Hum. Reprod. Advance Access originally published online on April 27, 2006
Human Reproduction 2006 21(8):2065-2074; doi:10.1093/humrep/del130
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Outcome of ICSI with ejaculated spermatozoa in a series of men with distinct ultrastructural flagellar abnormalities
1 Laboratoire de Spermiologie, CHRU-Faculté de Médecine, Lille cedex 2 Biologie de la Reproduction—CECOS, Rouen University Hospital, Rouen cedex 3 Biologie de la Reproduction et Génétique Médicale, CHIPS, UVSQ, Poissy 4 Clinique Gynécologique et Obstétricale, Rouen University Hospital, Rouen Cedex 5 Département de Génétique, Cytogénétique, Embryologie, Hôpital Pitié-Salpêtrière, INSERM U651, Paris and 6 Service d’Andrologie, CHU Kremlin-Bicêtre, France
7 To whom correspondence should be addressed at: Laboratoire de Spermiologie, Hôpital Albert Calmette, Boulevard du Professeur Jules Leclercq, CHRU-Faculté de Médecine, Lille cedex F-59037, France. E-mail: v-mitchell{at}chru-lille.fr
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BACKGROUND: Severe sperm motility impairment results in human infertility, which can be overcome by ICSI. Whether some particular, possibly genetic, flagellar abnormalities can influence embryonic development is a matter of debate. METHODS: Analysis of ultrastructural flagellar abnormalities and ICSI outcomes with ejaculated spermatozoa in a series of 21 infertile patients with asthenozoospermic or dyskinetic spermatozoa due to a primary and specific flagellar abnormality was carried out. RESULTS: Patients were sorted into six categories according to flagellar ultrastructural defects. Oocyte fertilization occurred in the 21 couples with a mean 2PN fertilization rate reaching 61.85%. No difference was observed in the kinetics of in vitro development or in the morphological quality of the embryos between the different types of flagellar abnormalities. Pregnancy occurred in 12 couples (57.1%) and delivery in nine couples (42.86%). Both the implantation rate and the clinical pregnancy rate per cycle were lower in type III abnormalities and in patients with an initial sperm motility less than 5%. CONCLUSIONS: The rate of ICSI success may be influenced by the type of flagellar abnormality. ICSI provides a suitable solution for patients with sperm flagellar defects but raises the question of the consequences of a specific (and primary flagellar) abnormality on oocyte fertilization, on embryo and fetal development as well as on live birth.
Key words: electron microscopy/flagella/ICSI/motility/ultrastructure
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Introduction |
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The sperm flagellum is composed of an axoneme and peri-axonemal structures (Inaba, 2003
). The axoneme consists of nine peripheral doublets arranged around two central microtubules and connected by multiprotein complexes. Among the protein complexes are the radial spokes, nexin links, central sheath and dynein arms. The dynein arms are attached to the peripheral doublets and generate motion through ATP-dependent reactions. The other components, mainly the central structures and radial spokes, provide the structural interface for transmitting regulatory signals to the arms (Ibanez-Tallon et al., 2003). The peri-axonemal structures include the mitochondrial sheath, the outer dense fibres and the fibrous sheath (Fawcett, 1975; Holstein, 1981).
Asthenozoospermia is a frequent cause of male infertility and could be explained by alterations of flagellum structure. These alterations may be non-specific and acquired affecting a variable number of spermatozoa, or primary and specific, observed in most spermatozoa (Chemes et al., 1998).
Numerous abnormalities of the axoneme and of the peri-axonemal structures are known in humans. An absence of both dynein arms revealed that these structures are essential to flagellar motion (Afzelius et al., 1975). Several other axonemal abnormalities have been found in infertile men, which could involve any of the axonemal components (Afzelius and Eliasson, 1979; Baccetti et al., 1981; Jouannet et al., 1983; Escalier and David, 1984; Baccetti et al., 2002). Abnormalities of the fibrous sheath, of the mitochondrial sheath and of the attachment of the flagellum to the nucleus have also been found to be responsible for male infertility (Chemes, 2000; Baccetti et al., 2001, 2002; Chemes and Rawe, 2003).
