RBMOnline - Vol No 346–352 Reproductive BioMedicine Online; www.rbmonline.com/Article/1038 on web September 2003 Article Results of preimplantation genetic diagnosis in patients with Klinefelter’s syndrome Dr Semra Kahraman Semra Kahraman graduated from Hacettepe University Medical School in Turkey and obtained her speciality degree in Obstetrics and Gynecology from Ankara University Medical School After training in the Haugesund Hospital ART Unit in Norway, she founded Sevgi Hospital ART Centre in Turkey in 1993 Currently she is the director of Istanbul Memorial Hospital Reproductive Endocrinology, ART and Genetics Centre, where more than 200 preimplantation genetic diagnosis (PGD) cycles are performed each year She has applied intracytoplasmic sperm injection (ICSI), surgical sperm retrieval techniques, embryo freezing and PGD for aneuploidy testing and single gene disorders for the first time in Turkey and obtained the first pregnancies with these procedures She is one of the founding members of the Preimplantation Genetic Diagnosis International Society and is a member of the Turkish Ministry of Health High Council on Assisted Reproductive Technology She has more than 250 publications S Kahraman1, N Findikli, H Berkil, E Bakircioglu, E Donmez, S Sertyel, A Biricik ART, Reproductive Endocrinology and Reproductive Genetics Unit, Istanbul Memorial Hospital, Piyalepasa Bulvari, 80270, Okmeydani, Istanbul, Turkey 1Correspondence: e-mail: skahraman@superonline.com Abstract With the application of preimplantation genetic diagnosis (PGD), a possible genetic contribution of spermatozoa obtained from 47,XXY non-mosaic Klinefelter patients on preimplantation embryos was analysed in eight couples Interpretable fluorescence in-situ hybridization results were obtained for 28 out of 33 embryos biopsied (84.8%) and 23 blastomeres were analysed for chromosomes 13, 18, 21, X and Y Nine out of 23 embryos were diagnosed as abnormal (39.1%) Five out of nine contained sex chromosome abnormalities (55.5%) Two were diagnosed as 47,XXY and three were found to have monosomy X Besides sex chromosomal abnormalities, other abnormalities detected were haploidy, triploidy, monosomy 13, monosomy 18 and trisomy 13 Five blastomeres were analysed for sex chromosomes only and all of them were found to be normal Overall, the rate of sex chromosome abnormality in biopsied embryos was found to be 17.8% (5/28) Moreover, among 33 embryos biopsied, five of the eight zygotes, which were classified as a poor prognosis group according to pronuclear morphology scoring, showed an impaired growth profile after biopsy and were found to be chromosomally abnormal Elimination of abnormal embyos and transfer of normal ones resulted in four pregnancies in eight cycles (50%) Two pregnancies, one singleton and one twins resulted in healthy births Two pregnancies, one singleton and one twins are continuing beyond the second trimester These results show that there is in fact elevated chromosomal abnormality for both sex chromosomes and autosomes in embryos developed from Klinefelter males Furthermore together with PGD, embryo scoring according to pronuclear morphology can give additional benefit for selecting chromosomally abnormal embryos Therefore, PGD should be recommended in cases with Klinefelter’s syndrome and this information should be discussed with the couple when genetic counselling is given Keywords: chromosomal abnormalities, Klinefelter’s syndrome, preimplantation genetic diagnosis, testicular sperm extraction Introduction 346 Klinefelter’s syndrome is the most frequent chromosomal disorder occurring with an incidence of 1/500 live male births (Rao and Rao, 1977) Approximately 90% of males with this syndrome show a 47,XXY karyotype, whereas in about 10% a 46,XY/47,XXY mosaic pattern is associated with a less severe phenotype Males having a pure 47,XXY chromosomal pattern are usually azoospermic but varying concentrations of spermatozoa have been reported for males with mosaic karyotypes (Laron et al., 1982; Harari et al., 1995) Application of testicular sperm extraction methods combined with an intracytoplasmic sperm