In Vitro Fertilization for Male Factor Infertility
Peter N. Schlegel and
Sarah K. Girardi
James Buchanan Brady Foundation, Department of Urology, The New
York Hospital-Cornell Medical Center, and The Population Council,
Center for Biomedical Research, New York, New York
Address correspondence and requests for reprints to: Peter N. Schlegel, M.D., Room F-905A, Department of Urology, The New York Hospital-Cornell Medical Center, 525 East 68th Street, New York, New York 10021.
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Introduction
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MALE FACTOR infertility is a general term
that describes couples in whom an inability to conceive is associated
with a problem identified in the male partner. This problem may be
associated with low sperm production (oligospermia), poor sperm
motility (asthenospermia), or abnormal morphology (teratospermia) (1).
Abnormal sperm function may also be evaluated with sperm function tests
that evaluate sperm interaction with cervical mucus (cervical mucus
penetration test), the zona pellucida surrounding the oocyte (hemi-zona
binding assay), or the oocyte itself (hamster-egg penetration assay)
(2). Male factor infertility also describes men with normal sperm
production but conditions that prevent sperm transport to the vagina
during intercourse (e.g. reproductive tract obstruction or
ejaculatory dysfunction). Obtaining serial semen analyses and
questioning of the male partner should be part of the initial survey of
an infertile couple. When a male factor is suspected during evaluation
of a couple for infertility, complete evaluation of the man is
warranted. If treatable conditions causing the male factor are found,
they should be corrected. If treatment is unsuccessful, or if the
couple still does not conceive, then assisted reproduction is
indicated. Assisted reproductive techniques include intrauterine
insemination (IUI), in vitro fertilization (IVF), and IVF
with micromanipulation. Micromanipulation refers to a series of
procedures that enhance the ability of sperm to fertilize an oocyte,
in vitro. In this review we will emphasize recent advances
in IVF, especially IVF with the advanced micromanipulation technique of
intracytoplasmic sperm injection (ICSI), as tools for treatment of the
infertile couple with male factor infertility.
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Evaluation of male factor infertility
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The cornerstones of evaluation of a subfertile man include a
comprehensive history, physical examination, multiple semen analyses,
and an endocrine evaluation. In specific circumstances, additional
testing may be indicated. For men with azoospermia or severe
oligospermia (sperm concentration < 5 x
106/cc), consideration of karyotypic abnormalities such as
Klinefelters syndrome is appropriate if clinically indicated. In
addition, up to 13% of men with azoospermia may have microdeletions of
the Y chromosome. Although routine evaluation for these microdeletions
is available at only a few U.S. academic centers in 1996, evaluation at
the SIMMY protocol referral center (Study of ICSI, Male Infertility and
Microdeletions on the Y chromosome) can provide free testing of
patients who are candidates for treatment with ICSI (3, 4, 5, 6). For men
with unilateral or bilateral congenital absence of the vas deferens,
cystic fibrosis transmembrane conductance regulator (CFTR) gene
analysis is important, as 5582% of men with congenital absence of
the vas deferens will carry detectable CFTR mutations (7). In addition,
patients with idiopathic epididymal obstruction have been estimated to
have a 47% chance of carrying a detectable CFTR mutation (8). For
couples with vasal or epididymal anomalies, testing of the female
partner for CFTR mutations is even more important, as not all CFTR
mutations are currently detectable in the man. In the case of any other
genetic condition, including treatment of men with Klinefelters
syndrome, and in the case of couples with a female partner over age 40
yr, genetic counselling is recommended before assisted reproduction
treatments.
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Treatment of male infertility
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Up to 75% of men with a male factor will have identifiable or
treatable conditions that affect their fertility (9, 10, 11). Nearly all
men with male factor infertility are treatable with assisted
reproductive techniques. Before applying more invasive techniques,
however, avoidance of specific gonadotoxic factors such as exogenous
heat, chemical gonadotoxins (e.g. sulfasalazine and
cimetidine), or medications that can adversely affect fertilization
(including calcium channel blockers) is appropriate. Treatment of
varicoceles, endocrine disturbances, symptomatic infections, and
obstructive azoospermia have all been demonstrated, using randomized or
other appropriately designed studies, to have a role in the management
of male infertility (12). Specific treatment of the man may be less
invasive, more successful, and more cost effective (13, 14) with lower
risk than IVF. In addition, it is worthwhile to remember that up to 1%
of men with subfertility have a potentially life threatening condition
associated with their fertility problem, (e.g. testis tumor)
(1, 15). Suffice it to say that evaluation and treatment of a man with
male factor is worthwhile, despite the recent advances in assisted
reproduction.