ICSI, successfully developed by Palermo et al. (1992), is considered as an efficient treatment to overcome male factor infertility. Initially proposed in severe oligozoospermia (Payne et al., 1994; Hamberger et al., 1998), ICSI has been applied with ejaculated spermatozoa in the presence of oligoasthenoteratozoospermia, and with epididymal and testicular spermatozoa in azoospermia, with fresh or frozen-thawed samples (for review Devroey and Van Steirteghem, 2004). Furthermore, fertilization and delivery were also obtained with ICSI using immotile but viable spermatozoa (Nijs et al., 1996; Barros et al., 1997; Kahraman et al., 1997; von Zumbusch et al., 1998).
Until now, ICSI outcomes have only been reported in rare cases of specific flagellar abnormalities responsible for immotile spermatozoa, such as absent dynein arms (Stalf et al., 1995; Olmedo et al., 1997, 2000; Papadimas et al., 1997; von Zumbusch et al., 1998; Cayan et al., 2001; Westlander et al., 2003; Peeraer et al., 2004), absent central microtubules (Vandervorst et al., 1997; Okada et al., 1999) or dysplasia of the fibrous sheath (Chemes et al., 1998). Only three studies reported the outcome of ICSI in a series of patients: four patients with absent central microtubules (Okada et al., 1999), six patients with dysplasia of the fibrous sheath (Chemes et al., 1998) and six patients with partial absence of inner dynein arms and dysplasia of the fibrous sheath (Olmedo et al., 2000). Most of these infertile patients with ultrastructural defects of their sperm flagella also exhibited clinical features suggesting abnormalities of their respiratory cilia, i.e. Kartagener’s syndrome, or chronic airway infections (Olmedo et al., 1997, 2000; Papadimas et al., 1997; Chemes et al., 1998; von Zumbusch et al., 1998; Cayan et al., 2001; Westlander et al., 2003; Peeraer et al., 2004).
The aim of this study was to investigate further the ICSI outcome in a series of 21 infertile asthenozoospermic patients with specific and primary flagellar abnormalities evaluated by a quantitative analysis of their flagella under electron microscopy. The outcome of ICSI, using ejaculated spermatozoa, was interpreted according to flagellar abnormalities, and patients with an extremely severe asthenozoospermia (initial motility <5%) were also explored separately.
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Patients
The 21 men included in the present study were recruited from four assisted reproductive technology (ART) centres. They belonged to primary infertile couples who were undergoing ICSI for male factor infertility. The procedure was approved by the institutional review board of these ART centres. The patients were informed of the exact nature and the goal of the investigations performed and provided written informed consent.
An electron microscopy analysis was performed for 60 patients with deficient sperm motility without necrozoospermia. Twenty-one of these patients underwent an ICSI programme and were selected for our study. The motility deficiency was a severe asthenozoospermia with only 0–15% progressive spermatozoa. In three patients, progressive spermatozoa had low amplitude of the flagellar beating. The selected patients had ultrastructural flagellar abnormalities in at least 50% of their spermatozoa. Only Patient 7 suffered from a respiratory disease (i.e. chronic bronchectasia). All patients presented a normal XY karyotype and a normal concentration range of testosterone and FSH. Among the 21 patients, seven patients belonged to three unrelated families (2, 3 and 2 brothers, see Table I). Patients 1 and 2 were born from consanguineous, first-cousin unions, and Patient 1 had a brother with asthenozoospermia related to flagellar abnormalities but who did not undergo ICSI.
