| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Andrology Lab Corner* |
From Molecular Assisted Reproduction and Genetics, Gracia 36, 18002 Granada, Spain.
| Correspondence to: Dr Jan Tesarik, MAR&Gen, Gracia 36, 18002 Granada, Spain (e-mail: cmendoza{at}ugr.es). |
| Received for publication January 13, 2003; accepted for publication January 13, 2003. |
From the Discovery of Spermatozoa to Artificial Insemination
When originally discovered by Antonij van Leeuwenhoek and his assistant,
Ludwig Hamm, in 1677 in the city of Delft
(Leeuwenhoek, 1678), spermatozoa were considered to be parasites of the male genital tract. The
concept of fertilization and the role the spermatozoon has in it was not
realized until the 19th century. This knowledge was being acquired in a
stepwise manner, owing to investigations conducted by Prevost and Dumas,
Peltier, and Dujardin between 1820 and 1840. Anyway, the reproductive
potential of semen was known before the formal recognition of the spermatozoon
as the oocyte fertilizing agent. The first documented successful attempt at
artificial insemination (AI) was performed in the dog by Spallanzani (1784),
and undocumented tales mention that Arabic horse breeders used AI as early as
the 13th century. The first attempts at human AI were made in 1785 by John
Hunter (1728-1793), a Scottish surgeon, resulting in the birth of a child in
the same year (Seibel, 1997).
At its beginnings, AI was performed with crude, untreated semen samples.
Because the introduction of seminal plasma into the uterus may cause infection
and produces painful spasms (Mastroianni
et al, 1957; Allen et al,
1985), semen samples were inseminated to the vagina or the
cervical canal rather than the uterine cavity.
From AI to In Vitro Fertilization
After the observation, in the early 1950s, that motile spermatozoa are not
fertile immediately after release from the male genital tract but require a
species-dependent amount of time to acquire fertilizing ability
(Austin, 1951;
Chang, 1951), the
time-dependent acquisition of sperm fertilizing ability has been termed
"capacitation" (Austin,
1952). In vivo spermatozoa are capacitated during their ascent
through the female genital tract toward the site of fertilization, but these
changes can be easily reproduced in vitro by sperm incubation in appropriate
media (Yanagimachi, 1994).
The optimal conditions for sperm in vitro capacitation differ from species to
species, and an understanding of them was a necessary prerequisite for the
development of in vitro fertilization (IVF).
Most of the knowledge required to successfully conduct human IVF was at hand in the early 1970s. This knowledge was essentially derived from animal research primarily aimed at the development of IVF as a tool in animal production (Brackett et al, 1982). However, IVF and preimplantation embryo development, followed by term pregnancy after embryo transfer to a recipient female, could not be obtained in any mammalian species until the 1970s. In fact, IVF was successful in the hamster (Yanagimachi and Chang, 1964), but the resulting embryos did not develop beyond the 1- to 2-cell stage, which also was the case for mice and rats. Rabbit embryos could be grown in vitro (Daniel, 1971), but IVF was not successful in this species because of special requirements for sperm in vitro capacitation. In domestic and agricultural animals, similar constraints were encountered. Compared to most animal species studied, the development of human IVF was relatively straightforward. Human IVF was introduced in 1969 (Edwards et al, 1969), and growth of in vitro–fertilized human embryos to the blastocyst stage was accomplished only 2 years later (Steptoe et al, 1971).
The idea of applying IVF in the treatment of human infertility initially met with much concern and adversity. From the technical point of view, in addition to the optimization of laboratory conditions for human IVF and early embryo culture (composition of culture media and gas phase, concentration of spermatozoa in the insemination medium, and evaluation of fertilization outcomes and embryo quality), it was necessary to develop efficient clinical protocols for controlled ovarian stimulation, oocyte recovery, and embryo transfer (Steptoe and Edwards, 1970). The first pregnancy achieved in the human by IVF was an extrauterine one (Steptoe and Edwards, 1976). Nevertheless, the birth of a healthy girl was achieved 2 years later by the same team of workers using the same methodology (Steptoe and Edwards, 1978). In the following years, human IVF was applied rapidly worldwide (eg, Trounson et al, 1981; Jones et al, 1984).
The finding that chicken sperm could be successfully frozen in solutions containing glycerol (Polge et al, 1949) was at the origin of the development of sperm cryopreservation protocols to be employed in different mammalian species and also in humans (Sherman, 1954). IVF with cryopreserved spermatozoa has been successful since 1985 (Cohen et al, 1985; Naz et al, 1985). In comparing IVF treatment attempts with fresh and cryopreserved spermatozoa from fertile donors, it was found that similar pregnancy rates were obtained between the 2 methods (Morshedi et al, 1990). Pregnancy rates with fresh and cryopreserved sperm from infertile patients were also similar in spite of slightly lower fertilization rates with cryopreserved sperm (Morshedi et al, 1990).
The clinical efficacy of IVF was further potentiated by the introduction of embryo cryopreservation. Because protocols of controlled ovarian stimulation usually lead to the recovery of numerous oocytes, embryo cryopreservation makes it possible to optimally benefit from the cohort of embryos available by transferring part of the embryos in the fresh state and cryopreserving supernumerary embryos for later transfer. The first successful cryopreservation protocols for human embryos used dimethyl sulfoxide as the cryoprotectant for embryos at early cleavage stages (Trounson and Mohr, 1983) and glycerol for blastocysts (Cohen et al, 1986). Later studies have shown that 1,2-propanediol is a better cryoprotectant than dimethyl sulfoxide for human early-cleaving embryos (Lassalle et al, 1985; Testart et al, 1986), and solutions containing this agent continue to be used for the cryopreservation of human embryos on days 2 or 3 after fertilization.
Laboratory and clinical experience achieved during IVF trials also helped improve the efficacy of more ancient assisted reproduction techniques, namely AI. The development of in vitro sperm washing methods made it possible to safely deliver adequate numbers of spermatozoa directly to the uterine cavity. In the 1980s, together with the rapid development of IVF, sperm isolation techniques combining the removal of seminal plasma with the selection of highly motile sperm subpopulations were applied to AI, leading to a substantial improvement in success rates (Marrs et al, 1983; Kerin et al, 1984; Sher et al, 1984; Byrd et al, 1987). With the use of these sperm preparation techniques, there is a wide consensus that intrauterine insemination (IUI) is superior to other AI techniques (Byrd et al, 1990; Patton et al, 1992; Wainer et al, 1995; Matorras et al, 1996).
Further improvement of IUI results was brought about by the introduction of ovarian stimulation (Kemman et al, 1987; Serhal et al, 1988; Chaffkin et al, 1991; Guzick et al, 1999). Compared to natural cycles, the inclusion of ovarian stimulation allows a better timing of the insemination procedure and the correction of subtle ovulatory disorders that might complicate the prediction of spontaneous ovulation (Dodson et al, 1987; Wallach, 1991). However, ovarian stimulation also increased the risk of multiple pregnancies after IUI (Navot et al, 1991; Farhi et al, 1996), which, however, can be minimized by the application of mild stimulation protocols, the careful monitoring of ovarian follicular development by vaginal ultrasonography, and the cancellation of treatment if too many follicles are recruited. Nevertheless, the optimal number of large preovulatory follicles to maintain acceptable cycle fecundity and minimize the risk of multiple pregnancy is not known. Nowadays, AI still has its place as a simple and inexpensive method in the treatment of certain types of infertility, in spite of the existence of more high-performance techniques.
The cryopreservation of sperm has further facilitated the use of donor sperm for both AI and IVF. The efficacy of using frozen sperm in donor AI was reported to be somewhat lower than in fresh sperm (Richter et al, 1984), but other studies did not find any significant differences (Bordsen et al, 1986; Keel and Webster, 1989).
Compared to AI, IVF represented a real breakthrough in the treatment of human infertility and strongly stimulated further development in this field. From the clinical viewpoint, it constituted an efficient treatment option for women suffering from tubal infertility, which was the originally advocated indication for this technique. Soon after its introduction, however, it became clear that the application sphere of IVF was much wider, including endometriosis and other types of female infertility, idiopathic infertility, and, particularly, various types of male infertility (Mahadevan et al, 1983).
A modification of IVF, whereby spermatozoa and oocytes were mixed together in vitro and then deposited in the fallopian tube, was also designed (Shettles, 1979). It was first successfully performed in 1982, during an attempt at microsurgical reconstruction of fallopian tubes, resulting in the birth of a healthy child (Tesarik et al, 1983). This technique was later adapted to be performed laparoscopically and became popularized under the term of "gamete intrafallopian transfer—GIFT" (Asch et al, 1984).
From the scientific viewpoint, the most important advancement resulting from the clinical application of IVF was doubtlessly the possibility of direct observation of human sperm–egg interaction. In addition to providing a number of new pathophysiological insights with numerous diagnostic implications, it became a new and powerful stimulus for further development of assisted reproduction techniques.
Development of In Vitro Pharmacological Treatments to Assist
Sperm–Egg Interaction
The clinical application of in vitro sperm capacitation revived the
interest in the behavior of spermatozoa in these conditions. In particular,
characteristics of sperm movement were no longer considered static parameters
merely reflecting the quality of fresh or cryopreserved sperm samples, but
they began to be analyzed as evolutive variables, subject to functionally
relevant modifications in the period between ejaculation and fertilization.