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Background: in vitro fertilization
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Male factor infertility was initially considered a
contraindication to IVF because abnormal sperm are less likely to
fertilize oocytes than normal sperm (16). However, subsequent
experience starting just over a decade ago indicated that
fertilizations and subsequent live births were possible despite
impaired sperm quality (17). IVF has had some success in the treatment
of these men, however it has been recognized that even normal
concentrations of sperm from oligozoospermic men, placed directly with
oocytes in culture, do not fertilize at the same rates as sperm from
otherwise normal men. In addition, adequate numbers of sperm cannot be
obtained from all men to allow insemination of oocytes with the usual
numbers of gametes (100,000 sperm/oocyte). Initial concerns, that
assisted fertilization with apparently defective sperm might lead to
the development of abnormal embryos and an increase in the number of
birth defects, have not been founded. In fact, once fertilization has
been achieved for male factor couples, implantation and subsequent
pregnancy appear to be just as likely, if not more likely, to occur
than in other cases of IVF (18).
Unless advanced age of the female partner is present, IVF is usually
indicated after specific treatment of male and female factors affecting
fertility has been unsuccessful and less invasive forms of assisted
reproduction (intrauterine inseminations) have been attempted. If
severe male factor infertility is present, direct treatment with IVF
and micromanipulation may be indicated. The technique of IVF is
described in greater detail elsewhere (19). Briefly, it involves
down-regulation of the womans pituitary function with GnRH agonists
given during the preceding luteal phase. This is followed by controlled
ovarian hyperstimulation using FSH or FSH-stimulating agents, to
increase the number of oocytes produced. Follicle development in the
ovary is evaluated directly with transvaginal ultrasound imaging of
follicular growth and by measurement of serial serum estrogen and
progesterone levels. Final oocyte maturation is induced with an
intramuscular dose of hCG (510,000 units) when optimal follicular
development is obtained. Retrieval of oocytes is performed by
transvaginal follicular aspiration using ultrasound guidance with
intravenous sedation. The transvaginal approach has obviated the need
for general anesthesia and laparoscopy to perform IVF. Many oocytes
(mean of 12 oocytes) can be obtained from otherwise normal women with
ovarian hyperstimulation. Morphologically mature, metaphase II oocytes
may then be inseminated with sperm. Human oocytes survive freezing
poorly since they are in metaphase; therefore, all retrieved and mature
oocytes are inseminated. Immature oocytes may be matured in
vitro and subsequently inseminated, although only anecdotal
pregnancies have been achieved after in vitro oocyte
maturation.
Sperm are washed free of seminal fluid and inseminated with oocytes at
a concentration of 100,000 or more sperm per oocyte in simulated human
(Fallopian) tubal fluid medium, and the oocytes that fertilize
(embryos) are usually allowed to divide up to the 8-cell stage before
embryo transfer. Embryo transfer back to the uterus is typically
performed after 23 days of incubation in vitro. Up to four
embryos may be transferred to the uterus, and excess embryos may be
frozen. An implantation rate can be calculated by dividing the number
of gestations (fetal heart on ultrasound) that result from embryo
transfer by the number of total number of embryos transferred to the
uterus in a population of treated patients. Implantation rates per
transferred embryo range from 1025% in most IVF programs.