injection (ICSI) technique are now being efficiently used for azoospermic Klinefelter patients However, some authors have expressed concern about the risk of genetic pathology transmission to the newborn Sperm fluorescence in-situ hybridization (FISH) analyses performed on cells of both non-mosaic and mosaic Klinefelter patients have revealed an increased percentage of 24,XY sperm cells compared with the normal population (Cozzi et al., 1994; Chevlet et al., 1996; Martini et al., 1996; Guttenbach et al., 1997; Hinney et al., 1997; Estop et al., 1998; Kruse et al., Articles - Preimplantation genetic diagnosis in Klinefelter s syndrome - S Kahraman et al 1998) Interestingly, this increase is not consistent with the abnormalities reported in neonates born to Klinefelter patients, 21 births have been reported so far and in only one case the fetus was detected as 47,XXY (Ron-El et al., 2000; Greco et al., 2001) Preimplantation genetic diagnosis (PGD) of aneuploidy (Kuliev and Verlinsky, 2002; Munné, 2002; Munné et al., 2003) is a well established technique used as an alternative to prenatal diagnosis and to improve the success rate of IVF (Verlinsky et al., 1998; Gianaroli et al., 1999; Munné et al., 1999; Kahraman et al., 2000) Several studies have reported the application of PGD in embryos developed from Klinefelter’s patients (Staessen et al., 1996; Reubinoff et al., 1998; Bielanska et al., 2000) However, they are very limited in the number of embryos analysed and the possible effect of Klinefelter’s on embryo development is not currently clear Documenting the possible genetic contribution of spermatozoa in the embryos can help increase understanding of the possible elimination mechanism(s) of aneuploidy and at which stage they come into effect This article, aims to analyse the outcome of PGD in eight cycles in which the male partner was diagnosed as non-mosaic Klinefelter’s syndrome Thirty-three embryos were biopsied and analysed by FISH procedure in order to determine their genetic constitution for a given set of chromosomes Results and possible outcomes for the treatment of Klinefelter patients are discussed Materials and methods Patients Eight couples in which male partners were diagnosed as having non-obstructive azoospermia with 47,XXY karyotypes were received at the clinic for infertility treatment All eight males were non-mosaic (47,XXY) Klinefelter’s syndrome patients They underwent genital examination and rectal ultrasonographic evaluation and their testicular volume was determined by Prader orchidometer Hormonal analysis for serum concentrations of FSH, LH and total testosterone was also carried out Cytogenetics For karyotype analysis, ml peripheral blood was sampled from both partners in ml lithium heparinized tubes A blood sample of 0.5 ml was added to ml Ham’s F-10 medium (Biochrom, Gründau, Germany) which contained 0.8 ml fetal bovine serum albumin (Sigma, MO, USA), 0.04 ml Lglutamine, 0.04 ml penicillin–streptomycin and 0.1 ml phytohaemagglutinin was added During 72 h incubation at 37°C, 50 μl ethidium bromide was added at the 68th hour and 100 μl colcemide (0.05 mg/ml) was added h later to harvest the cells at early metaphase, and the cells were prepared for GTG banding (Giemsa Trypsin G-banding) Twenty metaphases were analysed for each patient Testicular sperm extraction Microdissection testicular sperm extraction (TESE) procedures were done under general anaesthesia After the testis was exposed and examined at ×9 magnification under an operating microscope, a large horizontal incision was made on the tunica albuginea Direct examination of the testicular parenchyma was carried out at ×20–22 magnification to search for and identify seminiferous tubules that were larger and more opaque (whitish) than the others Biopsy pieces were minced in a Petri dish with pincers and 26-gauge needle to allow spermatozoa to be released from inside the tubules The suspension was evaluated in the operating room under a phase contrast microscope at ×200 power for the presence of spermatozoa If no spermatozoa were found in the examined sample, additional microscopic samples were obtained