The development of computer-assisted systems for the analysis of sperm motion
characteristics on a single-cell basis represented a significant technical
impetus for this approach. It became increasingly evident that not only
motility in its conventional sense (the percentage of sperm that move) but
also the speed and quality of movement of each motile spermatozoon were
important determinants of fertility.
These studies made it possible to determine the normal speed range and the characteristics of the trajectory of individual human spermatozoa at different time periods between ejaculation and fertilization as well as the effects of different components of the female genital tract on these parameters (Mendoza and Tesarik, 1990; Tesarik et al, 1990). It was also noted that abnormal sperm movement characteristics are associated with reduced fertilizing ability in vivo (Barratt et al, 1993) and in vitro (Jeulin et al, 1986; Liu et al, 1991). In many of these cases, sperm movement was enhanced and the fertilization rate was improved by sperm in vitro pretreatment with pentoxifylline, an inhibitor of phosphodiesterase that increases the intracellular concentration of cyclic adenosine monophosphate, before its use for IVF (Yovich et al, 1988, 1990; Rizk et al, 1995; Tarlatzis et al, 1995). Pentoxifylline has been shown to stimulate sperm velocity and the development of a special, capacitation-related change in sperm movement pattern, called "hyperactivation" (Tesarik et al, 1992b). Moreover, when used at adequate concentrations (around 1 mg/mL) and with appropriate incubation times (around 10 minutes), pentoxifylline also enhanced the response of spermatozoa to acrosome reaction–inducing stimuli (Tesarik et al, 1992a; Carver-Ward et al, 1994; Kay et al, 1994) and was used with success in patients with acrosome reaction insufficiency (Tesarik and Mendoza, 1993; Tasdemir et al, 1993). One study has also reported a beneficial effect of in vitro treatment with pentoxifylline on sperm binding to the zona pellucida in the hemizona assay (Yogev et al, 1995), but the mechanism of this effect is not known. Premature acrosome reaction may also occur spontaneously in some men with reduced fertility, and this condition can be alleviated by sperm pretreatment with egg yolk, which probably acts by stabilizing the abnormally fragile sperm plasma membrane in these patients (Tesarik and Mendoza, 1995).
After the advent of micromanipulation-assisted fertilization (see below), the pharmacological enhancement of sperm function lost much of its importance in attempts using freshly obtained ejaculated spermatozoa. However, it still maintains its place in assisted reproduction with poorly motile sperm samples, such as testicular spermatozoa from men with nonobstructive azoospermia (NOA) or cryopreserved sperm samples with poor prefreeze quality (Esteves et al, 1998). Pentoxifylline was also used with success in sperm preparation for IUI (Negri et al, 1996).
Development of Micromanipulation Techniques to Assist Sperm–Egg
Interaction
The development of micromanipulation-assisted fertilization was motivated
by the observation that failures of IVF were mostly associated with a failure
of sperm–zona pellucida binding or zona pellucida penetration. From the
technical point of view, it was facilitated by the increasing availability of
good-quality cell micromanipulators and growing experience with their use in
research studies with animal gametes. It was initially thought that problems
of sperm–zona pellucida interaction could be treated by the injection of
spermatozoa through the zona pellucida into the perivitelline space
(Laws-King et al, 1987).
These techniques, however, were burdened by the unpredictability of sperm–oolemma fusion in these artificial conditions, leading to a high incidence of both fertilization failure and polyspermic penetration. In fact, contrary to what was theoretically expected, most acrosome-reacted motile human spermatozoa proved incapable of fusing with the oolemma after subzonal insemination (Tesarik and Mendoza, 1994). It thus became clear that an efficient micromanipulation-assisted fertilization technique should not only ensure sperm passage through the zona pellucida, but it also should sever the oolemma as well in order to deposit a single spermatozoon directly in the oocyte cy-oplasm.
Attempts at direct intracytoplasmic sperm injection (ICSI) into human oocytes were first reported in 1988 (Lanzendorf et al, 1988), but the first term pregnancy after the transfer of embryos resulting from ICSI was achieved only 4 years later (Palermo et al, 1992). The technique then evolved rapidly and soon achieved the highest fertilization rates among all micromanipulation-assisted fertilization techniques (Van Steirteghem et al, 1993). The other types of manipulation were then progressively abandoned.
Evolution of ICSI and Development of ICSI-Derived Techniques Using
Immature Male Gametes
Extension of ICSI Indication—
Originally developed to be used for assisted reproduction in cases of
severe male infertility, ICSI has progressively become a treatment option for
other indications, too. These include not only male but also female
infertility. This development was essentially based on high and relatively
stable fertilization rates with ICSI
(Tournaye et al, 2002) and on
the independence of results on different sperm and oocyte functions, such as
sperm–zona pellucida recognition and binding, acrosome reaction,
sperm–oolemma binding and fusion, and the presence of antibodies on
gamete surfaces (Hamberger et al,
1998). Moreover, fertilization and pregnancy rates after ICSI with
ejaculated spermatozoa are not influenced by sperm cryopreservation, even in
patients with poor sperm quality
(Kuczynski et al, 2001). By
making it possible even for spermatozoa carrying various functional defects,
which otherwise would not be able to penetrate the egg vestments, to get
access to the oocyte cytoplasm, ICSI helped reveal sperm and oocyte functional
abnormalities that were not known before. Among these, the failure of oocyte
activation received the most attention.
Oocyte activation is a complex, sperm-initiated process whereby the mature oocyte, physiologically arrested at metaphase of the second meiotic division until fertilization, reactivates the cell cycle, completes the second meiosis, and, after nuclear syngamy, starts mitotic cell divisions of the early embryo. This process is essentially regulated by repetitive increases in free cytoplasmic Ca2+ concentrations (Ca2+ oscillations) that are triggered by the fertilizing sperm (Miyazaki et al, 1986). It has been shown that Ca2+ oscillations, similar to those observed after spontaneous sperm penetration into the oocyte (Taylor et al, 1993), also occur after ICSI (Tesarik et al, 1994). However, the mechanism whereby Ca2+ oscillations are triggered after ICSI is slightly different from normal (Tesarik and Mendoza, 1999). Because ICSI is usually performed with abnormal sperm samples, the content and quality of sperm factors responsible for oocyte activation can also be altered. Globozoospermia (round-headed sperm syndrome) is the best-known example of this deeficiency (Rybouchkin et al, 1996; Battaglia et al, 1997), but it is known that spermatozoa from some men consistently fail to activate the oocyte without presenting the typical round-head morphology. Moreover, repeated failures of oocyte activation after ICSI can also be due to anomalies of oocytes rather than spermatozoa in some infertile couples (Tesarik et al, 2002b). To improve ICSI results in cases of repeated oocyte activation failure, oocyte activation can be boosted by calcium ionophores (Rybouchkin et al, 1997; Kim et al, 2001), which were shown to substantially increase the fertilization rate (Tesarik and Sousa, 1995) and to potentiate the sperm-induced Ca2+ oscillations (Tesarik and Testart, 1994) of sperminjected human oocytes. However, a simple modification of the ICSI technique has recently been developed for cases of spermand oocyte-borne repeated oocyte activation failures without requiring recourse to the use of potentially toxic chemical agents (Tesarik et al, 2002b).
ICSI With Immature Sperm— The independence of fertilization by ICSI on sperm movement or on the presence of mechanisms required for oocyte penetration was at the origin of an increasing use of immature spermatozoa (epididymal and testicular) and even of sperm precursor cells for fertilization.
The association of ICSI with surgical sperm retrieval has revolutionized the treatment of azoospermic men. In cases of obstructive azoospermia (OA), sufficient numbers of spermatozoa for ICSI can usually be retrieved by epididymal sperm aspiration, whereas cases of NOA mostly require open testicular biopsy. Even with this more invasive approach, spermatozoa fail to be recovered in about a half of the cases of NOA in tissue samples obtained by multiple-site testicular biopsy (Kahraman et al, 1996; Westlander et al, 1999; Friedler et al, 2002). Unfortunately, a reliable test predicting successful sperm retrieval from the testes of men with NOA is still lacking.
There is a wide consensus that serum concentrations of follicle-stimulating hormone (FSH) do not predict sperm retrieval from men with NOA (Gil-Salom et al, 1995; Mulhall et al, 1997; Tournaye et al, 1997). Serum inhibin B levels, reflecting Sertoli cell function, were suggested to predict sperm retrieval by testicular biopsy from NOA patients (Ballesca et al, 2000; Brugo-Olmedo et al, 2001), but other studies (Von Eckardstein et al, 1999; Vernaeve et al, 2002) failed to corroborate this hypothesis. In the absence of more reliable criteria, the strongest indicator for finding sperm for ICSI in NOA patients is testicular histopathology (Tournaye et al, 1997; Mercan et al, 2000; Vicari et al, 2001; Friedler et al, 2002) and histochemistry (Anniballo et al, 2000), together with the detection of at least a few round spermatids in the ejaculate with the use of immunocytochemistry with antibodies to specific germline marker antigens (Ezeh et al, 1998). In some cases in which spermatozoa fail to be recovered, fertilization with sperm precursor cells can be attempted.
Epididymal Sperm— The first birth after fertilization with epididymal spermatozoa from a patient with OA was achieved with the use of conventional IVF (Silber et al, 1990). However, the application of ICSI substantially improved fertilization and pregnancy rates (Silber, 1994; Tournaye et al, 1994), which currently are similar to those for ICSI with ejaculated sperm (Van Steirteghem et al, 1998).