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Micromanipulation
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Gamete micromanipulation has enabled the embryologist to
circumvent inefficient steps in the fertilization process. Instead of
simply bringing sperm and oocyte together in vitro (IVF),
micromanipulation involves mechanical alteration of the oocyte in
vitro to increase the chance of fertilization of the oocyte by
sperm (Fig. 1
.) The three categories of assisted
fertilization by gamete micromanipulation that have been applied in
humans are illustrated in Fig. 2
. The first category
involves the creation of an opening in the zona pellucida, an acellular
layer surrounding the oocyte that serves as a major barrier to sperm
penetration. Subsequently, the micromanipulated oocyte is inseminated
according to standard IVF guidelines. These procedures have been
broadly termed "zona drilling." One variant of zona drilling
involving mechanical piercing of the zona pellucida has been successful
in male factor patients (20). This method has been called partial zona
dissection (PZD; Fig. 2A
). A second category of micromanipulation
techniques directed at facilitating sperm-oocyte interaction is the
subzonal insertion of sperm (SuZI). SuZI involves direct placement of
sperm into the perivitelline space between the zona pellucida and
oocyte, completely bypassing the zona pellucida (Fig. 2B
) (2, 21). The
third and most invasive form of microsurgical fertilization is the
microinjection of a single sperm into the cytoplasm of the oocyte,
referred to as intracytoplasmic sperm injection (ICSI; Fig. 2C
). This
technique for manipulation has a higher risk of oocyte injury than SuZI
or PZD, but overall higher fertilization and pregnancy rates (22). Most
importantly, only very few sperm are necessary for ICSI. The tremendous
superiority of fertilization and pregnancy rates after application of
ICSI when compared with PZD and SuZI have relegated both PZD and SuZI
to techniques of historical importance only. With micromanipulation,
fertilization and pregnancy rates appear to be independent of sperm
quality (23, 24), which is the opposite of what has been demonstrated
for both IUI and IVF (16).

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Figure 1. The structural components of an oocyte
important for micromanipulation are schematically illustrated.
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Figure 2. A, Schematic illustration of partial zona
dissection technique. B, Schematic illustration of subzonal insertion
procedure. C, The intracytoplasmic sperm injection technique is
schematically presented.
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Intracytoplasmic sperm injection
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Until recently, the clinical application of direct injection of a
single sperm into the cytoplasm of an oocyte during IVF had not been
feasible. The demonstration of fertilization and live births by Palermo
et al. (25) in 1993 was the first successful application of
ICSI. Since that time, ICSI has been performed extensively in multiple
centers to treat patients with severe male factor infertility. To date,
the success of ICSI procedures has been related to several factors: 1)
the viability of the spermatozoon, 2) the quality of the oocyte, 3)
effective activation of the oocyte, and 4) ability of the oocyte to
tolerate intracytoplasmic manipulation. Application of this treatment
is described below.
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Indications for ICSI
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To date, rigorous indications for ICSI are not universally agreed
upon. In general, any condition in which it is expected that oocyte
fertilization might be impaired, ICSI should be considered. Early
clinical series applied ICSI in cases where men had less than 500,000
motile sperm present in the ejaculate, less than 4% normal sperm forms
(with strict criteria evaluation), or where couples have failed to
fertilize any oocytes in an earlier IVF cycle. Sperm function tests (2)
may provide additional insight into specific sperm-oocyte interaction
defects that will define appropriate candidates for ICSI. We have
proposed the following minimal indications for micromanipulation
(26):
a. sperm concentration < 2 x 106 sperm/cc.
b. sperm motility < 5%
c. strict criteria normal morphology < 4%
d. use of surgically retrieved spermatozoa
e. failure of fertilization in a previous IVF cycle
Although fertilization and pregnancy rates with ICSI are similar
to or better than those achieved with normal sperm in other couples
undergoing IVF at the same center (27), couples with only minor semen
abnormalities have not been routinely treated with ICSI. Given the
relatively brief history of ICSI, and its potential effects on progeny,
it would seem prudent to avoid over-application of this new technology.
Therefore, ICSI should not be recommended to couples for whom there is
no documented benefit, as unknown risk to the embryo and resulting
fetus may still exist (28).
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Technique of ICSI
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Oocyte processing. Oocytes are prepared by removing the
cumulus mass and corona radiata with hyaluronidase. Intracytoplasmic
sperm injection is performed on all metaphase II oocytes. Metaphase II
oocytes have their diploid complement of chromosomes delicately
arranged on the metaphase plate near the polar body. Mechanical
disruption of the metaphase plate can occur by injury from the
injection pipette or by the presence of a motile sperm in the oocyte
cytoplasm. The oocyte is stabilized with a holding micropipette and
injected under an inverted microscope.
