High rate of premature chromosome condensation in human oocytes following microinjection with round-headed sperm: Case report

Hardi Schmiady1,3, Wolfgang Schulze2, Ingrid Scheiber1 and Bettina Pfüller1

1 Charité—Universitätsmedizin Berlin, Campus Mitte, Klinik für Frauenheilkunde und Geburtshilfe–Reproduktionsmedizin, Schumannstr. 20/21, 10117 Berlin and 2 Universitätsklinikum Hamburg–Eppendorf, Abteilung für Andrologie, Martinistraße 52, 20246 Hamburg, Germany

3 To whom correspondence should be addressed. Email: hardi.schmiady{at}charite.de


    Abstract
 Top
 Abstract
 Introduction
 Case report
 Results
 Discussion
 References
 
A young couple proceeded to three ICSI treatment cycles because of male infertility. The semen samples varied between 10 x 106 and 36 x 106/ml, 38 and 51% progressive motility but 0% normal morphology. Different types of sperm heads, mostly round-headed with varying spherical appearance (86%) were presented beside acephalic sperm (pinheads; 12%), both one- or two-tailed and the former also without a tail. Very few sperm (2%) exhibited slightly oval-shaped heads. Electron microscopy revealed the absence of the acrosome combined with disturbance of the chromatin condensation among the round-headed sperm. In all three cycles, the fertilization rate using the round-headed sperm fraction was very low with the best result of 2/18 (11%) two-pronucleate oocytes and one one-pronucleate oocyte obtained in the second ICSI cycle. The three oocytes cleaved and were transferred in the 3–4-cell stage without achieving a pregnancy. Of the 29 unfertilized and prepared oocytes from the last two cycles, 27 were informative and revealed the maternal metaphase II chromosomes in the haploid range and a high rate (85%) of premature chromosome condensation (PCC) of the sperm nucleus with remarkable variation in the degree of condensation. Thus, it appears that nearly all round-headed sperm from this patient were incapable of oocyte activation after ICSI, which could be due to non-release (or absence) of an activating factor. As a consequence, PCC was induced in the sperm nuclei by the chromosome condensing factors which were still active in the oopasm of the arrested oocytes.

Key words: ICSI/premature chromosome condensation/round-headed sperm/unfertilized oocytes


    Introduction
 Top
 Abstract
 Introduction
 Case report
 Results
 Discussion
 References
 
Patients with round-headed sperm in their semen (‘globozoospermia’) are considered sterile (Schirren et al., 1971Go) since these sperm are unable to bind to the zona pellucida and penetrate the oocyte during conventional IVF (Aitken et al., 1990Go; Schmiady et al., 1992Go). In this inherited condition, the sperm show severe gross and ultrastructural abnormalities, including the absence of the acrosome, but are capable of decondensation and pronuclear formation after direct injection into hamster oocytes (Lanzendorf et al., 1988Go).

With the introduction of ICSI (Palermo et al., 1992Go), 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., 1994Go; Liu et al., 1995Go; Trokoudes et al., 1995Go; Tasdemir et al., 1997Go; Kilani et al., 1998Go; Stone et al., 2000Go; Nardo et al., 2002Go; Zeyneloglu et al., 2002Go), followed—on the other hand—by low fertilization or fertilization failure (Battaglia et al., 1997Go; Edirisinghe et al., 1998Go; Khalili et al., 1998Go; Viville et al., 2000Go). 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., 1984Go).

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 cytogenetic–cytological study.


    Case report
 Top
 Abstract
 Introduction
 Case report
 Results
 Discussion
 References
 
A 25 year old woman (karyotype: 46,XX) and her 30 year old partner (karyotype: 46,XY) proceeded to a total of three ICSI treatment cycles because of male infertility. The number of sperm cells within the semen samples, also used for ICSI, varied between 10 x 106 and 36 x 106/ml with 38 and 51% progressive motility (WHO grades a+ b; World Health Organization, 1999Go) and 0% normal morphology. Semen smears were stained with Giemsa or according to Papanicolaou.

An ejaculate specimen was embedded for transmission electron microscopy (Holstein et al., 1988Go). 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 oxide–Epon (1:1; 1 h). The supernatant was removed, followed by another step in propylene oxide–Epon (1:3; overnight) which was pipetted off. Finally, the preparation was covered with the polymerization mixture of Epon (37°C, 2–6 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 1–12 or 225 IU day 1–10). 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 34–36 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., 1996Go).

The oocytes were examined 18–20 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)Go: 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.