from different areas in the testicular parenchyma If still no spermatozoa were detected then the contralateral testis was opened and the same procedures were performed Ovulation induction, oocyte recovery and embryo culture Ovulation induction and oocyte retrieval were performed as previously described (Kahraman et al., 2000) Immediately after ICSI, injected oocytes were cultured with G1.2 media (Vitrolife, Gothenburg, Sweden) until the morning of day Fertilization was assessed 16–18 h after injection Normal fertilization was determined when two clearly distinct pronuclei containing precursor nucleolar bodies (PNB) were present under an inverted microscope Pronuclear (PN) morphology scoring was carried out as previously described (Kahraman et al., 2002) Briefly, category A was defined as having a clear halo, well-defined PN borders and clear nucleolus localized on the same plane close to the intersection area A clear halo was observed in all categories from B to F; however, the distribution and size of PNB differed Category C pre-embryos had a clear halo, abutting PN, and large nucleoli scattered irregularly throughout the pronucleus Category D pre-embryos displayed a clear halo, abutting PN and nucleoli scattered irregularly Category E presented a clear halo, abutting PN and PNB irregular in size in the same hemisphere (big and small) or only large PNB in one PN and small in the other Category F exhibited a clear halo, abutting PN, and PNB irregular in size and distribution In category G, an ill-defined, blurred halo was the most distinctive characteristic, and the PN were either close or distinctly separate; the PNB were either unclear or not visible The PN could be either equal in size or significantly different In category J, the most significant features are early syngamy and clear halo, and small nucleoli irregular in distribution According to embryo development characteristics, these categories were further grouped as Group I (categories A to D) and Group II (categories E to J) After the assessment, the zygotes were transferred to fresh droplets of the corresponding medium The state of embryo cleavage and quality were assessed after a further 24, 48, 72 and 96 h of in-vitro culture The embryos were evaluated at ×600 magnification on an inverted microscope by at least two experienced embryologists according to the number of blastomeres present, blastomere size equality and the relative proportion of anucleate fragments On day 3, embryos were taken into G2.2 medium (Vitrolife) and kept in this medium until the day of embryo transfer Every 24 h, the culture media was changed 347 Articles - Preimplantation genetic diagnosis in Klinefelter s syndrome - S Kahraman et al Embryo biopsy Results On day of embryo development, embryos with seven or more blastomeres with good morphology (grade I or II) were selected for embryo biopsy Zona opening was carried out using a 1.48 μm diode laser (Fertilase®, MTM Medical Technologies, Montreux SA, Switzerland) Three shots of ms were sent for zona drilling and a 25–30 μm opening was made to allow placement of the blastomere biopsy pipette (Cook IVF, Queensland, Australia) Only blastomeres with a clear nucleus were biopsied After the biopsy, embryos were cultured at least overnight to observe their development and to allow a sufficient period for the FISH procedure Patient characteristics are summarized in Table All patients were identified as non-mosaic Klinefelter according to peripheral blood karyotypes The median age and serum FSH values of the males were 29.9 ± 2.4 years (range 26–33) and 30.9 ± 13.3 mIU/ml (range 24.6–60.0) respectively Testicular spermatozoa were retrieved with microdissection TESE procedures and in seven out of eight patients, spermatozoa were retrieved successfully and immediatelly used for ICSI In one cycle, no spermatozoa were obtained and round spermatid injection (ROSI) was applied according to the couple’s decision In this case a total of 18 MII oocytes were retrieved Seven of them were fertilized with two pronuclei (PN) after ROSI However only two zygotes presented C and D-scored PNB with two distinct pronuclei