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Microinjection
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Details of the preparation of microtools and protocols for ICSI
are described in detail elsewhere (27). Individual single sperm are
aspirated from a prepared semen specimen and directly injected into an
oocyte immobilized in a droplet of medium under paraffin oil. The polar
body is held at the 12 or 6 oclock position, and the injection
micropipette containing the single sperm is pushed through the zona
pellucida and oolemma into the cytoplasm of the oocyte at the 3
oclock position. Further handling of injected oocytes is similar to
that for oocytes in standard IVF.
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Results of ICSI
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One of the largest series reporting results using ICSI was from
Van Steirteghem et al. (22) at The Brussels Free University
in Brussels, Belgium. In their preliminary report on 150 couples who
underwent 150 consecutive treatment cycles, 1409 oocytes were injected
and 830 were successfully fertilized for a fertilization rate of 59
percent. A total clinical pregnancy rate of 35 percent was achieved.
Fertilization and pregnancy rates from their updated series (29) are
shown in Table 1
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In another large series, Palermo et al. (27) reported on 227
couples treated with ICSI for failed IVF cycles or for severe male
factor infertility. Fertilization and pregnancy rates were evaluated
relative to semen parameters and the origin of the semen samples. They
reported successful fertilization in 1,142/1,923 (59 percent) metaphase
II oocytes injected and ongoing pregnancies in 84/227 (37 percent)
couples.
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Factors affecting results of ICSI
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Spermatozoal factors. Nagy et al. (29) reviewed the
effect of spermatozoal factors on results of ICSI in 966 microinjection
cycles. Despite no normal forms in a semen preparation, virtual
azoospermia or essentially no motile sperm in the ejaculate, pregnancy
could still be achieved. Nagy et al. found that the only
absolute criterion for successful ICSI is the presence of at least one
viable spermatozoon to inject per oocyte in the prepared pellet of the
washed semen sample. The only category of semen parameters that had a
significantly adverse effect on fertilization and pregnancy rates with
ICSI was when there were no motile sperm (29). If no motility is
present, then viability is often impaired as well.
Female factors. Oehninger et al. (30) investigated
the role of female factors on ICSI results in a total of 92 couples,
where 1163 oocytes were injected, with an overall fertilization rate of
61 percent. Fertilization rates were unaffected by maternal age, but
pregnancy rates were significantly lower with increased maternal age.
Pregnancy rates were 49, 23, and 6 percent for couples in whom maternal
age was less than 34 yr, 3539 yr, and 40 yr or over. Similar results
were found by Sherins et al. (31), with a 30% pregnancy
rate for the youngest couples and a 13% pregnancy rate for the couples
with the oldest female partners. The rate of aneuploidy increased
dramatically for embryos derived from the oocytes of women over 40
compared with those from women less than 35 yr (32). Implantation of an
aneuploid embryo is highly unlikely. These observations suggest that
the chance of a metaphase II oocyte being fertilized with ICSI is
unrelated to female age, but the chance of a pregnancy occurring after
transfer of ICSI embryos dramatically decreases with increased female
age, especially female age over 40 yr.
Oocyte activation. Oocyte activation refers to the series of
events that occur after sperm-oocyte fusion during natural
fertilization, which result in the ability of the oocyte to complete
its nuclear maturation, to synthesize proteins and DNA. Because sperm
fusion with the oocyte is bypassed during ICSI, other approaches to
induce oocyte activation have been attempted. Tesarik and Sousa (33)
improved fertilization and pregnancy rates during ICSI by aggressive
aspiration and injection of the oocyte cytoplasm during injection of
sperm into the oocyte to induce oocyte activation. Vigorous cytoplasmic
aspiration resulted in an increase in fertilization rates per oocyte
from 3880% when compared with results achieved using only gentle
aspiration of the oocyte cytoplasm. Pregnancy rates increased up to
52% with aggressive aspiration/injection. Aggressive aspiration of
cytoplasm caused additional peaks of oocyte intracellular calcium
levels, when compared with gentle aspiration (33). Intracellular
calcium changes have long been thought to have a role in oocyte
activation, and these changes may constitute the mechanism by which
aggressive cytoplasmic aspiration improves fertilization rates.