    Results
 Top
 Abstract
 Introduction
 Case report
 Results
 Discussion
 References
 
The sperm of the husband showed in all three semen samples a variety of the same severe morphological defects indicating a permanent sperm abnormality: different types of sperm heads, most round-headed with varying size of the heads (86%) (Figure 1) beside acephalic forms (12%), both one- or two-tailed and also the former without a tail. Only 2% of the sperm exhibited slightly oval-shaped heads (Figure 1b).



View larger version (98K):
[in this window]
[in a new window]
 
Figure 1. Light micrographs of the stained semen smear. (a) Round-headed sperm with varying head size. The flagella are out of focus (original magnification x2000). (b) A spermatozoon exhibiting a slightly oval-shaped head (arrow) beside two round-headed sperm (original magnification x800).

 
Electron microscopy revealed a lack of the acrosome and a granular dispersion of the karyoplasm (Figure 2). In the mid-piece, the mitochondrial sheath is frequently disorganized.



View larger version (105K):
[in this window]
[in a new window]
 
Figure 2. Transmission electron micrograph of a spermatozoon from the globozoospermic man demonstrating the typical round head with vacuolated regions within the nucleus and absence of the acrosome. In addition, the karyoplasm displays a granular dispersion indicating an irregular chromatin condensation.

 
The couple went through three ICSI cycles with a low number of embryos for transfer and without achieving a pregnancy: 1/3 oocytes cleaved; however, the presence of two pronuclei (PN) was obscure but the cell cleaved and was transferred at the 4-cell stage; 2/18 oocytes (11%) were fertilized normally (2PN), another one showed 1PN; all three oocytes cleaved and were transferred at the 3–4-cell stage; only 1/16 oocytes exhibited 1PN, the presence of pronuclei in another oocyte remained obscure; however, both oocytes were transferred as 2- and 4-cell embryos.

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).



View larger version (97K):
[in this window]
[in a new window]
 
Figure 3. An unfertilized human oocyte prepared after ICSI. The regions numbered 1, 2 and 3 in the overall view (a; original magnification x200) are shown at higher magnification in insets (b–d; original magnification x2000) and represent the haploid set of metaphase II chromosomes (1), polar body chromatin (2) and premature chromosome condensation (PCC) of the sperm nucleus (3).

 


View larger version (62K):
[in this window]
[in a new window]
 
Figure 4. Sperm nuclei with varying degrees of decondensation and signs of premature chromosome condensation obtained after ICSI (a–c; original magnification x2000).

 

    Discussion
 Top
 Abstract
 Introduction
 Case report
 Results
 Discussion
 References
 
This case study has shown the variability that may exist in the sperm of a patient diagnosed as having round-headed sperm morphology. The results of the three ICSI cycles illustrated the extensive inability of such sperm to support oocyte activation after successful sperm injection. Only in the second treatment cycle did we achieve a low fertilization (2PN) rate of 11%. Although three embryos (another one developed from a 1PN oocyte) could be transferred, a pregnancy was not induced. In the case of a 1PN oocyte, the couple has been informed about the risk of a haploid chromosomal constitution of such a developing zygote (Sultan et al., 1995Go).

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., 1997Go; Viville et al., 2000Go) and Martin et al. (2003)Go discovered an elevated frequency of XY disomy in a case of globozoospermia. In contrast to these findings, Rybouchkin et al. (1997)Go 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., 1996Go) but corresponds exactly to the observations of Edirisinghe et al. (1998)Go 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., 1991Go), 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., 1996Go), 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)Go 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)Go 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, 1990Go). 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., 2001Go). 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 sperm–oocyte fusion (Swann et al., 1994Go; Dozortsev et al., 1995Go). In extracts of hamster sperm cells, one of these factors (oscillin, a glucosamine phosphate deaminase) has been identified and characterized (Parrington et al., 1996Go), 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., 1998Go). Recently, a novel sperm-specific phospholipase C, PLC{zeta}, has been identified that triggers Ca2+ oscillations in mouse oocytes indistinguishable from those at fertilization (Saunders et al., 2002Go). Even the PLC{zeta} content of a single sperm cell was sufficient to produce Ca2+ oscillations as well as normal embryo development. Because PLC{zeta} removal from sperm extracts abolished Ca2+ release in oocytes (Saunders et al., 2002Go), 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{zeta} 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{zeta} 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., 1997Go) and pregnancies were observed (Rybouchkin et al., 1997Go; Kim et al., 2001Go). 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., 1998Go) 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{zeta} could be applied at first in cases of 100% globozoospermia, where absence of PLC{zeta} 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{zeta}-activated oocytes, a fetal monitoring according to a risk pregnancy is highly recommended.


