamines, as well as with the formation
of disulphide bonds that stabilize the formation of disulphide
bonds that stabilize the chromatin structure (Nikolettos et al., 1999; de Kretser
et al., 1998; de Kretser and
Kerr, 1969;). As a result of DNA condensation, the cell size is reduced; thereby
less energy is required to support its mobility and the cell is better protected
against mechanical and chemical
damage (Nikolettos et al., 1999). Other important changes during spermiogenesis
include the process of genomic imprinting, the disappearance of the distal
centriole and the formation of the acrosome.
Spermatids as the only finding
of TESE
It is known that various pathological conditions can lead in
spermatogenetic abnormalities with resultant subfertility or infertility
(Martin-du Pan and Campana, 1993). In cases of non-obstractive azoospermia with
a lack of spermatozoa in the ejaculate, TESE is considered as the next step of
the treatment. For most of these cases, TESE results in retrieval of enough
spermatozoa to proceed in ICSI cycles, while in other cases spermatids are
completely absent. In the latter cases, round and elongated spermatids may be
present, indicating spermatogenesis arrest after the meiosis stage. In a large
series of TESE (N=364) performed at the Medical University of Lübeck, the
incidence of such cases was 3.9%:2.2% elongated spermatids were found, whereas
only 1.7% round spermatids were observed (Al-Hasani, unpublished data).
Although it is still controversial, it seems that in most of the cases the
spermatogenesis arrest happens in elongated spermatid stage. According to
Cremades et al. (1999) the recovery of only round spermatids is a frequent
finding in non-obstructive azoospermic patients with complete absence of
spermatozoa. On the other hand, Schulze et al. (1999) reported that from 1418
testicular biopsies (766 subfertile men), only in 26 samples spermatogenesis
arrest was in the round spermatid stage. There are also other investigators
supporting that maturation arrest is extremely rare in the round spermatid
stage (Silber and Johnson, 1998; Silber et al., 1997; 2000).
Isolation
and identification
In wet preparation, the identification of round spermatids has
many difficulties mainly due to their morphological similarities with small
lymphocytes (Vanderzwalmen et al., 1998; Silber et al, 2000). A considerable
experience is necessary for reliable identification of round spermatids and
avoidance of mistakes. Under the inverted microscope, without using any
specific staining methods, four different stages of spermatids can be
distinguished, according to their morphology: round spermatids (Sa, Sb1),
elongating spermatids (Sb2), elongated spermatids (Sc, Sd1) and late elongated
spermatids (Sd2) (Vanderzwalmen et al., 1998). Round spermatids appear as round
cells with a diameter of 7μmwhich are characterized by a dense, smooth and
dark nucleus positioned centrally or inclining towards the cell membrane. The
nucleus is surrounded by a continuous rim of cytoplasm. In some cells, the
early acrosomal vesicle or acrosomal cap is visible as a bright white spot or
sickle-shaped adjacent to the nucleus (Sousa et al., 1998; Verheyen et al., 1998;
Tesarik and Mendoza, 1996;). The distinction of elongating and elongated
spermatids is made on the basis of their shape and the size of the tail. The
mature spermatids (late elongated) appear to be similar to the ultimate sperm
morphology (Vanderzwalmen et al., 1998). The appearance of abnormal spermatid
forms in the preparation makes the identification procedure more difficult.
The assessment of spermatid
viability is a hard task, particularly of the round ones (Schoysman et al.,
1999). Aslam et al. (1998), using the Trypan blue exclusion test, found that
97% of the collected round spermatids were viable. However, distinguishing the
viable round spermatids from the non-viable ones, without staining or
destroying the cells, is not an easy task. Usually, the viability of round
spermatids is estimated during aspiration, according to their ability to
undergo a reversible deformation. The dead round spermatids are usually
subjected to lysis upon spiration (Vanderzwalmen et al., 1998; Tesarik and
Mendoza, 1996).
Spermatids injection in assisted reproduction
The concept of using round
spermatids in ICSI cycles was born in 1993, when Ogura and his collaborators
reported that the spermatids nuclei were able to duplicate their DNA and
participate in syngamy when incorporated into hamster or mouse oocyte either
by microsurgery or by electro fusion (Ogura and Yanagimachi, 1993; Ogura et
al.,
1993). A year later, Ogura et al (1994) reported the normal birth of four young
mice after electro fusion of oocytes with round spermatids; while, Sofikitis
et
al. (1994) reported successful pregnancy after injection of round spermatids
nuclei into rabbit oocytes. In 1995, the same research center reported that
mouse oocytes developed into normal offspring after injection with testicular
round spermatids (Kimura and Yanagimachi, 1995). Based on these animal tudies,
Edwards et al (1994) put forward the question: are spermatid injections into
human oocytes now mandatory? That suggestion along with the success of these
animal experiments was the (1995) reported the successful fertilization of an
oocyte after injection with late stage testicular spermatid and during the next
years several papers presented pregnancies achieved after spermatid injection
(Table I).