A sperm factor may also have a role in oocyte activation. This
cytoplasmic sperm factor may need to diffuse through the sperm plasma
membrane to induce post-ICSI events in the oocyte that facilitate
pronuclear formation, including formation of the sperm aster by the
paternally-derived centrosome and mitosis of the embryo. Aggressive
immobilization of spermatozoa involves mechanical crushing of the sperm
tail between the injection micropipette and the bottom of the petri
dish containing the spermatozoon. Gerris et al. (34)
reported an increase in the percentage of normally fertilized oocytes
from 3660% with aggressive immobilization. Palermo et al.
(35) found the effect of aggressive sperm immobilization on
fertilization rates was seen primarily in immature spermatozoa that
were surgically retrieved from the epididymis and testis. For
epididymal sperm, Palermo et al. demonstrated an increase in
fertilization rates, from 5184% per oocyte, with an associated
improvement in pregnancy rates from 5182% (35).
It appears that aggressive immobilization of immature spermatozoa may
increase sperm membrane permeability, which enhances release of
cytosolic sperm factors that facilitate oocyte activation (36).
Alternatively, it is possible that the increased sperm membrane
permeability results in leakage of toxic factors, such as reactive
oxygen species, out of the cytoplasmic droplet of immature spermatozoa.
Oocyte activation must be induced for optimal success with ICSI.
Cytoplasmic sperm factors (37) as well as mechanical stimulation of the
oocyte are helpful in inducing oocyte activation.
Cytoplasmic injection/oocyte injury. Disruption of the oocyte
sufficient to cause oocyte demise may occur during ICSI. Results from
some of the major centers performing ICSI show rates of oocyte loss
after injection of 714%. Although the precise reasons for oocyte
injury are not known, it is thought to occur as a result of plasma
membrane and ultrastructural disturbances associated with injection,
damage to the meiotic spindle during injection, and/or extrusion of the
oocyte cytoplasm following injection. In addition, other factors such
as changes in temperature have been reported to cause irreversible
changes in the meiotic spindle of the human oocyte. Clearly, there is a
learning curve for embryologists performing the ICSI procedure. As
greater expertise is gained over the first 50100 oocyte injections,
the oocyte injury rate decreases (38).
Palermo et al. (39) have described an oocyte membrane
response of "sudden breakage" during attempted ICSI. The oocytes
with this response did not form a normal funnel during attempted
penetration of the injection pipette, but suddenly separated, spilling
the oocyte cytoplasm. With sudden breakage, a 14% oocyte injury rate
was seen, compared with a 4% injury rate for other oocytes. Oocytes
demonstrating sudden breakage were more likely to be retrieved from
women treated with higher gonadotropin treatment doses, with lower
serum estradiol levels at retrieval, or where the oocytes were
immature, requiring maturation in vitro. These observations
suggest that ovarian hyperstimulation may affect the ability of oocytes
to survive ICSI (39).
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Risks of ICSI
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Risks of ICSI include general risks of IVF as well as the specific
risks related to the micromanipulation procedure of ICSI. One of the
most significant risks associated with stimulation of the ovaries is
the ovarian hyperstimulation syndrome (OHSS). This can manifest as
massive ovarian enlargement, peritoneal irritation caused by follicular
rupture or hemorrhage, ovarian torsion, ascites, pleural effusion,
oliguria, electrolyte imbalance, hypercoagulability (40), and sometimes
death (41). The syndrome occurs in a moderate form for 34% percent
of initiated cycles, and in a severe form for 0.10.2% of the
population (42) undergoing controlled ovarian hyperstimulation. Other
reported complications of ovarian hyperstimulation are pituitary
hemorrhage, endometriotic bloody ascites, and genital cancer (43).