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Semen samples and transmission electron microscopy studies
Semen samples were obtained by masturbation after 3–5 days of abstinence. After liquefaction (30 min after ejaculation), 0.5 ml of each ejaculate was used for routine seminal analysis according to the protocol of the World Health Organization laboratory manual (WHO, 1999
). Semen characteristics are summarized in Table I. The remaining semen was fixed in 2.5% v/v glutaraldehyde in 0.1 phosphate buffer, washed for 15 min in fresh buffer containing 4% w/v sucrose and embedded in 2% agar. Post-fixation lasted 1 h in 1% w/v osmic acid in phosphate buffer. After dehydration in a graded series of ethanol, small pieces of agar containing sperm cells were embedded in Epon resin (Polysciences Inc., Warrington, PA, USA). Sections were cut on a Reichert OmU2 ultramicrotome (Reichert-Jung AG, Wien, Austria) using a diamond knife. Sections (70 nm) were collected on nickel grids and stained with uranyl acetate (4% in 70% ethanol, 20 min) and Reynolds lead citrate (10 min), and examined using a JEOL JEM100 CXII transmission electron microscope (Jeol Ltd, Tokyo, Japan) operated at 80 kV.
For each patient, the results of quantitative analysis using electron microscopy were expressed as follows: the quantitative study of morphological abnormalities of the sperm head was performed on at least 30 longitudinal sections; the percentages of misassembled and of absent flagella were first determined in the entire flagella population; the percentage of the flagellar abnormalities was determined in flagella with a recognizable axoneme (at least 50 transverse sections of the principal piece of the flagellum).
Ovarian stimulation and ICSI procedures
The ICSI procedure and the media conditions were similar to routine ICSI performed with motile spermatozoa. Ovarian stimulation was induced after pituitary desensitization with gonadotrophin-releasing hormone agonist (Enantone®, Takeda, Puteaux, France, or Decapeptyl®, IPSEN, Paris, France) at the beginning of the menstrual cycle. Serum estradiol concentration and negative vaginal ultrasonographic scans were used to define ovarian quiescence (J15). Follicular development was then stimulated with recombinant FSH (Puregon®, Organon, Puteaux, France, or GonalF®, Serono, Boulogne, France). According to sonographic and hormonal criteria, oocyte maturation was induced by the administration of 5000 IU hCG (Endo®, Organon, Puteaux, France), and oocytes were collected using vaginal ultrasound-guided puncture 35 h later.
The cumulus-corona cells were removed by incubation for about 20 s in IVF medium (MediCult, Lyon, France) with 30 IU hyaluronidase/ml (MediCult) and aspiration with a glass pipette with 150–300 µm diameter openings. The oocytes were rinsed several times in IVF medium and observed with an inverted microscope at x400 magnification, and the presence or absence of the first polar body was noted. ICSI occurred in a Petri dish (Falcon 1008, Becton-Dickinson, Grenoble, France) containing one 30-µl droplet of HEPES–IVF medium, for spermatozoa, one 10-µl droplet of polyvinylpyrrolidone (PVP, MediCult) in the centre of the Petri dish and three 10-µl droplets of HEPES–IVF medium for oocytes. The Petri dish was surrounded with lightweight detoxified paraffin oil. A single spermatozoon was selected, aspirated tail first in the injection pipette (J.C.D. International, Lyon, France) and transferred to the PVP droplet. When present, motile spermatozoa were used. For samples with absence of sperm motility, sperm viability was assessed with the hypo-osmotic swelling test (HOST) (WHO, 1999). For each spermatozoon, even if it was immotile, the tail was damaged by compression with the injection pipette against the bottom of the dish before the injection into the oocyte. Culture of injected oocytes and embryos was performed in IVF medium (MediCult) under 5% CO2 at 37°C. Assessment of fertilization was done by observing the pronuclei 20 h after sperm injection. Embryos were evaluated 48 h after ICSI and graded according to their morphology (A: blastomeres of equal size, no cytoplasmic fragment; B: blastomeres of equal size, less than 20% cytoplasmic fragments; C: blastomeres of unequal sizes, 30–50% cytoplasmic fragments).