    References
 Top
 Abstract
 Introduction
 Case report
 Results
 Discussion
 References
 
Aitken RJ, Kerr L, Bolton V and Hargreave T (1990) Analysis of sperm function in globozoospermia: implications for the mechanism of sperm–zona interaction. Fertil Steril 54, 701–707.[ISI][Medline]

Alberio R, Zakhartchenko V, Motlik J and Wolf E (2001) Mammalian oocyte activation: lessions from the sperm and implications for nuclear transfer. Int J Dev Biol 45, 797–809.[ISI][Medline]

Battaglia DE, Koehler JK, Klein NA and Tucker MJ (1997) Failure of oocyte activation after intracytoplasmic sperm injection using round-headed sperm. Fertil Steril 68, 118–122.[CrossRef][ISI][Medline]

Calafell JM, Badenas J, Egozcue J and Santaló J (1991) Premature chromosome condensation as a sign of oocyte immaturity. Hum Reprod 6, 1017–1021.[Abstract]

Dozortsev D, Rybouchkin A, De Sutter P, Qian C and Dhont M (1995) Human oocyte activation following intracytoplasmic injection: the role of the sperm cell. Hum Reprod 10, 403–407.[Abstract]

Edirisinghe WR, Murch AR, Junk SM and Yovich JL (1998) Cytogenetic analysis of unfertilized oocytes following intracytoplasmic sperm injection using spermatozoa from a globozoospermic man. Hum Reprod 13, 3094–3098.[Abstract]

Holstein AF, Roosen-Runge EC, Schirren C (1988) Illustrated pathology of human spermatogenesis. Grosse, Berlin.

In't Veld PA, Broekmans FJ, de France HF, Pearson PL, Pieters MHEC and van Kooij RJ (1997) Intracytoplasmic sperm injection (ICSI) and chromosomally abnormal spermatozoa. Hum Reprod 12, 752–754.[Abstract]

Khalili MA, Kalantar SM, Vahidi S and Ghafour-Zadeh M (1998) Failure of fertilization following intracytoplasmic injection of round-headed sperm. Ann Saudi Med 18, 408–411.[ISI]

Kilani ZM, Shaban M, Ghunaim SD, Keilani SS and Dakkak AI (1998) Triplet pregnancy and delivery after intracytoplasmic injection of round-headed spermatozoa. Hum Reprod 13, 2177–2179.[Abstract]

Kim ST, Cha YB, Park JM and Gye MC (2001) 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 75, 445–447.[CrossRef][ISI][Medline]

Lanzendorf S, Maloney M, Ackerman S, Acosta A and Hodgen G (1988) Fertilizing potential of acrosome-defective sperm following microsurgical injection into eggs. Gamete Res 19, 329–337.[CrossRef][ISI][Medline]

Liu J, Nagy Z, Joris H, Tournaye H, Devroey P and Van Steirteghem A (1995) Successful fertilization and establishment of pregnancies after intracytoplasmic sperm injection in patients with globozoospermia. Hum Reprod 10, 626–629.[Abstract]

Lundin K, Sjogren A, Nilsson L and Hamberger L (1994) Fertilization and pregnancy after intracytoplasmic microinjection of acrosomeless spermatozoa. Fertil Steril 62, 1266–1267.[ISI][Medline]

Martin RH, Greene C and Rademaker AW (2003) Sperm chromosome aneuploidy analysis in a man with globozoospermia. Fertil Steril 79 (Suppl 3), 1662–1664.[CrossRef][ISI][Medline]

Nardo LG, Sinatra F, Bartoloni G, Zafarana S and Nardo F (2002) Ultrastructural features and ICSI treatment of severe teratozoospermia: report of two human cases of globozoospermia. Eur J Obstet Gynecol Reprod Biol 104, 40–42.[CrossRef][ISI][Medline]

Nurse P (1990) Universal control mechanism regulating onset of M-phase. Nature 344, 503–508.[CrossRef][ISI][Medline]

Palermo G, Joris H, Devroey P and Van Steirteghem AC (1992) Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 340, 17–18.[CrossRef][ISI][Medline]

Parrington J, Swann K, Shevchenko VI, Sesay AK and Lai FA (1996) Calcium oscillations in mammalian eggs triggered by a soluble sperm protein. Nature 379, 364–368.[CrossRef][ISI][Medline]

Rybouchkin A, Dozortsev D, Pelinck MJ, De Sutter P and Dhont M (1996) Analysis of the oocyte activating capacity and chromosomal complement of round-headed human spermatozoa by their injection into mouse oocytes. Hum Reprod 11, 2170–2175.[Abstract]