Complications of ovarian retrieval have been reported for transvaginal
aspiration as well as laparoscopic aspiration. Complications associated
with transvaginal aspiration have been reported in 0.33% of cases
and include bleeding, pelvic infections, and abdominal viscera
perforation (44). Laparoscopic complications include hemorrhage,
intestinal perforation, infection, and carbon dioxide embolism. The
laparoscopic risks are no higher in ovarian retrieval procedures than
in other laparoscopic applications.
Finally, pregnancies resulting from ovarian stimulation are at risk for
spontaneous abortion (45), ectopic pregnancy (46), and multiple
gestations (47, 48, 49). The rate of spontaneous abortion after achieving a
biochemical pregnancy with assisted reproduction is approximately 25%.
These losses are attributed to a) advanced maternal age and the
associated increased prevalence of chromosomal abnormalities; b) a
higher rate of pregnancy loss resulting from multiple gestations, and
c) early recognition of these pregnancies because of close monitoring.
After achieving a clinical pregnancy (the presence of at least one
fetal heart beat on ultrasound), the chance of a spontaneous abortion
occurring for ICSI cycles ranges from 1016%. Ectopic pregnancies
occur in up to 35.5% of gestational cycles and can be life
threatening. The etiology is usually pelvic adhesions and tubal damage
from pelvic inflammatory disease or previous surgery (46). Multifetal
pregnancies occur in 22% of cases of IVF with embryo transfer (47),
and 4446% of ICSI cases (27, 30) in the United States. Multifetal
pregnancies are considered a complication of assisted reproductive
techniques because of the associated increased incidence of
preeclampsia, placenta previa, placental abruption, premature rupture
of membranes, and postpartum hemorrhage (48). Most importantly,
multiple gestations are almost universally associated with prematurity
and the associated complications to offspring, including cerebral palsy
and intracranial hemorrhage with mental retardation or blindness. To
prevent multifetal pregnancies and their attendant complications, it
would be preferable to avoid assisted reproduction unless it is
specifically indicated and to limit the number of embryos transferred.
Where there is government regulation of IVF, including England,
Australia, and France, transfer of only three or fewer embryos is
allowed, and multifetal pregnancies are less common (49).
Unfortunately, there is significant pressure to transfer more than
three embryos by couples in the United States who are desperate to
conceive. In general, for women less than 35 yr of age, only three
embryos should be transferred.
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Birth defects after assisted reproduction
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The bypass of natural barriers to fertilization, possible genetic
defects in men with severe male infertility, and the use of severely
abnormal sperm for intracytoplasmic sperm injection has engendered
concern over the impact of ICSI on the genetic complement of the
offspring (28). Previous studies have suggested no increase in birth
defect rates when IVF alone was used to induce conception (50). Van
Steirteghem (51) reported no increase in the congenital malformation
rate in their center after ICSI when compared with the general
population. Of 877 children born after ICSI procedures, 23 (2.6
percent) had major congenital malformations compared with 2.02.8% in
the general population and 1.92.9% of children resulting from IVF
without ICSI (51).
Sex chromosome abnormalities have also been reported in ICSI cases.
Int Veld et al. (52) reported on 12 patients with ICSI
pregnancies who underwent prenatal diagnosis for advanced maternal age.
Three of the 12 women had twin pregnancies for a total of 15 diagnostic
procedures by amniocentesis or chorionic villus sampling. A total of 5
chromosomal abnormalities were detected: 2 cases of 47 XXY, 1 complex
mosaic 45,X/46,X.dic(Y)(q11)/46,X.del(Y)(q11), and 2 cases of 45 XO.
This high rate of sex chromosome abnormalities has not been
corroborated by other studies. The Brussels group reported on a total
of 585 prenatal diagnoses performed in pregnancies established by ICSI.
A total of 6 sex chromosome abnormalities (1.0 percent) were detected
compared with 0.2 percent in the general population (53). This
difference did not achieve statistical significance. Govaerts et
al. (54) reported on 55 karyotypes obtained by amniocentesis or
chorionic villus sampling in pregnancies from ICSI and found no sex
chromosome abnormalities. When sex chromosome abnormalities have been
identified it has been unclear whether they were related to the ICSI
procedure, underlying paternal cytogenetic defects, or advanced
maternal age. What is reassuring is that the rates of non-sex
chromosomal abnormalities in the ICSI population published to date do
not exceed the rates seen in the general population.