Embryo transfer
Transcervical embryo transfer was performed between 48 and 72 h after oocyte retrieval, using a Frydman cannula (Prodimed, Neuilly, France). When possible, two grade A embryos were transferred, or one grade A and one grade B, or two grade B when no grade A embryo was obtained. 
-hCG plasma level determinations were done 2 weeks later, and uterine ultrasonography was performed 4 weeks after embryo transfer.
Statistical analysis
Chi-square tests, with Yates’ correction or Fischer’s exact test for categorical variables and Kruskall–Wallis test for continuous variables, taking into account the size of our series, were performed using Statview® for Windows 95 (Abacus Concepts, Inc., Berkeley, CA, USA), and a P-value less than 0.05 was considered to be significant. When possible, the values were noted as mean ± SE.
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Semen analysis under light microscopy
Results of the semen analysis under light microscopy, performed the same day as the electron microscopy study, are summarized in Table I. Total motility of spermatozoa varied from 0 to 45%. There was no progressive motility in 14 patients, and progressive motility was lower than normal (<5–15%) in seven patients. Three patients (Patients 18–20) had a characteristic sperm flagella pattern with low-wave amplitude of the flagellum. The predominant flagellar abnormalities were short or absent flagella in 12 patients (Patients 1–12) and coiled flagella in five patients (Patients 13–17). Three patients (Patients 18–20) with sperm dyskinesia had generally normal sperm flagella, with some spermatozoa with truncated flagella. The last patient’s sperm (Patient 21) displayed elongated (giant) middle pieces. The tail defects were often seen in combination with head defects, such as a small acrosomal area or no acrosome at all, with variable proportions between patients (Table I).
Quantitative electron microscopy analysis
The 21 patients of our study were sorted into six types of flagellar abnormalities according to their characteristic ultrastructural defect (Table II): type I, absence of outer dynein arms and associated axonemal abnormalities (Figure 1a); type II, absence of radial spokes (Figure 1b); type III, both flagella without axonemal central structures (also called ‘9+0’ axoneme) (Figure 1c) and flagella with structure misassembly (Figure 1d); type IV, absence of axonemal doublets (Figure 1e), an abnormality often present in coiled flagella (Figure 1f); type V, abnormalities of the longitudinal columns of the fibrous sheath, which were mislocated and/or contained only one column (Figure 1g) and type VI, elongated (giant) mitochondrial sheath (Figure 1h). However, besides the specific flagellar structures that allowed to distinguish type I–III, type I and II presented, as in type III, flagella with ‘9+0’ axoneme, axoneme growth failure and flagella with misassembly of the axonemal and peri-axonemal structures (Table II). The percentage of sperm heads with abnormalities was highly variable between patients (Table II).
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ICSI data
The outcomes of ICSI treatment cycles are shown in Table III, taking into account the different types of flagellar ultrastructural abnormalities and in Table IV, according to initial sperm motility (group A <5%; group B >5%).
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The 21 couples underwent a total of 53 ICSI treatment cycles, leading to the following: (i) 52 fresh and 14 cryopreserved embryo transfers, (ii) 12 (fresh embryos) and 2 (cryopreserved embryos) pregnancies and (iii) 9 (fresh embryos) and 2 (cryopreserved embryos) deliveries. The mean age of the male and the female partners did not differ significantly between the different types of flagellar ultrastructural anomalies and between groups A and B.
ICSI allowed fertilization for all types of spermatozoa ultrastructural defects. The fertilization rate with a mean value of 61.85% did not significantly vary according to sperm motility and flagellar morphology, even if in type I abnormality, the fertilization rate seems to be lower compared with the mean group value (43.21% versus 61.85%). The number of patients included in type I abnormalities was too low to conclude. Most of the fertilized oocytes lead to cleaved embryos (mean rate: 96.01%). Only one complete cleavage failure occurred in Patient 4 during the first ICSI attempt.