Epididymal sperm can also be cryopreserved for later use in ICSI. Comparing the efficacy of fresh and frozen-thawed epididymal sperm in ICSI, most authors report similar fertilization and pregnancy rates (Silber et al, 1997; Friedler et al, 1998; Tournaye et al, 1999; Janzen et al, 2000; Cayan et al, 2001), although one study reported a significantly lower clinical pregnancy rate with cryopreserved vs fresh epididymal sperm in spite of similar fertilization rates (Palermo et al, 1999).
Testicular Sperm— Since the first successful attempts at ICSI with testicular sperm (Devroey et al, 1995), the technique has been perfected to give fertilization rates that are close to those obtained by ICSI with ejaculated sperm (Van Steirteghem et al, 1998). However, several studies have reported a dependence of ICSI outcomes on the underlying etiology of azoospermia. The ESHRE ICSI Task Force, analyzing data from the time period 1993–95, reported a lower fertilization rate in patients with NOA than in those with OA but no differences in the pregnancy rate, delivery rate, and perinatal outcome between the 2 groups of patients (Tarlatzis and Bili, 1998). Similar fertilization and pregnancy rates for patients with OA and NOA were also reported by others (Silber et al, 1997; Vicari et al, 2001). However, several authors have noted lower fertilization (Mansour et al, 1997; Palermo et al, 1999), pregnancy (Kahraman et al, 1996; Aboulghar et al, 1997; Mansour et al, 1997; Ghazzawi et al, 1998), and implantation (Ubaldi et al, 1999) rates and a higher abortion rate (Vicari et al, 2001) in ICSI attempts using testicular sperm from men with NOA compared to ICSI with epididymal or testicular sperm from men with OA.
No differences were found in fertilization and pregnancy rates with fresh and cryopreserved testicular sperm from patients with OA or NOA (Ben-Yosef et al, 1999; Habermann et al, 2000; Friedler et al, 2002).
Sperm Precursor Cells— The first birth after fertilization with elongated spermatids was reported in 1995 (Fishel et al, 1995). The ability of elongated spermatids to act as male gametes after injection into human oocytes (elongated spermatid injection [ELSI]) was further confirmed by different groups (Araki et al, 1997; Vanderzwalmen et al, 1997; Bernabeu et al, 1998; Kahraman et al, 1998; Sofikitis et al, 1998; Al-Hasani et al, 1999; Sousa et al, 1999; Tesarik et al, 2000). However, most of these reports did not mention the exact phase of spermiogenesis of the elongated spermatids that were used. Accordingly, there was an overlap between what some authors reported as ELSI and others as ICSI with testicular spermatozoa (Tesarik, 1997). Deeper analysis of the evolution of spermatid developmental potential during spermiogenesis (Sousa et al, 1999) showed that the chance of obtaining a normal embryo is relatively acceptable with spermatids at late stages of spermiogenesis (Sd1 and Sd2), but it drops markedly when less mature stages are used.
The original enthusiasm inspired by reports of births (Tesarik et al, 1995; Vanderzwalmen et al, 1997; Barak et al, 1998; Gianaroli et al, 1999) and ongoing pregnancies (Antinori et al, 1997a,b) after fertilization with round spermatids was subsequently tempered by the low success rates obtained in a larger series of round spermatid injection (ROSI) attempts (Al-Hasani et al, 1999; Tesarik et al, 2000). On the basis of a meta-analysis of data reported by different groups (255 treatment attempts), the overall fertilization, pregnancy, and confirmed birth rate after ROSI were 30.5%, 5.1%, and 2.0%, respectively (Tesarik et al, 2000). The results of ROSI were deceiving, especially as to the low birth rate, which resulted not only from the low implantation potential of embryos developing from oocytes fertilized with round spermatids but also from the unusually high rate of early wastage of spermatid-derived pregnancies (Amer et al, 1997).
Some improvement of ROSI success rates was achieved by the application of germ cell in vitro culture (see below), but the clinical efficacy still remains well below that of ELSI with late elongated spermatids or ICSI with testicular spermatozoa.
There also is an isolated report on a pregnancy achieved with embryos conceived by intracytoplasmic injection of secondary spermatocytes into human oocytes (Sofikitis et al, 1998). However, this experience has not been further confirmed either by the same or by another group.
Germ Cell In Vitro Culture and its Clinical Applications— Several early studies described the occurrence of spermatogenic events during in vitro culture of pieces of animal and human testicular tissue (reviewed in Tesarik et al, 2000). However, these attempts acquired potential clinical importance only in the last decade, when micro-manipulation methods capable of delivering poorly motile sperm and sperm precursor cells to the interior of the oocyte became available. Currently, in vitro culture methods are used for 2 different purposes: to overcome in vivo maturation arrest and to select against spermatozoa carrying DNA damage.
Overcoming In Vivo Maturation Arrest by In Vitro Culture— It has been shown that spermatids from men with normal spermatogenesis can be stimulated to undergo isolated spermatogenic events (nuclear condensation and protrusion as well as flagellar growth) at a highly accelerated speed when cultured in vitro (Aslam and Fishel, 1998; Tesarik et al, 1998b; Cremades et al, 1999). The presence of high concentrations of FSH and testosterone in culture media further enhances the differentiation rate (Tesarik et al, 1998b).
When in vitro culture conditions were applied to germ cells obtained from men with in vivo maturation arrest, a reactivation of spermiogenesis, presenting the same morphological patterns, was observed in some but not all men with postmeiotic arrest at the round spermatid stage, and the development of late elongated spermatids (Sd stage) was achieved even in some men with meiotic arrest at pachytene, but not at earlier stages, of the first meiotic division (primary spermatocyte stage) (Tesarik et al, 1999a, 2002a). These in vitro–developed spermatids were able to fertilize human oocytes, and term pregnancies were achieved after the transfer of the resulting embryos, including the first birth after fertilization with germ cells from a man with in vivo maturation arrest at the primary spermatocyte stage (Tesarik et al, 1999a).
Selection Against Sperm Carrying Damaged DNA— A high incidence of DNA fragmentation in round spermatids from men with complete maturation arrest at the round spermatid stage (Tesarik et al, 1998a; Jurisicova et al, 1999) is suspected to be at the origin of the high prevalence of assisted reproduction failure when round spermatids are used for fertilization (Tesarik et al, 2000). However, a high incidence of DNA fragmentation is also often found in mature spermatozoa from some patients with complete spermatogenesis (Gandini et al, 2000; Muratori et al, 2000). Given the observation that in vitro culture facilitates the selection against spermatids with fragmented DNA (Tesarik et al, 1999b), the same approach has been suggested to improve ICSI outcomes in such cases. A subsequent pilot study confirmed this working hypothesis (Tesarik et al, 2001a). Consequently, sperm samples from men with repeated failure of assisted reproduction can now be tested for DNA fragmentation, and ICSI with in vitro cultured sperm can be proposed in an attempt to increase the chance of success. However, a testicular biopsy is needed to recover whole portions of seminiferous tubules for in vitro culture, even in cases in which spermatozoa are present in the ejaculate.
Safety Concerns About Micromanipulation-Assisted Fertilization
Techniques
Concerns About the Use of ICSI for Fertilization—
An analysis of the obstetric outcomes of 904 pregnancies after ICSI
(Wisanto et al, 1996) did not
suggest any increase in the risk of pregnancy loss, clinical abortion,
pregnancy complications, and chromosomal aberrations (on the basis of a
prenatal diagnosis of 64.4% of the clinical pregnancies) related to the use of
the ICSI technique. However, one report suggested an increase in the incidence
of sex chromosomal abnormalities among the ICSI babies
(In't Veld et al, 1995). A
slight but significant increase in chromosomal abnormalities (1.2%) compared
to the general newborn population, partly due to de novo abnormalities, was
confirmed by a larger prospective follow-up study of children born after ICSI
(Bonduelle et al, 1996).
Several hypotheses have been formulated to explain the increased risk of chromosomal abnormalities after ICSI. In the first place, the prevalence of chromosomal abnormalities in couples undergoing ICSI was higher than in general population and, somewhat surprisingly, this concerned not only the males but also the female partners of the couples (Meschede et al, 1998). An increased incidence of numerical chromosomal abnormalities was also found in spermatozoa from patients treated by ICSI (Macas et al, 2001). The prevalence of sperm chromosomal abnormalities was found to be linked with gonadal failure (high serum FSH levels) in men undergoing ICSI (Levron et al, 2001). Surprisingly, chromosomal analysis of spontaneous abortions did not reveal any difference in the incidence of chromosomal abnormalities between ICSI and conventional IVF (Causio et al, 2002). Also, the ICSI technique itself has been suspected to contribute to the development of de novo chromosomal abnormalities in ICSI-derived embryos (Tesarik, 1995). This possibility remains to be further examined.
In addition to chromosomal abnormalities, the risk of mild delays in development (Bowen et al, 1998) and major birth defects (Ludwig and Katalinic, 2002) also appears to be slightly elevated after ICSI compared with spontaneously conceived children. However, another recent study has shown that the twofold increased risk of major birth defects compared with naturally conceived infants was not strictly related to the ICSI technique but concerned births after conventional IVF as well (Hansen et al, 2002).