Recently, results for the development of blastocysts after
testicular round spermatid injection were published. Balaban et al. (2000)
reported that 34% of the embryos derived from round spermatids injections
reached the blastocyst stage, but none of them hatched. Urman et al. (2002)
also presented results from transfer of blastocysts derived from injection with
testicular round spermatids: with a fertilization rate of 19.7%, the blastocyst
stage reached by only a few embryos (7.6%), which failed to implant.
It is clear that the reproductive potency of round spermatids
is inferior to that of elongated spermatids. So far, the fertilization rate
with round spermatids appears to be low ranging from 20% to 25% in most of the
reported cycles, while the fertilisation rate with elongated spermatids is
significantly higher, being between 40% to 60%. Correspondingly, the
implantation rate is extremely low with round spermatids, while it is higher
with the elongated ones.
In-vitro
maturation attempts
Obviously, the aforementioned poor results reduced the previous enthusiasm
for the use of spermatids in assisted reproduction procedures. However it is
true
that while the outcome of round spermatid-ICSI cycles is totally disappointing,
the use of elongated spermatids resulted in better fertilization and pregnancy
rates. Consequently, attempts for the in-vitro
maturation of spermatids were made in order to solve the problem. Tesarik et
al. (2000,1999, 1998) presented some encouraging results on in-vitro maturation
of primary spermatocytes and round spermatids, especially using media
supplemented with rFSH. Cremades et al. (1999) managed to obtain elongating
spermatids and a few mature spermatozoa, after a prolonged co-culture on Vero
cells, in four cases. Aslam and Fishel (1999) found out that although short
term in-vitro culture of the spermatogenetic cells has a positive effect; it
does not improve the incidence of fertilization significantly. They pointed out
that spermatid maturation, which takes about 16 days to complete in-vivo, can
not be accelerated in- vitro, especially within 48 z(Aslam and Fishel,
1999).
Problems and Concerns
Which are the main problems responsible for the poor
outcome of spermatid injection, especially the round one?
Table I: Published papers reporting pregnancies achieved after round
(ROS) or elongated (ELS) spermatid injection.
|
Type of spermatid
|
No of cycles
|
No of injected oocytes
|
No (%) of 2PN oocytes
|
No of transferred embryos
|
No of pregnancies
|
Outcome
|
Tesarik et al. (1995)
|
ROS
|
|
39
|
14 (36)
|
>7
|
2
|
Born (first world birth)
|
Fishel et al. (1995)
|
ELS
|
1
|
10
|
1 (10)
|
1
|
1
|
Born
|
Mansour et al. (1996)
|
ROS/ELS
|
15
|
105
|
40 (38)
|
32
|
1
|
Born
|
Antinori et al. (1997a)
|
ROS
|
29
|
211
|
117 (55.4)
|
81
|
4
|
2 ongoing
|
|
ELS
|
34
|
229
|
158 (69)
|
119
|
7
|
4 ongoing, 1 born
|
Antinori et al. (1997b)
|
ELS
|
9
|
116
|
_
|
_
|
3
|
Born (1 twin, 2 singletons)
|
Amer et al. (1997)
|
ROS
|
31
|
251
|
63 (25)
|
_
|
4
|
Biochemical
|
|
ELS
|
3
|
34
|
19 (56)
|
_
|
2
|
Ongoing
|
Araki et al. (1997)
|
ELS
|
9
|
116
|
_
|
_
|
3
|
Born (1 twin, 2 singletons)
|
Vanderzwalmen et al. (1997)
|
ROS
|
32
|
260
|
57 (22)
|
_
|
1
|
Born
|
|
ELS
|
5
|
21
|
15 (71)
|
_
|
4
|
2 born, 1 ongoing
|
Barak et al. (1998)
|
ROS
|
8
|
37
|
10 (27)
|
_
|
1
|
Born
|
Barros et al. (1998)
|
ELS
|
7
|
_
|
_
|
_
|
3
|
1 born, 2 ongoing
|
Bernabeu et al. (1998)
|
ELS
|
1
|
7
|
3 (43)
|
3
|
1
|
Born
|
Kahraman et al. (1998)
|
ROS
|
20
|
199
|
51 (26)
|
32
|
1
|
Biochemical
|
|
ELS
|
3
|
31
|
22 (71)
|
11
|
2
|
1 born (twins)
|
Sofikitis et al. (1998)
|
ELS
|
13
|
79
|
52 (66)
|
41
|
2
|
Born
|
Al-Hasani et al. (1999a, 1999b)
|
ELS
|
2
|
18
|
10 (56)
|
6
|
2
|
Ongoing
|
Gianaroli et al. (1999)
|
ROS
|
1
|
5
|
2 (40)
|
2
|
1
|
Born
|
Tesarik et al. (1999)
|
ELS
|
1
|
6
|
|
|
|
Born (twin)
|
Saremi et al. (2002)
|
ROS
|
|
|
|
|
1
|
Born
|
Sousa et al. (2002)
|
ELS (cases with hypoplasia)
|
10
|
73
|
34 (49.3)
|
|
4
|
3 born, 1 ongoing
|
|
ELS (cases with Matuation)
|
16
|
140
|
79 (59.8)
|
|
5
|
2 born, 2 biochemical, 1 ongoing
|
|
ELS (cases with sertoli cell-only syndrome)
|
7
|
48
|
21 (51.2)
|
|
3
|
2 born, 1 ongoing
|
Khalili et al.