The percentages of grade A, B and C embryos were comparable in the different types of flagellar ultrastructural abnormalities and between groups A and B. All the 21 couples underwent at least one fresh embryo transfer (mean value of 2.48 fresh embryo transfers per couple). Furthermore, nine couples underwent 14 cryopreserved embryo transfers (Couples 2, 3, 5, 7, 10, 11, 14, 18 and 21). After the 52 fresh embryo transfers, 12 pregnancies were detected using -hCG plasma level determinations 2 weeks after the fresh embryo transfer and concerned ten couples. The number of pregnancies significantly differed according to flagellar abnormalities (P = 0.03) and to initial sperm motility (P = 0.04; Table IV). In the type III abnormality, pregnancy (14.82%), delivery (14.82%) and implantation (8.33%) rates were lower compared to the series mean value (26.98, 20.64 and 15.23% respectively; Table III). The same observation could be addressed for group A patients with severe motility impairment (motility <5%) compared to group B patients (motility >5%). One pregnancy turned out to be biochemical (Couple 17). Two women had a spontaneous abortion, one during the first trimester (Couple 20) and the other one during the third trimester, with in utero fetal death attributed to a placentitis (Couple 10). Two pregnancies also occurred after frozen-thawed embryo transfer (Patients 5 and 18).
Delivery of healthy children was obtained for nine of the 21 couples (42.86%) after fresh (seven couples, Couples 2, 8, 12, 15, 17, 19, 20) and cryopreserved (two couples, Couples 5 and 18) embryo transfers. Two couples (Couples 13 and 14) obtained two pregnancies and two deliveries after two consecutive ICSI treatment cycles.
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Discussion |
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In the present study, we reported the ICSI outcome in a series of 21 patients with severe deficiency of their motility related to different types of flagellar ultrastructural defects. Data on the ICSI outcomes for patients with immotile spermatozoa and/or short flagella have been previously reported (Stalf et al., 1995
; Kahraman et al., 1996, 1997; Nijs et al., 1996; Abu-Musa et al., 1999; Favero et al., 1999; Viville et al., 2000; Alosilla Fonttis et al., 2002; Evirgen et al., 2003). In this discussion, we focus particularly on reports that included sperm flagella ultrastructural analysis.
Six types of specific ultrastructural defects of the flagella, which have been previously described in humans (Afzelius and Eliasson, 1979; Escalier and David, 1984; Escalier and Serres, 1985; Baccetti et al., 2004), were represented in our series. Types I to III flagella correspond to the known short/stump flagella, as seen in light microscopy (Baccetti et al., 1993). Short/stump flagellum is a complex sperm phenotype, also known as ‘Dysplasia of the Fibrous Sheath’ (Chemes et al.; 1998; Chemes, 2000). The severe failure of the axonemal growth leads to accumulation on several layers of the fibrous sheath (i.e. dysplasia) and is frequently associated with the absence of the central axonemal structures (i.e. ‘9+0’). Moreover, sperm with these flagellar abnormalities also contain various percentages of flagella with a total misassembly of the flagellar structures. This series of patients shows that, in a low number of cases with all these abnormalities related to short/stump flagella, additional axonemal abnormalities are present, such as the absence of the outer dynein arms (type I) and of the radial spokes (type II).