A recent report on 2 children conceived after ICSI who developed Angelman syndrome (Cox et al, 2002) has suggested an increased risk of imprinting defects, but the possible relationship between this type of epigenetic anomaly and the ICSI technique remains to be evaluated.
Concerns About the Use of Immature Sperm for Fertilization— In spite of the fact that all postmeiotic germ cells, from round spermatids to ejaculated spermatozoa, have the same nuclear DNA content, the form of DNA organization undergoes essential changes during postmeiotic maturation. It was pointed out that incomplete nuclear condensation in immature spermatids makes DNA more vulnerable to damage by nucleases and other factors (Tesarik et al, 1998c). In agreement with this concept, high frequencies of spermatids carrying apoptosisrelated DNA fragmentation were found in patients suffering from maturation arrest at the round spermatid stage (Tesarik et al, 1998a; Jurisicova et al, 1999). This finding can at least partly explain the high incidence of implantation failure and early pregnancy wastage after ROSI (Tesarik et al, 2000).
The possible risk associated with genomic imprinting anomalies in the immature germ cells used for fertilization has also been discussed (Tesarik and Mendoza, 1996), but this risk remains purely hypothetical because no children with anomalies attributable to genomic imprinting have yet been born in these cases.
All babies resulting from ROSI were normal and healthy. This contrasts with a report on 4 pregnancies after the transfer of embryos resulting from ELSI with late elongated spermatids, 2 of which developed major fetal malformations (hydrocephalus associated with trisomy 9 and Arnold Chiari Syndrome type II, respectively; Zech et al, 2000). However, as mentioned earlier in this paper, many centers report cases of ELSI with late elongated spermatids similar to those described by Zech et al (2000) as ICSI with testicular sperm (Tesarik, 1997). Consequently, this isolated report on a small patient group must be interpreted with caution. A multicenter study employing exact staging of germ cells used for fertilization is needed to evaluate the real risks of ELSI and ICSI with testicular sperm.
Future Challenges
Spermatogenesis in a Host Testis—
Pioneering experiments from Ralph Brinster's laboratory showed that mouse
spermatogonial stem cells can repopulate the seminiferous tubules of the host
mouse testis and subsequently undergo complete spermatogenesis
(Brinster and Zimmermann,
1994; Russell et al,
1996), that the spermatozoa having developed in the host testis
are fertile (Ogawa et al,
2000), and that even cryopreserved mouse spermatogonial stem cells
are able to generate spermatogenesis in recipient seminiferous tubules
(Avarbock et al, 1996).
Further studies were aimed at the modification of this system to be used for
heterospecific transplantations. These experiments attempted to achieve rat
spermatogenesis (Clouthier et al,
1996; Russell and Brinster,
1996), hamster spermatogenesis
(Ogawa et al, 1999), rabbit
and dog spermatogenesis (Dobrinski et al,
1999), and boar, bull, and stallion spermatogenesis
(Dobrinski et al, 2000) after
the transplantation of spermatogonial stem cells from each of these species to
the mouse testis. Spermatogenesis after these xenotransplantations led to the
formation of mature spermatozoa in the rat-to-mouse system
(Clouthier et al, 1996;
Russell and Brinster, 1996) but was more or less incomplete in the other donor–host
combinations.
Recolonization of seminiferous tubules with transplanted spermatogonial stem cells was also achieved in a nonhuman primate (cynomolgus monkey), but spermatogenesis progressed only to a very limited extent in this system (Schlatt et al, 1999). Xenogeneic transplantation of human spermatogonia to the mouse testis failed to ensure survival and differentiation of the transplanted cells (Reis et al, 2000). However, a recent study has demonstrated that an alternative approach, based on grafting tissue from immature testes under the back skin of castrated immunodeficient animals, can be successful in achieving complete xenogeneic spermatogenesis of phylogenetically (relatively) distant species (pig and goat) in the mouse host (Honaramooz et al, 2002).
Fertilization With Haploidized Somatic Cells— Mammalian metaphase II oocytes have an extraordinary capacity to rapidly drive any injected cell nucleus to metaphase, irrespective of the actual cell cycle phase of the cell from which the nucleus has been removed (Tesarik, 2002). Owing to this capacity, if a nucleus that has not yet undergone the DNA synthetic phase is introduced into a metaphase II oocyte, it is driven to a premature metaphase without previous DNA replication, thus bypassing a step that otherwise is obligatory in any mammalian cell type. This makes it possible to reduce the DNA content of injected cell nuclei by half. This phenomenon has been called "somatic cell haploidization" and has been suggested as a tool for replacing gametes with somatic cells in the fertilization process (Tesarik, 2002).
Somatic cell haploidization was initially suggested as a means for oocyte reconstruction in women with ovarian failure (Tsai et al, 2000). Being able to achieve chromosomal segregation between the reconstituted oocyte that resulted from the injection of a somatic cell (cumulus cell) nucleus into a previously enucleated metaphase II donor oocyte and a pseudo-second polar body during subsequent oocyte activation led to the development of 4-cell stage embryos that resulted from fertilization by ICSI of these oocytes has been documented recently (Tesarik et al, 2001b). A similar approach, but one using the somatic cell (fibroblast) nucleus as a substitute for the male gamete genome, has been tried in the mouse model, resulting in fertilization and early cleavage divisions in some of the treated oocytes (Lacham-Kaplan et al, 2001).
Because the techniques using somatic cells as substitutes for the male gamete must also circumvent the lack of sperm as the oocyte-activation trigger and the source of the microtubule organizing center, they are expected to represent a more difficult challenge than the use of somatic cells for oocyte reconstitution (Tesarik and Mendoza, 2003). Notwithstanding, this technique represents an exciting challenge to future research because it could be used in yet untreatable cases of total germline absence (complete Sertoli cell–only syndrome) and maturation arrest at the early stages of spermatogenesis.
Footnotes
* Andrology Lab Corner welcomes the submission of unsolicited
manuscripts, requested reviews, and articles in a debate format. Manuscripts
will be reviewed and edited by the Section Editor. Papers appearing in this
section are not considered primary research reports and are thus not subjected
to peer review. All submissions should be sent to the Journal of
Andrology Editorial Office. Letters to the editor in response to
articles as well as suggested topics for future issues are
encouraged. 
References
Aboulghar M, Mansour RT, Serour GI, Fahmy I, Kamal A, Tawab NA, Amin YM. Fertilization and pregnancy rates after intracytoplasmic sperm injection using ejaculated semen and surgically retrieved sperm. Fertil Steril.1997; 68:108 –111.[Medline]
Al-Hasani S, Ludwig M, Palermo I, et al. Intracytoplasmic injection
of round and elongated spermatids from azoospermic patients: results and
review. Hum Reprod.1999; 14(suppl 1):97
–107.
Allen NC, Herbert CM, Maxson WS, Rogers BJ, Diamond MP, Wentz AC. Intrauterine insemination: a critical review. Fertil Steril. 1985; 44:569 –580.[Medline]
Amer M, Soliman E, El-Sadek M, Mendoza C, Tesarik J. Is complete spermiogenesis failure a good indication for spermatid conception? Lancet. 1997;350:116 .[Medline]
Anniballo R, Ubaldi F, Cobellis L, Sorrentino M, Rienzi L, Greco E,
Tesarik J. Criteria predicting the absence of spermatozoa in the Sertoli
cell–only syndrome can be used to improve success rates of sperm
retrieval. Hum Reprod.2000; 15:2269
–2277.
Antinori S, Versaci C, Dani G, Antinori M, Pozza D, Selman HA.
Fertilization with human testicular spermatids: four successful pregnancies.
Hum Reprod.1997a; 12:286
–291.
Antinori S, Versaci C, Dani G, Antinori M, Selman HA. Successful
fertilization and pregnancy after injection of frozen-thawed round spermatids
into human oocytes. Hum Reprod.1997b; 12:554
–556.
Araki Y, Motoyama M, Yoshida A, Kim SY, Sung H, Araki S. Intracytoplasmic injection with late spermatids: a successful procedure in achieving childbirth for couples in which the male partner suffers from azoospermia due to deficient spermatogenesis. Fertil Steril. 1997;67:559 –561.[Medline]
Asch RH, Ellsworth LR, Balmaceda JP, Wong PC. Pregnancy after translaparoscopic gamete intrafallopian transfer. Lancet. 1984;2:1034 –1035.[Medline]
Aslam I, Fishel S. Short-term initro culture and cryopreservation
of spermatogenic cells used for human initro conception. Hum
Reprod. 1998;13:634
–638.
Austin CR. Observations on the penetration of the sperm into the mammalian egg. Aust J Sci Res B.1951; 4:581 –596.[Medline]
Austin CR. The "capacitation" of the mammalian sperm. Nature. 1952; 170:326 .[Medline]
Avarbock MR, Brinster CJ, Brinster RL. Reconstitution of spermatogenesis from frozen spermatogonial stem cells. Nat Med. 1996;2:693 –696.[Medline]
Ballesca JL, Balasch J, Calafell JM, Alvarez R, Fabregues F,
Martinez de Osaba MJ, Ascaso C, Vanrell JA. Serum inhibin B determination is
predictive of successful testicular sperm extraction in men with
nonobstructive azoospermia. Hum Reprod.2000; 15:1734
–1738.