The source of sex chromosomal abnormalities in offspring after ICSI may
be related to abnormalities in testicular germ cells. In addition to
disomic sex chromosome abnormalities, several investigators have
reported that up to 13% of men with azoospermia or severe oligospermia
may have deletions of 15,000200,000 base pair lengths of the Y
chromosome (3, 4, 5, 6). At least one gene (DAZ; deleted in azoospermia) is
deleted in 13% of patients with nonobstructive azoospermia and some
men with severe oligospermia. Of greater concern is the possibility
that additional unknown genetic problems may be present in infertile
men who have never been able to conceive in the past, but can now
become fathers with ICSI. Evaluation of 32 father-son pairs after ICSI
showed that 3 (9%) ICSI-derived sons had microdeletions of the DAZ
region in one study (6), possibly because of germ line mosaicism for Y
chromosome microdeletions in the fathers of 2 of these boys. The third
father had a Y chromosome microdeletion detected on peripheral
leukocyte evaluation that was inherited by his son. ICSI-derived sons
may be at increased risk of infertility or other abnormalities because
of transmission of the genetic defects that are associated with male
infertility.
Although chromosomal abnormality rates in offspring after assisted
reproductive procedures have not exceeded those in the general
population, experience with these techniques is brief. Genetic
counseling, preimplantation genetic diagnosis, and state of the art
prenatal diagnosis must also be available to couples enrolled in
assisted reproductive programs. All couples undergoing
micromanipulation procedures are strongly urged to have prenatal
diagnosis with amniocentesis or chorionic villus sampling. The need for
prenatal diagnosis is dependent on whether the couple would consider
terminating the pregnancy if the results are abnormal. If the couple
would carry a pregnancy to term regardless of the results of prenatal
diagnosis, then the procedure of prenatal intervention would carry
risks to the fetus without benefit and therefore cannot be
required.
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Application of ICSI: epididymal and testicular sperm
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The application of ICSI has allowed treatment of couples who until
very recently were considered sterile and untreatable. Men with
bilateral congenital absence of the vas deferens and other
unreconstructable obstructions of the male reproductive tract are good
candidates for ICSI. In these men, microsurgical retrieval as well as
cryopreservation of sperm is possible despite the fact that the sperm
are immature (i.e. have not traversed most of the excurrent
duct system.) Percutaneous aspiration of sperm from the epididymis or
testis can also provide sperm for ICSI cycles, although the sperm are
often not of adequate quality for cryopreservation; therefore a repeat
sperm retrieval procedure mighty be needed with each ICSI attempt.
Using ICSI and simultaneous open surgical sperm retrieval, clinical
pregnancy rates per sperm and oocyte retrieval attempt range from
4582% at established centers (35, 55, 56).
For men with nonobstructive azoospermia, sperm retrieval from the
testis is often successful using an open biopsy technique. Even if a
small diagnostic testis biopsy reveals a Sertoli cell-only pattern in a
man with small volume testes and an elevated FSH level, sperm retrieval
might still be possible with a more extensive biopsy. Taken together,
5070% of men with nonobstructive azoospermia can have sperm
retrieved surgically from the testis, with a biopsy performed
simultaneous to an IVF-ICSI procedure (57, 58, 59). Up to 20% or more of
attempts at sperm retrieval-ICSI for nonobstructive azoospermia will
result in a clinical pregnancy (57, 58, 59).
Even for men who have no sperm production in their testes, immature
germ cells (spermatids or spermatocytes) might have application in
micromanipulation procedures with some chance of contributing to
pregnancies. In an animal model, Kimura and Yanigamachi (60) have
demonstrated induction of pregnancies by injecting the nucleus of a
secondary spermatocyte from a normal animal into an oocyte and
activating the egg with an electrical pulse. Transfer of resulting
embryos to the uterus of a recipient animal resulted in normal
offspring. In humans, round spermatids retrieved from the semen of
seven men were microinjected into oocytes of their female partners,
with ongoing pregnancies for two couples, including delivery of a
normal child for one couple (61). The potential for future application
of these techniques is difficult to predict. In our experience, we have
rarely found spermatids in testicular biopsy specimens, unless more
mature spermatozoa are present.