Only one patient had a respiratory disease, which could explain why this series did not include two flagellar phenotypes usually associated with primary ciliary dyskinesia (PCD), such as an absence of both dynein arms, and the association of inner arm, nexin link and radial spoke flagella defects (Afzelius and Eliasson, 1979; Escudier et al., 1990). Cases with type III (‘9+0’ axoneme) rarely presented respiratory deficiency, as shown in another series of 14 patients with this flagellar abnormality that did not have PCD or a Kartagener’s syndrome (Escalier and David, 1984). Absence of outer arms alone also was not represented in our series. This later sperm phenotype is difficult to identify using routine sperm analysis because these spermatozoa can appear morphologically normal, and only a careful observation of their progression pattern enables to detect their low sperm velocity (Jouannet et al., 1983). To our knowledge, we also report, for the first time, ICSI outcomes in patients with rare flagellar abnormalities not associated with PCD. These abnormalities include absence of radial spokes (Escalier and David, 1984; Escudier et al., 1990), a giant middle piece (Sauvalle et al., 1983; Baccetti et al., 2004) and the mislocation of the longitudinal columns (Escalier and Serres, 1985; Escalier, 2003). This latter flagellar abnormality is characterized by an abnormal motility pattern in what appears as normal flagella under light microscopy (Feneux et al., 1985).
The high frequency of type III flagella is in accordance with a previous study showing that in patients with totally immotile spermatozoa, 40% exhibited a ‘9+0’ axoneme (Okada et al., 1999). Men with an absence of sperm’s peripheral doublets and coiled flagella represent the second most frequent group in our series. This abnormality, commonly observed in fertile men, is considered as abnormal only when it concerns a certain proportion of spermatozoa (Escalier and David, 1984). Patient 17 had an unusual sperm profile with only 31% absent doublets and a high proportion of coiled flagella with a normal axoneme.
A good fertilization rate was obtained for most of our patients (>50% in 14 of 21 patients), despite an abnormal ultrastructural morphology in almost all spermatozoa. A good fertilization rate, varying from 54 to 63%, depending on the flagellar abnormality, has already been reported in cases of severe asthenozoospermia (Chemes and Rawe, 2003). This fertilization rate does not differ from the rate usually reported after ICSI with ejaculated spermatozoa from oligozoospermic males (61.8 and 62.9% according to the national French results in 2001 and 2002 respectively; http://www.fivfrance.com). The pregnancy rate obtained in our series (23%) was also comparable to the rate obtained with ICSI in cases of oligozoospermia (23 and 23.8% respectively in 2001 and 2002, according to http://www.fivfrance.com). Besides, the rate of couples who obtained children in our series (9 of 21 couples, 42.8%) is comparable to the rate reported in previous reports on ICSI outcomes in cases of flagellar ultrastructural abnormalities (references in Table V, births of healthy children in 12 of 24 couples, 50%). However, only the patients with ‘9+0’ axoneme of our series can be compared with those previously published (Table V). This specific flagellar abnormality may impair ICSI outcomes and may reduce the chance to conceive: 33% (three of nine couples, Couples 5, 8 and 12) and 50% (six of 12 couples, Couples 2, 15, 17, 18, 19, 20) for the other specific flagellar abnormalities (types I, II, IV, V and VI) conceived a child, compared with only 20% of couples (one of five couples) in previously published data (Vandervorst et al., 1997; Okada et al., 1999). Contrary to other studies, our series does not comprise patients with an absence of the two dynein arms, of only the outer dynein arms and of the inner dynein arms and ‘dysplasia’ of the fibrous sheath (Table V). Considering the previously published cases with abnormalities of the dynein arms (Table V, whatever the type of dynein arms abnormalities), 61.5% (eight of 13 couples) of these couples obtained children. This delivery rate is higher than our results obtained in the type III flagella.
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Even though our series of 21 patients with distinct flagellar abnormalities is large, it does not allow searching for statistical correlations between the flagellar phenotype and the outcomes of ICSI. Moreover, the very low number of patients of flagellar types I, II and VI, which have not yet been evaluated in other studies, does not allow for ICSI prognosis. However, data suggest that the delivery rate per cycle of healthy children could be higher in types IV and V of flagellar abnormalities, because three of five (Patients 13, 14 and 15 with fresh embryos) and two of three (Patient 19 with fresh embryos and Patient 18 with cryopreserved embryos) patients, respectively, fathered a child. To our knowledge, there are no reports on ICSI outcomes for men with these types of flagellar abnormalities, and further investigation of patients with similar abnormalities is necessary to confirm whether types IV and V flagellar abnormalities are of better prognosis.