Barak Y, Kogosowski A, Goldman S, Soffer Y, Gonen Y, Tesarik J. Pregnancy and birth after transfer of embryos that developed from singlenucleated zygotes obtained by injection of round spermatids into oocytes. Fertil Steril.1998; 70:67 –70.[Medline]
Barratt CLR, Tomlinson MJ, Cooke ID. Prognostic significance of computerized motility analysis for in vivo fertility. Fertil Steril. 1993;60:520 –525.[Medline]
Battaglia DE, Koehler JK, Klein NA, Tucker MJ. Failure of oocyte activation after intracytoplasmic sperm injection using round-headed sperm. Fertil Steril.1997; 68:118 –122.[Medline]
Ben-Yosef D, Yogev L, Hauser R, Yavetz H, Azem F, Yovel I, Lessing
JB, Amit A. Testicular sperm retrieval and cryopreservation prior to
initiating ovarian stimulation as the first line approach in patients with
azoospermia. Hum Reprod.1999; 14:1794
–1801.
Bernabeu R, Cremades N, Takahashi K, Sousa M. Successful pregnancy
after spermatid injection. Hum Reprod.1998; 13:1898
–1900.
Bonduelle M, Wilikans A, Buysse A, Van Assche E, Wisanto A, Devroey
P, Van Steirteghem AC, Liebaers I. Prospective follow-up study of 877 children
born after intracytoplasmic sperm injection (ICSI), with ejaculated,
epididymal and testicular spermatozoa and after replacement of cryopreserved
embryos obtained after ICSI. Hum Reprod.1996; 11(suppl 4):131
–155.
Bordsen BL, Ricci E, Dickey RP. Comparison of fecundability with fresh and frozen semen in therapeutic donor insemination. Fertil Steril. 1986;46:466 –469.[Medline]
Bowen JR, Gibson FL, Leslie GI, Saunders DM. Medical and developmental outcome at 1 year for children conceived by intracytoplasmic sperm injection. Lancet.1998; 351:1529 –1534.[Medline]
Brackett BG, Bousquet D, Dressel NA. In vitro sperm capacitation and in vitro fertilization with normal development in the rabbit. J Androl. 1982;3:402 –411.[Abstract]
Brinster RL, Zimmermann JW. Spermatogenesis following male germcell
transplantation. Proc Natl Acad Sci U S A.1994; 91:11298
–11302.
Brugo-Olmedo S, De Vincentiis S, Calamera JC, Urrutia F, Nodar F, Acosta A. Serum inhibin B may be a reliable marker of the presence of testicular spermatozoa in patients with nonobstructive azoospermia. Fertil Steril.2001; 76:1124 –1129.[Medline]
Byrd W, Ackerman GA, Carr BR, Edman CD, Guzick DS, McConnell JD. Treatment of refractory infertility by transcervical intrauterine insemination of washed spermatozoa. Fertil Steril.1987; 48:921 –927.[Medline]
Byrd W, Bradshaw K, Carr B, Edman C, Odom J, Ackerman G. A prospective randomized study of pregnancy rates following intrauterine and intracervical insemination using frozen donor sperm. Fertil Steril. 1990;53:521 –527.[Medline]
Carver-Ward JA, Jaroudi KA, Einspenner M, Parhar RS, al-Sedairy ST,
Sheth KV. Pentoxifylline potentiates ionophore (A23187) mediated acrosome
reaction in human sperm: flow cytometric analysis using CD46 antibody.
Hum Reprod.1994; 9:71
–76.
Causio F, Fischetto R, Sarcina E, Geusa S, Tartagni M. Chromosome analysis of spontaneous abortions after in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Eur J Obstet Gynecol Reprod Biol. 2002;10:44 –48.
Cayan SLD, Conaghan J, Givens CA, Ryan IP, Schriock ED, Turek PJ. A
comparison of ICSI outcomes with fresh and cryopreserved epididymal
spermatozoa from the dame couples. Hum Reprod.2001; 16:495
–499.
Chaffkin LM, Nulsen JC, Luciano AA, Metzger DA. A comparative analysis of the cycle fecundity rates associated with combined human menopausal gonadotropin (hMG) and intrauterine insemination (IUI) versus either hMG or IUI alone. Fertil Steril.1991; 55:252 –257.[Medline]
Chang MC. Fertilizing capacity of spermatozoa deposited into fallopian tubes. Nature.1951; 168:697 –698.[Medline]
Clouthier DE, Avarbock MR, Maika SD, Hammer RE, Brinster RL. Rat spermatogenesis in mouse testis. Nature.1996; 381:418 –421.[Medline]
Cohen J, Edwards RG, Fehilly CB, et al. In vitro fertilization using cryoreserved donor semen in cases where both partners are infertile. Fertil Steril.1985; 43:570 –574.[Medline]
Cohen J, Simmons RS, Fehilly CB, Edwards RG. Factors affecting survival and implantation of cryopreserved human embryos. J In Vitro Fert Embryo Transfer.1986; 3:46 –52.[Medline]
Cox GF, Burger J, Lip V, Mau UA, Sperling K, Wu BL, Horsthemke B. Intracytoplasmic sperm injection may increase the risk of imprinting defects. Am J Hum Genet.2002; 71:162 –164.[Medline]
Cremades N, Bernabeu R, Barros A, Sousa M. In-vitro maturation of
round spermatids using co-culture on Vero cells. Hum
Reprod. 1999; 14:1287
–1293.
Daniel J. Methods in Mammalian Embryology. San Francisco, CA: WH Freeman; 1971.
Devroey P, Liu J, Nagy Z, Goessens A, Tournaye H, Camus M, Van
Steirteghem A, Silber S. Pregnancies after testicular sperm extraction and
intracytoplasmic sperm injection in non-obstructive azoospermia.
Hum Reprod.1995; 10:1457
–1460.
Dobrinski I, Avarbock MR, Brinster RL. Transplantation of germ
cells from rabbits and dogs into mouse testes. Biol
Reprod. 1999;61:1331
–1339.
Dobrinski I, Avarbock MR, Brinster RL. Germ cell transplantation from large domestic animals into mouse testes. Mol Reprod Dev. 2000;57:270 –279.[Medline]
Dodson WC, Whitesides DB, Hughes CL, Easley HA, Haney AF. Superovulation with intrauterine insemination in the treatment of infertility: a possible alternative to gamete intrafallopian transfer and in vitro fertilization. Fertil Steril.1987; 48:441 –445.[Medline]
Edwards RG, Bavister BD, Steptoe PC. Early stages of fertilization in vitro of human oocytes matured in vitro. Nature. 1969;221:632 –635.[Medline]
Esteves SC, Sharma RK, Thomas AJ Jr, Agarwal A. Cryopreservation of
human spermatozoa with pentoxifylline improves post-thaw agonistinduced
acrosome reaction rate. Hum Reprod.1998; 13;3384
–3389.
Ezeh UI, Martin M, Cooke ID, Moore HD. Correlation of testicular
pathology and sperm extraction in azoospermic men with ejaculated spermatids
detected by immunofluorescent localization. Hum
Reprod. 1998;13:3061
–3065.
Farhi J, West C, Patel A, Jacobs HS. Treatment of unovulatory
infertility: the problem of multiple pregnancy. Hum
Reprod. 1996;11:429
–434.
Fishel S, Green S, Bishop M, Thornton S, Hunter A, Fleming S, al-Hassan S. Pregnancy after intracytoplasmic injection of spermatid. Lancet. 1995;345:1641 –1642.[Medline]
Friedler S, Raziel A, Soffer Y, Strassburger D, Komaroversusky D,
RonEl R. The outcome of intracytoplasmic injection of fresh and cryopreserved
epididymal spermatozoa in patients with obstructive azoospermia—a
comparative study. Hum Reprod.1998; 13:1872
–1877.
Friedler S, Raziel A, Strassburger D, Schachter M, Soffer Y, RonEl
R. Factors influencing the outcome of ICSI in patients with obstructive and
non-obstructive azoospermia: a comparative study. Hum
Reprod. 2002;17:3114
–3121.
Gandini L, Lombardo F, Paoli D, Caponecchia L, Familiari G,
Verlengia C, Dondero F, Lenzi A. Study of apoptotic DNA fragmentation in human
spermatozoa. Hum Reprod.2000; 15:830
–839.
Ghazzawi IM, Saraf MG, Taher MR, Khalifa FA. Comparison of
fertilizing capability of spermatozoa from ejaculates, epididymal aspirates
and testicular biopsies using intracytoplasmic sperm injection. Hum
Reprod. 1998;13:348
–352.
Gianaroli L, Selman HA, Magli MC, Colpi G, Fortini D, Ferraretti AP. Birth of a healthy infant after conception with round spermatids isolated from cryopreserved testicular tissue. Fertil Steril.1999; 72:539 –541.[Medline]
Gil-Salom M, Remohi J, Minguez Y, Rubio C, Pellicer A. Pregnancy in an azoospermic patient with markedly elevated serum follicle-stimulating hormone levels. Fertil Steril.1995; 64:1218 –1220.[Medline]
Guzick DS, Carson SA, Coutifaris C, et al. Efficacy of superovulation and intrauterine insemination in the treatment of infertility. N Engl J Med.1999; 340:177 –183.[Medline]
Habermann H, Seo R, Cieslak J, Niederberger C, Prins GS, Ross L. In vitro fertilization outcomes after intracytoplasmic sperm injection with fresh or frozen-thawed testicular spermatozoa. Fertil Steril. 2000;73:955 –960.[Medline]
Hamberger L, Lundin K, Sjögren A, Söderlund B.