Rybouchkin AV, Van der Straeten F, Quatacker J, De Sutter P and Dhont M (1997) Fertilization and pregnancy after assisted oocyte activation and intracytoplasmic sperm injection in a case of round-headed sperm associated with deficient oocyte activation capacity. Fertil Steril 68, 1144–1147.[CrossRef][ISI][Medline]

Sakkas D, Urner F, Bianchi PG, Bizzaro D, Wagner I, Jaquenoud N, Manicardi G and Campana A (1996) Sperm chromatin anomalies can influence decondensation after intracytoplasmic sperm injection. Hum Reprod 11, 837–843.[Abstract]

Saunders CM, Larman MG, Parrington J, Cox LJ, Royse J, Blayney LM, Swann K and Lai FA (2002) PLC {zeta}: a sperm-specific trigger of Ca2+ oscillations in eggs and embryo development. Development 129, 3533–3544.[Abstract/Free Full Text]

Schirren CG, Holstein AF and Schirren C (1971) Über die Morphogenese rund-köpfiger Spermatozoen des Menschen. Andrologia 3, 117–125.

Schmiady H, Radke E and Kentenich H (1992) Round-headed spermatozoa—contraindication for IVF. Geburtshilfe Frauenheilkd 52, 301–303.[ISI][Medline]

Schmiady H, Tandler-Schneider A and Kentenich H (1996) Premature chromosome condensation of the sperm nucleus after intracytoplasmic sperm injection. Hum Reprod 11, 2239–2245.[Abstract]

Stone S, O'Mahony F, Khalaf Y, Taylor A and Braude P (2000) A normal livebirth after intracytoplasmic sperm injection for globozoospermia without assisted oocyte activation. Hum Reprod 15, 139–141.[Abstract/Free Full Text]

Sultan KM, Munné S, Palermo GD, Alikani M and Cohen J (1995) Chromosomal status of uni-pronuclear human zygotes following in-vitro fertilization and intracytoplasmic sperm injection. Hum Reprod 10, 132–136.[Abstract]

Swann K, Homa S and Carroll J (1994) An inside job: the results of injecting whole sperm into eggs supports one view of signal transduction at fertilization. Hum Reprod 9, 978–980.[ISI][Medline]

Syms AJ, Johnson AR, Lipshultz LI and Smith RG (1984) Studies on human spermatozoa with round head syndrome. Fertil Steril 42, 431–435.[ISI][Medline]

Tarkowski AK (1966) An air-drying method for chromosome preparations from mouse eggs. Cytogenetics 5, 394–400.[ISI]

Tasdemir I, Tasdemir M, Tavukcuoglu S, Kahraman S and Biberoglu K (1997) Effect of abnormal sperm head morphology on the outcome of intracytoplasmic sperm injection in humans. Hum Reprod 12, 1214–1217.[CrossRef][ISI][Medline]

Trokoudes KM, Danos N, Kalogirou L, Vlachou R, Lysiotis T, Georghiades N, Lerios S and Kyriacou K (1995) Pregnancy with spermatozoa from a globozoospermic man after intracytoplasmic sperm injection treatment. Hum Reprod 10, 880–882.[Abstract]

Viville S, Mollard R, Bach M-L, Falquet C, Gerlinger P and Warter S (2000) Do morphological anomalies reflect chromosomal aneuploidies? Hum Reprod 15, 2563–2566.[Abstract/Free Full Text]

Wolosker H, Kline D, Bian Y, Blackshaw S, Cameron AM, Fralich TJ, Schnaar RL and Snyder SH (1998) Molecularly cloned mammalian glucosamine-6-phosphate deaminase localizes to transporting epithelium and lacks oscillin activity. FASEB J 12, 91–99.[Abstract/Free Full Text]

World Health Organization (1999) Laboratory Manual for the Examination of Human Semen and Semen–Cervical Mucus Interaction, 4th edn. Cambridge University Press, New York.

Zeyneloglu HB, Baltaci V, Duran HE, Erdemli E and Batioglu S (2002) Achievement of pregnancy in globozoospermia with Y chromosome microdeletion after ICSI. Hum Reprod 17, 1833–1836.[Abstract/Free Full Text]

Submitted on December 3, 2004; resubmitted on January 10, 2005; accepted on January 17, 2005.





This Article
Abstract
Full Text (PDF )
All Versions of this Article:
20/5/1319    most recent
deh792v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Request Permissions
Google Scholar
Articles by Schmiady, H.
Articles by Pfüller, B.
PubMed
PubMed Citation
Articles by Schmiady, H.
Articles by Pfüller, B.