In our series, clinical pregnancy did not occur in 10 couples, despite of several fresh and frozen-thawed embryo transfers in nine of them. The quality of the cleaved embryos was extremely variable between the different types of flagellar abnormalities. Only four patients did not obtain grade A embryos (Patients 15, 17, 19 and 20). However, four pregnancies occurred in these couples that have led to two healthy children. Furthermore, in type III flagellar abnormality, the distribution between the different grades of embryos did not differ from that observed in all the series (Table III). Specific and primary flagellar abnormalities may not specifically affect embryo quality at early stages but may impair embryo developmental potential resulting in (i) reduced and abnormal blastomere mitotic division ability, (ii) failure of implantation or (iii) spontaneous abortion. Two women from our series endured spontaneous abortion, one during the first trimester and the other one during the third trimester attributed to a placentitis. The frequent reports of miscarriages following ICSI with abnormal spermatozoa indicate that it could be linked to a male genetic defect, but this remains to be established (Kahraman et al., 1996, 1997; Nijs et al., 1996; Lundin et al., 1997; Olmedo et al., 2000; Viville et al., 2000; Peeraer et al., 2004). Furthermore, aneuploid embryos may also be generated in patients with flagellar abnormalities, not solely because of instability of blastomere mitotic division, but also after fertilization with aneuploid spermatozoa. It is worth noting that patients with flagellar abnormalities can exhibit an increased rate of spermatozoa aneuploidy, most of the time, XX and YY hyperhaploid or diploid nuclei (Rives et al., 2005).
The ICSI outcome is not satisfying in the patients of type III, i.e. ‘9+0’ axoneme, although grade A embryos were obtained for the nine couples. Pregnancy (14.82%), implantation (8.33%) and delivery (14.82%) rates were very low compared to the mean of the series. Embryo implantation failed in six of these nine couples, as estimated by the -hCG plasma level analysis. These data question, in type III flagella, of a possible dysfunction of the sperm centriole leading to an early arrest of the zygote development. In humans, the flagellar distal centriole is modified to give rise to the axoneme, but the proximal centriole is conserved, contrary to what happens in rodents. During fertilization, the sperm centriole forms the sperm aster responsible for the male pronuclear movement for the union of the male and female genomes (Sathananthan et al., 1991). Electron microscopy has revealed that a sperm aster is formed after the centriole duplicates at the pronuclear stage (Sathananthan et al., 1996, 2001; Sathananthan, 1998). Moreover, it has been shown that the sperm centrosome controls the first mitotic divisions after fertilization (Palermo et al., 1994). These findings have led to postulate that a sperm centrosomal dysfunction could lead to an aberrant embryonic development and that sperm with impaired motility could present centriolar defects (Palermo et al., 1997). This hypothesis has been supported by studies on heterospecific ICSI using spermatozoa with abnormal head–tail attachment and fibrous sheath dysplasia microinjected into bovine oocytes. The initial study reported insufficient sperm aster formation, lack of syngamy and cleavage, or defective embryos and early abortions (Rawe et al., 2002). Low rates of sperm aster formation (Terada, 2004) and of oocyte activation were found in two other studies (Terada et al., 2004). It should be noted that, in our series, embryos have been obtained whatever the type of flagellar abnormalities, indicating that the sperm centrioles were functional.
Apart from type III flagellar abnormalities, another sperm parameter seems to affect ICSI results in our series. Patients presenting an initial sperm motility less than 5% (Table IV) obtained a very reduced rate of clinical pregnancy (11.11%), delivery (11.11%) and implantation (7.41%), even if fertilization rate, embryo cleavage and quality were not affected. Apart from asthenozoospermia or teratozoospermia, the other semen parameters were normal: spermatozoa viability was preserved, except for Patients 1 and 13, this parameter is known to severely affect ICSI outcomes (Nagy et al., 1995). Sperm count revealed oligozoospermia in only one patient (Patient 1). Furthermore, most of the patients of group A presented type III flagellar abnormalities (five of nine patients, 55.6%). The other patients of this group were distributed in the other types of flagellar abnormalities (types I, IV and VI). Thus, type III flagellar abnormality may impair not solely spermatozoa motility but also embryo potential development.