Indications for intracytoplasmic sperm injection. Hum
Reprod. 1998;13(suppl 1):128
–133.
Hansen M, Kurinczuk JJ, Bower C, Webb S. The risk of major birth defects after intracytoplasmic sperm injection and in vitro fertilization. N Engl J Med.2002; 346:725 –730.[Medline]
Honaramooz A, Snedaker A, Boiani M, Schöler H, Dobrinski I, Schlatt S. Sperm from neonatal mammalian testes grafted in mice. Nature. 2002;418:778 –781.[Medline]
In't Veld P, Brandenburg H, Verhoeff A, Dhont M, Los F. Sex chromosomal abnormalities and intracytoplasmic sperm injection. Lancet. 1995;346:773 .[Medline]
Janzen N, Goldstein M, Schlegel PN, Palermo GD, Rozenwaks Z. Use of electively cryopreserved microsurgically aspirated epididymal sperm with IVF and intracytoplasmic sperm injection for obstructive azoospermia. Fertil Steril.2000; 74:696 –701.[Medline]
Jeulin C, Feneux D, Serres C, Jouannet P, Guillet-Rosso F,
Belaisch-Allart J, Frydman R, Testart J. Sperm factors related to failure of
human in vitro fertilization. J Reprod Fertil.1986; 76:1
–11.
Jones HW Jr, Acosta AA, Andrews MC, et al. Three years of in vitro fertilization at Norfolk. Fertil Steril.1984; 42:826 –834.[Medline]
Jurisicova A, Lopes S, Meriano J, Oppedisano L, Casper RF, Varmuza
S. DNA damage in round spermatids of mice with a targeted disruption of the
Pp1cgamma gene and in testicular biopsies of patients with non-obstructive
azoospermia. Mol Hum Reprod.1999; 5:323
–330.
Kahraman S, Ozgur S, Alatas C, et al. High implantation and
pregnancy rates with testicular sperm extraction and intracytoplasmic sperm
injection in obstructive and non-obstructive azoospermia. Hum
Reprod. 1996;11:673
–676.
Kahraman S, Polat G, Samli M, Sozen E, Ozgun OD, Dirican K, Ozbicer
T. Multiple pregnancies obtained by testicular spermatid injection in
combination with intracytoplasmic sperm injection. Hum
Reprod. 1998;13:104
–110.
Kay VJ, Coutts JR, Robertson L. Effects of pentoxifylline and
proges terone on human sperm capacitation and acrosome reaction.
Hum Reprod.1994; 9:2318
–2323.
Keel BA, Webster BW. Semen analysis data from fresh and cryopreserved donor ejaculates: comparison of cryoprotectants and pregnancy rates. Fertil Steril.1989; 52:100 –105.[Medline]
Kemmann E, Bohrer R, Shelden R, Fiasconaro G, Beardsley L. Active ovulation management increases the monthly probability of pregnancy occurrence in ovulatory women who receive intrauterine insemination. Fertil Steril. 1987;48:916 –920.[Medline]
Kerin JFP, Peek J, Warnes GM, Kirby C, Jeffrey R, Matthews CD. Improved conception rate after intrauterine insemination of washed spermatozoa from men with poor quality semen. Lancet.1984; i:533 –535.
Kim ST, Cha YB, Park JM, Gye MC. Successful pregnancy and delivery from frozen-thawed embryos after intracytoplasmic sperm injection using round-headed spermatozoa and assisted oocyte activation in a globozoospermic patient with mosaic Down syndrome. Fertil Steril.2001; 75:445 –447.[Medline]
Kuczynski W, Dhont M, Grygoruk C, Grochowski D, Wolczynski S,
Szamatowicz M. The outcome of intracytoplasmic injection of fresh and
cryopreserved ejaculated spermatozoa—a prospective randomized study.
Hum Reprod.2001; 16:2109
–2113.
Lacham-Kaplan O, Daniels R, Trounson A. Fertilization of mouse oocytes using somatic cells as male germ cells. Reprod Biomed Online. 2001; 3:205 –211.[Medline]
Lanzendorf SE, Malony MK, Veeck LL, Slusser J, Hodgen GD, Rosenwaks Z. A preclinical evaluation of pronuclear formation by microinjection of human spermatozoa into human oocytes. Fertil Steril.1988; 49:835 –842.[Medline]
Lassalle B, Testart J, Renard JP. Human embryo features that influence the success of cryopreservation with the use of 1,2 propanediol. Fertil Steril.1985; 44:645 –651.[Medline]
Laws-King A, Trounson A, Sathananthan H, Kola I. Fertilization of hu man oocytes by microinjection of a single spermatozoon under the zona pellucida. Fertil Steril.1987; 48:637 –642.[Medline]
Leeuwenhoek A. De natis è semine genitali animalculis. R Soc (Lond) Philos Trans.1678; 12:1040 –1043.
Levron J, Aviran-Goldring A, Madgar I, Raviv G, Barkai G, Dor J. Sperm chromosome abnormalities in men with severe male factor infertility who are undergoing in vitro fertilization with intracytoplasmic sperm injection. Fertil Steril.2001; 76:479 –484.[Medline]
Liu DY, Clarke GN, Baker HGW. Relationship between sperm motility
assessed with the Hamilton Thorne motility analyzer and fertilization rates in
vitro. J Androl.1991; 12:231
–239.
Ludwig M, Katalinic A. Malformation rate in fetuses and children con ceived after ICSI: results of a prospective cohort study. Reprod Biomed Online.2002; 5:171 –178.[Medline]
Macas E, Imthurn B, Keller PJ. Increased incidence of numerical
chromosome abnormalities in spermatozoa injected into human oocytes by ICSI.
Hum Reprod.2001; 16:115
–120.
Mahadevan MM, Trounson AO, Leeton JF. The relationship of tubal blockade, infertility of unknown cause, suspected male infertility, and endometriosis to success of in vitro fertilization and embryo transfer. Fertil Steril.1983; 40:755 –762.[Medline]
Mansour RT, Kamal A, Fahmy I, Tawab N, Serour GI, Aboulghar MA.
Intracytoplasmic sperm injection in obstructive and non-obstructive
azoospermia. Hum Reprod.1997; 12:1974
–1979.
Marrs RP, Vargyas JM, Saito H, Gibbons WE, Berger T, Mishell DR. Clinical applications of techniques used in human in vitro fertilization research. Am J Obstet Gynecol.1983; 146:477 –481.[Medline]
Mastroianni L, Laberge JL, Rock J. Appraisal of the efficacy of artificial insemination with husband's sperm and evaluation of insemination technics. Fertil Steril.1957; 8:260 –266.[Medline]
Matorras R, Gorostiaga A, Diez J, Corcostegui B, Pijoan JI, Ramon O, Rodriguez-Escudero FJ. Intrauterine insemination with frozen sperm increases pregnancy rates in donor insemination cycles under gonadotropin stimulation. Fertil Steril.1996; 65:620 –625.[Medline]
Mendoza C, Tesarik J. Effect of follicular fluid on human sperm movement characteristics. Fertil Steril.1990; 54:1135 –1139.[Medline]
Mercan R, Urman B, Alatas C, Aksoy S, Nuhoglu A, Isiklar A, Balaban
B. Outcome of testicular sperm retrieval procedures in non-obstructive
azoospermia: percutaneous aspiration versus open biopsy. Hum
Reprod. 2000;15:1548
–1551.
Meschede D, Lemcke B, Exeler JR, De Geyter C, Behre HM, Nieschlag
E, Horst J. Chromosome abnormalities in 447 couples undergoing
intracytoplasmic sperm injection-prevalence, types, sex distribution and
reproductive relevance. Hum Reprod.1998; 13:576
–582.
Miyazaki S, Hashimoto N, Yoshimoto Y, Kishimoto T, Igusa Y, Hiramoto Y. Temporal and spatial dynamics of the periodic increase in intracellular free calcium at fertilization of golden hamster eggs. Dev Biol.1986; 118:259 –267.[Medline]
Morshedi M, Oehninger S, Veeck LL, Ertunc H, Bocca S, Acosta AA. Cryopreserved/thawed semen for in vitro fertilization: results from fertile donors and infertile patients. Fertil Steril.1990; 54:1093 –1099.[Medline]
Mulhall JP, Burgess CM, Cunningham D, Carson R, Harris D, Oates RD. Presence of mature sperm in testicular parenchyma of men with nonobstructive azoospermia: prevalence and predictive factors. Urology. 1997;49:91 –96.[Medline]
Muratori M, Piomboni P, Baldi E, et al. Functional and ultrastructural features of DNA-fragmented human sperm. J Androl. 2000;21:903 –912.[Abstract]
Navot D, Goldstein N, Mor-Josef S, Simon A, Relou A, Birkenfeld A.
Multiple pregnancies: risk factors and prognostic variables during induction
of ovulation with human menopausal gonadotrophins. Hum
Reprod. 1991;6:1152
–1155.
Naz RK, Littman BA, Stillman RJ. Successful human pregnancy following in vitro fertilization using frozen semen. J In Vitro Fert Embryo Transfer. 1985;2:143 –145.[Medline]
Negri P, Grechi E, Tomasi A, Fabbri E, Capuzzo A. Effectiveness of pentoxifylline in semen preparation for intrauterine insemination. Hum Reprod.1996; 1:1236 –1239.