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Associated procedures
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Micromanipulation procedures can also be used to analyze and
select embryos with specific genetic, chromosomal, or biochemical
characteristics before the transfer of those embryos. Analysis of the
embryo is performed at the four- or eight-cell stage by extracting an
individual cell (blastomere) for evaluation. Chromosome-specific
sequences can be identified using fluorescent hybridization probes or,
alternatively, polymerase chain reaction (PCR) amplification of
individual alleles on the chromosomes themselves may be applied to
identify the genotype of the biopsied embryo. These techniques can
allow sex selection to avoid transmission of X-linked diseases such as
hemophilia A or von Willebrands disease. In addition, specific
genetic defects such as the homozygous
F508 mutation of the CFTR
gene, associated with the development of a severe form of cystic
fibrosis, can also be identified. These techniques have been applied
for couples known to be at high risk of having children with specific
genetic diseases. Biopsied embryos have been successfully transferred,
resulting in pregnancies and live births (62). These micromanipulation
techniques are highly labor intensive and carry some potential
pitfalls. For example, if both male and female partners are
heterozygous for the
F508 CFTR mutation, then an individual embryo
has a one-in-four chance of being homozygous for that gene mutation.
However, differential amplification of either the normal or mutated
allele may result in a false positive or negative result by
preimplantation diagnosis (63).
For sex chromosome analysis, this evaluation is more accurately
performed (up to 95.5% efficiency) using different colored fluorescent
probes (64). A test for both X- and Y-specific sequences is possible
and provides further confirmation of the results of these tests. Given
the extensive manpower needed for single-day biopsy and evaluation of
the results of embryo biopsy, this technique is limited to those cases
where life threatening genetic defects can be reliably detected before
embryo transfer to prevent the potential termination of a fetus later
in development. In addition, chromosomal mosaicism is common in
embryos, limiting the accuracy of single blastomere biopsy to
approximately 80%.
 |
Summary
|
---|
Since the first U.S. report of a successful delivery from in
vitro fertilization in 1982 (65), progress in the field of
assisted reproduction and micromanipulation has been truly dramatic.
Perhaps the most exciting advances have been in the area of male factor
infertility. Couples who previously would have been offered donor
insemination or adoption are now achieving pregnancies despite severe
impairments in semen quality, the presence of only single numbers of
sperm in the ejaculate, or unreconstructable reproductive tract
obstruction. Techniques of micromanipulation that were revolutionary
less than five yr ago are now obsolete, replaced by even more
successful methods. Even nonobstructive azoospermia resulting from
maturation arrest or other impairments in germ cell development have
been added to the list of treatable factors in male infertility, as
sperm can frequently be extracted directly from testicular parenchyma
that is aspirated or surgically biopsied. For patients without sperm in
the testicular parenchyma, round spermatid or secondary spermatocyte
injections are at least theoretically possible.
Several important questions remain with regard to IVF-ICSI. 1) What
should be the specific indications for IVF and IVF-ICSI? Should IVF
alone ever be used for male factor infertility? 2) What are the reasons
for failure to achieve pregnancy after ICSI, which still represent over
half of our attempts at achieving ongoing pregnancies? 3) Can we be
certain that using severely impaired or less mature sperm will not
result in significant birth defects or in genetic abnormalities that
could affect the offspring in adolescence or adulthood? 4) What is the
most successful and cost effective approach for the infertile couple
with impaired semen parameters?
For couples with male factor infertility, careful evaluation and
treatment of the man should be considered before assisted reproduction,
including ICSI. Contemporary application of ICSI for severe male factor
infertility can allow pregnancy rates up to 52% (33), with ongoing
pregnancy and live delivery rates as high as 37% per IVF cycle attempt
(27). As long as viable sperm are present in the ejaculate or
retrievable from the reproductive tract, then ICSI procedures can be
applied.
Received March 1, 1996.
Revised July 11, 1996.
Revised October 18, 1996.
Accepted October 28, 1996.
 |
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