(2002)
|
ROS
ELSI
|
7
3
|
42
15
|
9
9
|
6
8
|
-
1
|
-
ongoing
|
It is possible that the immaturity of spermatids may be the
most important factor to impair fertilization capacity in various. This can be
due to an incomplete histone/protamine transition, since it can lead to
chromatin instability and sensitivity, making spermatids more vulnerable to
denaturing stress. This can further lead to DNA fragmentation and apoptosis.
In
addition, a lack of protamines may enhance the cell cycle imbalance between
spermatid and oocyte resulting in premature chromatin condensation, with a
consequent failure of the transformation of spermatid nucleus into male
pronucleus (Aslam and Fishel, 1999; Sousa et al., 1998). Aneuploidy is also a
major consideration in spermatid injection. Oppedisano et al. (2002) studied
the rate of aneuploidy/diploidy in spermatids from three different sterile mice
strains showing pathological and histological similarities to human idiopathic
non-obstructive males. They found that round spermatids had elevated level of
numerical chromosomal abnormalities in two out of three different sterile
strains. Their results support the hypothesis that abnormal testicular
environment can adversely affect meiosis (Oppedisano et al., 2002).
Nevertheless, one of the most important considerations in DNA
which is developed essentially during gametogenesis.spermatid injection is the
incomplete or abnormal genomic imprinting. This process is an allele-specific
modification of Genomic imprinting results in the expression or repression of
maternal or paternal alleles of certain genes (Sleutels et al., 2000; Brannan
and Bartolomei, 1999; Reik and Walter, 1998). Disruption of the imprinting
mechanism is associated with disordered growth and development, especially
prenatal, as well as with certain clinical syndromes (Preece and Moore, 2000).
The status of genomic imprinting has been studied in mouse embryos derived by
round spermatid injection (Shamanski et al., 1999). In this study, the
expression of imprinted genes did not differ significantly from controls,
indicating that paternal genes underwent proper imprinting by the round
spermatids (Shamanski et al., 1999). However, the problem still exists since
there are no studies from primates or human embryos available. It is worth to
note that defects on genomic imprinting process may be manifested relatively
late in postnatal life (stavik et al., 2003; Cox et al., 2002; Preece and
Moore, 2000).
At the present time, there is not
enough data on the rate of malformation of the children born after spermatid
injection. In fact, an extremely small number of such births have been reported
(Saremi et al., 2002; Sousa et al., 2002; Zech et al., 2002; Al-Hasani et al.,
1999a, 1999b; Gianaroli et al., 1999; Tesarik et al., 1999; Barak et al.,
1998; Barros et al., 1998; Bernabeu et al., 1998; Kahraman et al., 1998;
Sofikitis et al., 1998; Amer et al., 1997; Antinori et al., 1997a, 1997b;
Mansour et al., 1996; Fishel et al., 1995; Tesarik et al., 1995). A recent
report described two out of four cases of pregnancies obtained through
injection with elongated spermatids, in which congenital malformations were
observed (Zech et al., 2002). The authors decided to postpone injection with
spermatids due to unpromising results and to potentially high rates of
malformation (Zech et al., 2002).
Last but not least, the methodology for
isolation, identification and assessment of viability, especially of the round
ones, are also important factors affecting the outcome of spermatid injection
(Silber et al., 2000; Vanderzwalmen et al., 1998).
Conclusion
Spermatid injection, as an assisted reproduction technique,
concerns a small number of the cases of male infertility. It presents significant
difficulties in methodology, regarding the isolation, identification and
assessment of the viability of spermatids, especially the round ones. The
fertilization and the implantation rates are extremely low with round
spermatids, while they are higher with elongated spermatids. Until today, only
a few pregnancies have been achieved. The risk for genetic abnormalities of the
offspring has not been estimated yet which could be high, mainly because of
incomplete biochemical transitions in the nucleus and of possible high rates of
aneuploidy. Unfortunately, there is a complete lack of experimental studies in
primates and there are no large clinical studies available.
Taking these facts into consideration, it is a question
whether spermatids injection should be considered even as a treatment of last
choice. According to our opinion, for the time being, round spermatid injection
should be faced as an interesting technique for animal experiments. In that
way, the methodology will be developed, the fertilization, implantation and
pregnancy rates will be improved, and there will be a better estimation of the
possible genetic risks. The injections with elongated spermatids seem to be
more efficient, theoretically more secure and consequently could be considered
as a treatment of last choice in cases of azoospermia; however, after the
couples have received appropriate counselling for the possible risks of the
procedure.
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