Ogawa T, Dobrinski I, Avarbock MR, Brinster RL. Xenogeneic
spermatogenesis following transplantation of hamster germ cells to mouse
testes. Biol Reprod.1999; 60:515
–621.
Ogawa T, Dobrinski I, Avarbock MR, Brinster RL. Transplantation of male germ line stem cells restores fertility in infertile mice. Nat Med. 2000;6:29 –34.[Medline]
Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet. 1992;340:17 –18.[Medline]
Palermo GD, Schlegel PN, Hariprashad JJ, Ergun B, Mielnik A,
Zaninovic N, Veeck LL, Rozenwaks Z. Fertilization and pregnancy outcome with
intracytoplasmic sperm injection for azoospermic men. Hum
Reprod. 1999;14:741
–748.
Patton PE, Burry KA, Thurmond A, Novy MJ, Wolf DP. Intrauterine insemination outperforms intracervical insemination in a randomized, controlled study with frozen, donor semen. Fertil Steril. 1992;57:559 –564.[Medline]
Polge C, Smith AU, Parkes AS. Revival of spermatozoa after vitrification and dehydration at low temperature. Nature. 1949;164:666 .[Medline]
Reis MM, Tsai MC, Schlegel PN, Feliciano M, Raffaelli R, Rosenwaks Z, Palermo GD. Xenogeneic transplantation of human spermatogonia. Zygote. 2000;8:97 –105.[Medline]
Richter MA, Haning RW Jr, Shapiro SS. Artificial donor insemination: fresh versus frozen semen: the patient as her own control. Fertil Steril.1984; 41:277 –280.[Medline]
Rizk B, Fountain S, Avery S, Palmer C, Blayney M, Macnamee M, Mills C, Brinsden P. Successful use of pentoxifylline in male-factor infertility and previous failure of in vitro fertilization: a prospective randomized study. J Assist Reprod Genet.1995; 12:710 –714.[Medline]
Russell LD, Brinster RL. Ultrastructural observations of
spermatogenesis following transplantation of rat testis cells into mouse
seminiferous tubules. J Androl.1996; 17:615
–627.
Russell LD, Franca LR, Brinster RL. Ultrastructural observations of
spermatogenesis in mice resulting from transplantation of mouse spermatogonia.
J Androl. 1996;17:603
–614.
Rybouchkin A, Dozortsev D, Pelinck MJ, De Sutter P, Dhont M.
Analysis of the oocyte activating capacity and chromosomal complement of
round-headed human spermatozoa by their injection into mouse oocytes.
Hum Reprod.1996; 11:2170
–2175.
Rybouchkin AV, Van der Straeten F, Quatacker J, De Sutter P, Dhont M. Fertilization and pregnancy after assisted oocyte activation and intrcytoplasmic sperm injection in a case of round-headed sperm associated with deficient oocyte activation capacity. Fertil Steril. 1997;68:1144 –1147.[Medline]
Schlatt S, Rosiepen G, Weinbauer GF, Rolf C, Brook PF, Nieschlag E.
Germ cell transfer into rat, bovine, monkey and human testes. Hum
Reprod. 1999;14:144
–150.
Seibel MM, ed. Infertility: A Comprehensive Text. Stamford, Conn: Appleton & Lane;1997 .
Serhal PF, Katz M, Little V, Woronowski H. Unexplained infertility—the value of Pergonal superovulation combined with intrauterine insemination. Fertil Steril.1988; 49:602 –606.[Medline]
Sher G, Knutzen VK, Stratton CJ, Montakhab MM, Allenson SG. In vitro capacitation and transcervical intrauterine insemination for the treatment of refractory infertility. Fertil Steril.1984; 41:260 –265.[Medline]
Sherman JK. Freezing and freeze-drying of human spermatozoa. Fertil Steril.1954; 5:357 –371.[Medline]
Shettles LB. Ova harvest with in vivo fertilization. Am J Obstet Gynecol. 1979;133:845 .[Medline]
Silber SJ. The use of epididymal sperm in assisted reproduction. In: Tesarik J, ed. Male Factor in Human Infertility. Rome: Ares-Serono Symposia; 1994:335 –368.
Silber SJ, Nagy Z, Devroey P, Camus M, Van Steirteghem AC. The
effect of female age and ovarian reserve on pregnancy rate in male
infertility: treatment of azoospermia with sperm retrieval and
intracytoplasmic sperm injection. Hum Reprod.1997; 12:2693
–2700.
Silber SJ, Ord T, Balmaceda J, Patrizio P, Asch RH. Congenital absence of the vas deferens. The fertilizing capacity of human epididymal sperm. N Engl J Med.1990; 323:1788 –1792.[Medline]
Sofikitis N, Mantzavinos T, Loutradis D, Yamamoto Y, Tarlatzis V, Miyagawa I. Ooplasmic injections of secondary spermatocytes for nonobstructive azoospermia. Lancet.1998; 351:1177 –1178.[Medline]
Sousa M, Barros A, Takahashi K, Oliveira C, Silva J, Tesarik J.
Clinical efficacy of spermatid conception: analysis using a new spermatid
classification scheme. Hum Reprod.1999; 14:1279
–1286.
Steptoe PC, Edwards RG. Laparoscopic recovery of preovulatory human oocytes after priming of ovaries with gonadotrophins. Lancet. 1970; 1:683 –689.[Medline]
Steptoe PC, Edwards RG. Reimplantation of human embryos with subsequent tubal pregnancy. Lancet.1976; 1:880 –882.[Medline]
Steptoe PC, Edwards RG. Birth after re-implantation of a human embryo. Lancet.1978; 2:366 .[Medline]
Steptoe PC, Edwards RG, Purdy JM. Human blastocysts grown in culture. Nature.1971; 229:132 –133.[Medline]
Tarlatzis B, Bili H. Survey on intracytoplasmic sperm injection:
report from the ESHRE ICSI Task Force. Hum Reprod.1998; 13(suppl 1):165
–177.
Tarlatzis BC, Kolibianakis EM, Bontis J, Tousiou M, Lagos S, Mantalenakis S. Effect of pentoxifylline on human sperm motility and fertilizing capacity. Arch Androl.1995; 34:33 –42.[Medline]
Tasdemir M, Tasdemir I, Kodama H, Tanaka T. Pentoxifylline-enhanced
acrosome reaction correlates with fertilization in vitro. Hum
Reprod. 1993;8:2102
–2107.
Taylor CT, Lawrence YM, Kingsland CR, Biljan MM, Cuthbertson KS.
Oscillations in intracellular free calcium induced by spermatozoa in human
oocytes at fertilization. Hum Reprod.1993; 8:2174
–2179.
Tesarik J. Sex chromosome abnormalities after intracytoplasmic sperm injection. Lancet.1995; 346:1096 .[Medline]
Tesarik J. Sperm or spermatid conception? Fertil Steril. 1997;68:214 –216.[Medline]
Tesarik J. Reproductive semi-cloning respecting biparental embryo
or gin: embryos from syngamy between a gamete and a haploidized somatic cell.
Hum Reprod.2002; 17:1933
–1937.
Tesarik J, Bahceci M, Özcan C, Greco E, Mendoza C. Restoration of fertility by in-vitro spermatogenesis. Lancet.1999a; 353:555 –556.[Medline]
Tesarik J, Greco E, Cohen-Bacrie P, Mendoza C. Germ cell apoptosis
in men with complete and incomplete spermiogenesis failure. Mol Hum
Reprod. 1998a;4:757
–762.
Tesarik J, Greco E, Mendoza C. Assisted reproduction with
in-vitro–cultured testicular spermatozoa in cases of severe germ cell
apoptosis: a pilot study. Hum Reprod.2001a; 16:2640
–2645.
Tesarik J, Guido M, Mendoza C, Greco E. Human spermatogenesis
in vitro: respective effects of follicle-stimulating hormone and
testoster one on meiosis, spermiogenesis, and Sertoli cell apoptosis.
J Clin Endocrinol Metab.1998b; 83:4467
–4473.
Tesarik J, Mendoza C. Sperm treatment with pentoxifylline improves the fertilizing ability in patients with acrosome reaction insufficiency. Fertil Steril.1993; 60:141 –148.[Medline]
Tesarik J, Mendoza C. Most living acrosome-reacted spermatozoa do not fuse with the oocyte when inserted into the perivitelline space. Fertil Steril.1994; 61:529 –535.[Medline]
Tesarik J, Mendoza C. Alleviation of acrosome reaction prematurity by sperm treatment with egg yolk. Fertil Steril.1995; 63:153 –157.[Medline]
Tesarik J, Mendoza C. Genomic imprinting abnormalities: a new
potential risk of assisted reproduction. Mol Hum
Reprod. 1996;2:295
–298.
