1 CharitéUniversitätsmedizin Berlin, Campus Mitte, Klinik für Frauenheilkunde und GeburtshilfeReproduktionsmedizin, Schumannstr. 20/21, 10117 Berlin and 2 Universitätsklinikum HamburgEppendorf, Abteilung für Andrologie, Martinistraße 52, 20246 Hamburg, Germany
3 To whom correspondence should be addressed. Email: hardi.schmiady{at}charite.de
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Abstract |
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Key words: ICSI/premature chromosome condensation/round-headed sperm/unfertilized oocytes
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Introduction |
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With the introduction of ICSI (Palermo et al., 1992), a new treatment could be offered to couples with severe male infertility. However, in cases with round-headed sperm, the results were quite contrary: on the one hand fertilization, pregnancy and live birth have been reported (e.g. Lundin et al., 1994
; Liu et al., 1995
; Trokoudes et al., 1995
; Tasdemir et al., 1997
; Kilani et al., 1998
; Stone et al., 2000
; Nardo et al., 2002
; Zeyneloglu et al., 2002
), followedon the other handby low fertilization or fertilization failure (Battaglia et al., 1997
; Edirisinghe et al., 1998
; Khalili et al., 1998
; Viville et al., 2000
). The variability in fertilization rates may be a result of varying percentages of sperm with round-headed forms seen in the ejaculate of different men with this problem (Syms et al., 1984
).
In this report, we describe the results of three ICSI cycles in a couple with the husband's semen samples showing a mixture of different abnormal sperm heads, round-headed forms with varying spherical appearance beside acephalic sperm (pinheads). Electron microscopy was performed on a semen sample to confirm the absence of the acrosome.
Moreover, the failed ICSI oocytes from the last two cycles were prepared for a cytogeneticcytological study.
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Case report |
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An ejaculate specimen was embedded for transmission electron microscopy (Holstein et al., 1988). Briefly, 0.5 ml of ejaculate was fixed with 5.5% glutaraldehyde (30 min at room temperature). After centrifugation the sediment was postfixed with 1% osmium tetroxide (1 h at 4°C) and centrifuged again. The sediment was then dehydrated in an ascending alcohol series (ethanol 50%, 70%, 96%, 100% twice), propylene oxide twice. Each step was centrifuged (15 min, 800g) and the supernatant discarded. The specimen was covered with propylene oxideEpon (1:1; 1 h). The supernatant was removed, followed by another step in propylene oxideEpon (1:3; overnight) which was pipetted off. Finally, the preparation was covered with the polymerization mixture of Epon (37°C, 26 h; 45°C, 24 h; 60°C, 24 h). The polymerized sediment-blocks were used for semithin/ultrathin sectioning.
The motile sperm fraction for the ICSI procedure was isolated using the swim-up method.
The couple were informed about lower fertilization, embryo development and pregnancy rates.
Three treatment cycles were performed during a period of 7 months. The ovaries were stimulated using a long protocol of GnRH analogue (Synarela; Heumann Pharma, Germany) and recombinant FSH (Gonal F; Serono, Germany; 150 IU day 112 or 225 IU day 110). Ovulation was induced with 10 000 IU hCG (Pregnesin; Serono, or Predalon; Organon, Germany) when the leading follicle had reached a diameter of 20 mm.
Follicles were aspirated transvaginally under ultrasound guidance 3436 h after hCG administration. Six, 20 and 18 oocytes were retrieved and preincubated for 4 h. Three, 18 and 16 oocytes exhibited a polar body and were injected with round-headed sperm, which were immobilized by touching vehemently the flagellum with the injection pipette. The ICSI procedure was performed as described previously (Schmiady et al., 1996
).
The oocytes were examined 1820 h after ICSI. Unfertilized oocytes from the last two cycles were prepared 40 h after microinjection (when they were still in metaphase II of the meiotic division) according to the method of Tarkowski (1966)
: hypotonic treatment with 1% sodium citrate for 5 min, transfer to a marked glass slide followed by fixation (cold methanol:acetic acid =3:1), air-drying and staining with Giemsa.
At genetic counselling, the pedigree of the family revealed that there is a degree of relationship between both partners because their great-grandparents were cousins. Moreover, the husband has a young brother (16 years old) whose fertility could not be proven because of the impossibility of obtaining a semen sample for analysis. Although this information is missing, the possibility that this condition is an autosomal recessive disorder cannot be excluded.
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Results |
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The couple was informed about the problem and the risk of transferring 1PN oocytes.
Of the 29 unfertilized and prepared oocytes from the last two cycles, 27 were informative and revealed in both cycles the maternal metaphase II chromosomes and a high rate (85%) of premature chromosome condensation (PCC) of the sperm nucleus with remarkable variation in the degree of decondensation (Figures 3 and 4).