Familial cases (with similar ultrastructural flagellar abnormalities) were found in types I, III and V (see Table II): absence of outer dynein arms (one brother, not included because he did not undergo ICSI), absence of axonemal central structures (two and three brothers) and mislocation of the longitudinal columns (two brothers). Therefore, these flagellar abnormalities are likely to be of genetic origin and could impair embryo development or could be transmitted to descendants. In fact, both the presence of parental consanguinity in patients with short flagella (Baccetti et al., 2001), which could correspond to type I and III flagellar abnormalities, and of family cases with similar flagellar abnormalities in our series suggest that type I, III and V flagellar abnormalities could be of genetic origin. This notion is also supported by the studies of Chlamydomonas mutants with ‘9+0’ axoneme (similar to type III) (Inaba, 2003) and of knockout mice with mislocation of the longitudinal columns (similar to type V) (Escalier, 2003). It is worth noting that seven of the 12 patients with short flagella (I–III types) are from regions with a common practice of consanguineous marriage, as already observed in eight of 13 patients with short flagella and consanguinity (Baccetti et al., 2001). In addition, 11 of these 12 patients were from North Africa, a geographical clustering observed a long time ago (Bisson et al., 1979).
It remains to be evaluated whether there is a genetic risk when performing ICSI with spermatozoa that carry abnormal genes, in particular by a follow-up of children fathered by men with sperm flagellar ultrastructural defects. ICSI offspring have an increased risk of chromosomal abnormalities, especially for those of sex chromosomes, and the risk has been reported to correlate with the motility of the father’s spermatozoa (Bonduelle et al., 2002). It is therefore necessary to inform couples of the possible risks and to study the genetic basis of these diseases, in order to determine whether these children may turn out to be healthy carriers of a genetic abnormality or not.
In conclusion, the present study of the ICSI outcomes is the first to evaluate a large series of patients bearing a wide panel of well-characterized flagellar abnormalities. Our study shows that different axonemal defects are found in spermatozoa with short flagella and raises the question of each of their childbearing potential. Similarly, the systematic screening of flagellar motility disorders and a careful analysis using electron microscopy allowed us to report, for the first time, data on the ICSI outcomes in the particular cases of radial spoke abnormalities (type II), mislocation of the longitudinal columns (type V) and giant midpiece (type VI). However, further investigations are warranted to describe ICSI outcomes for type II and VI flagellar abnormalities. Data in the literature indicate that the prognosis of ICSI outcome is poor for ‘9+0’ flagellar abnormalities (which is consistent with the results of our study), whereas two of two patients with a Kartagener’s syndrome and absence of both dynein arms in sperm flagella have obtained a child (Table V). Our data demonstrate a good prognosis of ICSI outcome in cases of absence of axonemal doublets (type IV) and mislocation of the longitudinal columns (type V). However, our data also suggest that a flagellar phenotype with a good prognosis for ICSI (i.e. mislocation of the longitudinal columns) did not exclude a genetic risk for the descendants, as suggested by their familial incidence.
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We are grateful to the gynaecological and IVF technical staff for their participation in the ICSI procedures. We wish to thank Mrs F. Carpentier for the sperm preparation for electron microscopy and Dr E. Broneer for reviewing the English text. This work was supported by funds from the Institute of Rare Diseases (INSERM, GIS 0334) and the French National Research Agency (ANR-05-MRAR-022).
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References |
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Submitted on December 22, 2005; resubmitted on March 3, 2006; resubmitted on March 23, 2006; accepted on April 3, 2006.
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