Tesarik J, Mendoza C. In vitro fertilization by intracytoplasmic sperm injection. BioEssays.1999; 21:791 –801.[Medline]
Tesarik J, Mendoza C. Somatic cell haploidization: an update. Reprod Biomed Online.2003; 6:60 –65.[Medline]
Tesarik J, Mendoza C, Carreras A. Effects of phosphodiesterase inhibitors caffeine and pentoxifylline on spontaneous and stimulus-induced accrosome reactions in human sperm. Fertil Steril.1992a; 58:1185 –1190.[Medline]
Tesarik J, Mendoza C, Greco E. In vitro culture facilitates the selection of healthy spermatids for assisted reproduction. Fertil Steril. 1999b; 72:809 –813.[Medline]
Tesarik J, Mendoza C, Greco E. Immature germ cell conception-in vitro germ cell manipulation. Baillieres Best Pract Res Clin Endocrinol Metab. 2000;14:437 –452.[Medline]
Tesarik J, Mendoza C, Testart J. Viable embryos from injection of round spermatids into oocytes. N Engl J Med.1995; 333:525 .[Medline]
Tesarik J, Mendoza Oltras C, Testart J. Effect of the human cumulus
oophorus on movement characteristics of human capacitated spermatozoa.
J Reprod Fert.1990; 88:665
–675.
Tesarik J, Nagy P, Abdelmassih R, Greco E, Mendoza C. Pharmacological concentrations of follicle-stimulating hormone and testosterone improve the efficacy of in vitro germ cell differentiation in men with maturation arrest. Fertil Steril.2002a; 77:245 –251.[Medline]
Tesarik J, Nagy ZP, Sousa M, Mendoza C, Abdelmassih R. Fertilizable oocytes reconstructed from patient's somatic cell nuclei and donor ooplasts. Reprod Biomed Online.2001b; 2:160 –164.[Medline]
Tesarik J, Pilka L, Dvorak M, Travnik P. Oocyte recovery, in vitro insemination, and transfer into the oviduct after its microsurgical repair at a single laparotomy. Fertil Steril.1983; 39:472 –475.[Medline]
Tesarik J, Rienzi L, Ubaldi F, Mendoza C, Greco E. Use of a modified intracytoplasmic sperm injection technique to overcome sperm-borne and oocyte-borne oocyte activation failures. Fertil Steril. 2002b;78:619 –624.[Medline]
Tesarik J, Sousa M. More than 90% fertilization rates after intracytoplasmic sperm injection and artificial oocyte activation with calcium ionophore. Fertil Steril.1995; 63:343 –349.[Medline]
Tesarik J, Sousa M, Greco E, Mendoza C. Spermatids as gametes:
indications and limitations. Hum Reprod.1998c; 13(suppl 3):89
–107.
Tesarik J, Sousa M, Testart J. Human oocyte activation after
intracytoplasmic sperm injection. Hum Reprod.1994; 9:511
–518.
Tesarik J, Testart J. Treatment of sperm-injected human oocytes with Ca2+ ionophore supports the development of Ca2+ oscillations. Biol Reprod.1994; 51:385 –391.[Abstract]
Tesarik J, Thébault A, Testart J. Effect of pentoxifylline
on sperm movement characteristics in normozoospermic and asthenozoospermic
specimens. Hum Reprod.1992b; 7:1257
–1263.
Testart J, Lassalle B, Belaisch-Allart J, Hazout A, Forman R, Rainhorn JD, Frydman R. High pregnancy rate after early human embryo freezing. Fertil Steril.1986; 46:268 –272.[Medline]
Tournaye H, Merdad T, Silber S, Joris H, Verheyen G, Devroey P, Van
Steirteghem AC. No differences in outcome after intracytoplasmic sperm
injection with fresh or with frozen-thawed epididymal spermatozoa.
Hum Reprod.1999; 14:90
–95.
Tournaye H, Verheyen G, Albano C, Camus M, Van Landuyt L, Devroey P, Van Steirteghem A. Intracytoplasmic sperm injection versus in vitro fertilization: a randomized controlled trial and a meta-analysis of the literature. Fertil Steril.2002; 78:1030 –1037.[Medline]
Tournaye H, Verheyen G, Nagy P, Ubaldi F, Goossens A, Silber S, Van
Steirteghem AC, Devroey P. Are there any predictive factors for successful
testicular sperm recovery in azoospermic patients? Hum
Reprod. 1997;12:80
–86.
Trounson AO, Leeton JF, Wood C, Webb J, Wood J. Pregnancies in
humans by fertilization in vitro and embryo transfer in the
controlled ovulatory cycle. Science.1981; 212:681
–682.
Trounson AO, Mohr L. Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature.1983; 305:707 –709.[Medline]
Tsai MC,Takeuchi T, Bedford JM, Reis MM, Rosenwaks Z, Palermo GD.
Alternative sources of gametes: reality or science fiction? Hum
Reprod. 2000;15:988
–998.
Ubaldi F, Nagy ZP, Rienzi L, Tesarik J, Anniballo R, Franco G,
MenchiniFabris F, Greco E. Reproductive capacity of spermatozoa from men with
testicular failure. Hum Reprod.1999; 14:2796
–2800.
Vanderzwalmen P, Zech H, Birkenfeld A, et al. Intracytoplasmic
injection of spermatids retrieved from testicular tissue: influence of
testicular pathology, type of selected spermatids and oocyte activation.
Hum Reprod.1997; 12:1203
–1213.
Van Steirteghem A, Nagy P, Joris H, et al. Results of
intracytoplasmic sperm injection with ejaculated, fresh and frozen-thawed
epididymal and testicular spermatozoa. Hum Reprod.1998; 13(suppl 1):134
–142.
Van Steirteghem AC, Nagy Z, Joris H, Liu J, Staessen C, Smitz J,
Wisanto A, Devroey P. High fertilization and implantation rates after
intracytoplasmic sperm injection. Hum Reprod.1993; 8:1061
–1066.
Vernaeve V, Tournaye H, Schiettecatte J, Verheyen G, Van
Steirteghem A, Devroey P. Serum inhibin B cannot predict testicular sperm
retrieval in patients with non-obstructive azoospermia. Hum
Reprod. 2002; 17:971
–976.
Vicari E, Grazioso C, Burrello N, Cannizzaro M, D'Agata R, Calogero AE. Epididymal and testicular sperm retrieval in azoospermic patients and the outcome of intracytoplasmic sperm injection in relation to the etiology of azoospermia. Fertil Steril.2001; 75:215 –216.[Medline]
Von Eckardstein S, Simoni M, Bergmann M, Weinbauer GF, Gassner P,
Schepers AG, Nieschlag E. Serum inhibin B in combination with serum
follicle-stimulating hormone (FSH) is a more sensitive marker than serum FSH
alone for impaired spermatogenesis in men, but cannot predict the presence of
sperm in testicular tissue samples. J Clin Endocrinol
Metab. 1999;84;2496
–2501.
Wainer R, Merlet F, Ducot B, Bailly M, Tribalat S, Lombroso R.
Prospective randomized comparison of intrauterine and intracervical
insemination with donor spermatozoa. Hum Reprod.1995; 10:2919
–2922.
Wallach EE. Gonadotropin treatment of the ovulatory patient—the pros and cons of empiric therapy for infertility. Fertil Steril.1991; 55:478 –451.[Medline]
Westlander G, Hamberger L, Hanson C, Lundin K, Nilsson L, Söderlund B, Werner C, Bergh C. Diagnostic epididymal and testicular sperm recovery and genetic aspects in azoospermic men. Fertil Steril. 1999; 14:118 –122.
Wisanto A, Bonduelle M, Camus M, Tournaye H, Magnus M, Liebaers I,
Van Steirteghem A, Devroey P. Obstetric outcome of 904 pregnancies after
intracytoplasmic sperm injection. Hum Reprod.1996; 11(suppl 4):121
–129.
Yanagimachi R. Mammalian fertilization. In: Knobil E, Neill D, eds. The Physiology of Reproduction. 2nd ed. New York, NY: Raven Press; 1994:189 –317.
Yanagimachi R, Chang MC. In vitro fertilization of golden hamster ova. J Exp Zool.1964; 156:361 –376.[Medline]
Yogev L, Gamzu R, Botchan A, Homonnai ZT, Amit A, Lessing JB, Paz G, Yavetz H. Pentoxifylline improves sperm binding to the zona pellucida in the hemizona assay. Fertil Steril.1995; 64:146 –149.[Medline]
Yovich JM, Edirisinghe WR, Cummins JM, Yovich JL. Preliminary results using pentoxifylline in a pronuclear stage tubal transfer (PROST) programme for severe male factor infertility. Fertil Steril. 1988;50:179 –185.[Medline]
Yovich JM, Edirisinghe WR, Cummins JM, Yovich JL. Influence of pentoxifylline in severe male factor infertility. Fertil Steril. 1990;53:715 –722.[Medline]
Zech H, Vanderzwalmen P, Prapas Y, Lejeune B, Duba E, Schoysman R.
Congenital malformations after intracytoplasmic injection of spermatids.
Hum Reprod.2000; 15:969
–971.
This article has been cited by other articles:
|
|
 |
|
  R. Ciriminna, M.L. Papale, P.G. Artini, M. Costa, L. De Santis, L. Gandini, L. Parmegiani, G. Ragni, A. Revelli, L. Rienzi, et al. Impact of Italian legislation regulating assisted reproduction techniques on ICSI outcomes in severe male factor infertility: a multicentric survey Hum. Reprod., September 1, 2007; 22(9): 2481 - 2487. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Greco, F. Ubaldi, L. Rienzi, M. Iacobelli, S. Ferrero, G. Minasi, S. Romano, C. Litwicka, and J. Tesarik Limiting the number of injected oocytes to three impairs ICSI outcomes in patients with nonobstructive azoospermia Hum. Reprod., June 1, 2006; 21(6): 1521 - 1524. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