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Discussion |
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It could not be excluded that chromosomal aneuploidies could be a cause of the low fertilization rate because a highly elevated aneuploidy/diploidy rate has been reported in cases with macrocephalic sperm (In't Veld et al., 1997; Viville et al., 2000
) and Martin et al. (2003)
discovered an elevated frequency of XY disomy in a case of globozoospermia. In contrast to these findings, Rybouchkin et al. (1997)
demonstrated the presence of normal karyotypes in round-headed sperm.
However, the most distinctive finding in this study was the very high rate of PCC (85%) in the sperm nucleus, which was about 3-fold higher than in an earlier ICSI study (Schmiady et al., 1996
) but corresponds exactly to the observations of Edirisinghe et al. (1998)
who performed ICSI in a case with 100% round-headed sperm. The prerequisite for the induction of PCC is that after sperm injection the oocytes do not become activated but remain arrested at metaphase II and possess the chromosome condensing factors in an active form. Although PCC may be associated with some degree of oocyte immaturity (Calafell et al., 1991
), it is unlikely that from three cycles all the oocytes could be immature, because they were preincubated to complete cytoplasmic maturation before ICSI started. The remarkable variation in the degree of condensation of the prematurely condensed sperm chromosomes has been discussed recently (Schmiady et al., 1996
), but to date it is not known whether this variation would also reflect some degree of sperm chromatin disturbances as revealed by our electron microscope analysis. Recently, Sakkas et al. (1996)
found an association between the failure of fertilization after ICSI and the number of sperm that remained condensed, and postulated that poor chromatin packaging and/or damaged DNA may contribute to failure of sperm decondensation.
Rybouchkin et al. (1996) showed that round-headed sperm failed to activate mouse oocytes following ICSI. It was concluded that these sperm are deficient in the oocyte activation, either due to the absence or down-regulation of the sperm-associated oocyte-activating factor. We agree with this kind of interpretation. Furthermore, this deficiency seems to be independent of the variation in morphology of the sperm heads.
The failure of oocyte activation, which itself is caused by the lack of activating protein(s) and the consequent failure of the oocyte to complete meiosis II (MII) is likely to contribute to the high rate of PCC seen in the present case. The MII arrest is characterized by a high maturation promoting factor (MPF) activity (Nurse, 1990). Normally, the sperm induces the release from the meiotic arrest during fertilization by a signal transduction pathway at which MPF and its stabilizing molecules as well as calcium play a determinant role during oocyte activation (for review: Alberio et al., 2001
). The release of free calcium ions from intracellular stores in the oocyte is essential for this process and is mediated, possibly through the introduction of a soluble factor(s) after spermoocyte fusion (Swann et al., 1994
; Dozortsev et al., 1995
). In extracts of hamster sperm cells, one of these factors (oscillin, a glucosamine phosphate deaminase) has been identified and characterized (Parrington et al., 1996
), but although oscillin has been shown to induce transient calcium ion fluctuations after injection into oocytes, new data corroborated that this protein was not the mammalian sperm Ca2+ oscillogen (Wolosker et al., 1998
). Recently, a novel sperm-specific phospholipase C, PLC
, has been identified that triggers Ca2+ oscillations in mouse oocytes indistinguishable from those at fertilization (Saunders et al., 2002
). Even the PLC
content of a single sperm cell was sufficient to produce Ca2+ oscillations as well as normal embryo development. Because PLC
removal from sperm extracts abolished Ca2+ release in oocytes (Saunders et al., 2002
), one might speculate that in sperm from our globozoospermic patient this protein could be either absent or not be released. We can imagine that injection of PLC
in addition to the non-activating sperm cell could offer a chance to trigger Ca2+ oscillations and achieve normal fertilization. Analysis of human sperm PLC
may help to reveal the molecular mechanisms in such cases of male factor infertility.
When oocyte activation was assisted by calcium ionophore treatment after ICSI in cases with round-headed sperm, high fertilization rates (Battaglia et al., 1997) and pregnancies were observed (Rybouchkin et al., 1997
; Kim et al., 2001
). Nevertheless, to recommend the use of calcium ionophore treatment routinely to assist oocyte activation is debatable because there is less information regarding the direct effects that this drug may have on embryo development. Although single sperm cells could contribute to normal fertilization without assisted oocyte activation, the successful selection of such sperm does not seem possible.
Together, the high rate of PCC in this study of round-headed sperm is not specific to this case as it corresponds to another case published recently (Edirisinghe et al., 1998) and leading to the assumption that PCC is associated with fertilization failure which itself is caused by the lack of an oocyte-activating sperm factor.
A combination of ICSI and assisted oocyte activation should be verified in a pilot study. PLC could be applied at first in cases of 100% globozoospermia, where absence of PLC
has been confirmed. The local ethics committee should be consulted and patients should be informed that this treatment is still at an experimental stage. At the moment, it is not possible to make a prognosis on risk factors. In the few cases reported on successful oocyte activation by calcium ionophore treatment, healthy pregnancies have been induced.
When a pregnancy has been achieved after transfer of PLC-activated oocytes, a fetal monitoring according to a risk pregnancy is highly recommended.
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Submitted on December 3, 2004; resubmitted on January 10, 2005; accepted on January 17, 2005.
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