The remaining five zygotes showed J score with overlapping pronuclei indicating an early syngamy Three embryos with seven and eight blastomeres with clearly visible nuclei were biopsied for PGD One of them had apoptotic nuclei, one had normal chromosomal constitution for chromosomes 13, 18, 21, X and Y and one embryo had monosomy X and monosomy 18 Only one embryo was transferred but it resulted in no pregnancy Blastomere preparation and FISH procedure Biopsied embryos were first transfered onto a clean slide with a minimum amount of media and the position was marked After a brief wash with hypotonic medium, the cytoplasmic parts were removed with Carnoy’s solution (acetic acid/methanol, 3:1) The digestion of the cytoplasm continued until appearance of the nucleus The slides were then dried at room temperature for one hour After the initial denaturation for at 73˚C the slides were hybridized with μl of probe mix at 37˚C FISH analyses were performed using DNA probes specific for chromosomes 13, 18, 21, X and Y The assisted reproduction treatment outcomes of eight cycles are shown in Table The mean female age was 25.9 ± 4.1 years (range 20–32) Apart from one case, all females were normal responders On average, 12.5 ± 5.6 MII oocytes were collected per patient in eight cycles with an overall fertilization rate of 56.0% After selection of normal embryos with PGD, transfer of normal embryos to corresponding females resulted in three pregnancies (one twins and two singletons) Embryo transfer and pregnancy test Embryo transfer was performed on the fourth (n = 6) or fifth (n = 2) day of embryo culture according to the developmental potential of the transferrable embryos and the results of the FISH procedure Pregnancy was first evaluated by serum HCG 12 days after embryo transfer Clinical pregnancy was assessed by the presence of a gestational sac, and heart beats by echographic screening at approximately weeks after the embryo replacement procedure Blastomere biopsy was performed on 33 embryos and for 28 of them interpretable FISH results were obtained, 23 blastomeres were analysed for chromosomes 13, 18, 21 X and Y and nine embryos were detected as chromosomally abnormal Types of chromosomal abnormalities were detected Table Characteristics of non-mosaic Klinefelter’s patients 348 Patient Age (years) Karyotype Testis volume (ml) Serum FSH (IU/l) LH (mIU/ml) Serum testosterone (pg/ml) Sperm retrieval procedure Spermatozoa present 30 47,XXY 3.0 24.6 13.0 2.2 Yes 31 47,XXY 2.0 60.0 11.7 1.9 26 47,XXY 3.0 27.7 18.0 2.3 32 47,XXY 3.0 20.7 14.7 3.6 30 47,XXY 0.5 27.6 19.7 0.4 33 47,XXY 2.0 20.9 22.3 2.4 30 47,XXY 3.0 25.3 15.7 2.1 27 47,XXY 3.0 40.3 14.3 1.3 Microdissection TESE Microdissection TESE Microdissection TESE Microdissection TESE Microdissection TESE Microdissection TESE Microdissection TESE Microdissection TESE Yes Yes Yes Round spermatid Yes Yes Yes Articles - Preimplantation genetic diagnosis in Klinefelter s syndrome - S Kahraman et al Table Cycle outcomes of couples undergoing PGD for non-mosaic Klinefelter’s syndrome Patient number Female age (years) Number of MII oocytes Number of embryos fertilized Number of embryos biopsied Day of embryo transfer Pregnancy results 24 28 20 31 24 32 25 23 13 18 17 13 18 12 11 11 7 5 4 4 5 No No Yes (singleton) Yes (singleton) No No No Yes (twins) Table Further embryo development of abnormal embryos Embryo number PNMSa score Day of development Number Grade of cells Day of development Stage/number of cells FISH result Grade II II 1 Arrested Compacted I >8 I Compacted II I I II 8 1 Arrested 8 II Haploid Triploid (69,XXY) Klinefelter (47,XXY) Klinefelter (47,XXY) Monosomy 13 Monosomy X Monosomy X Monosomy X, monosomy 18 Trisomy 13 aPNMS = pronuclear morphology scoring a b and their corresponding embryo development is shown in Table Among nine embryos detected as abnormal, two were diagnosed as having Klinefelter chromosomal constitution (47,XXY; Figure 1a), two were detected as having only one X chromosome (monosomy X; Figure 1b) and one embryo was detected as complex aneuploid, showing monosomy X together with monosomy 18 in a case with ROSI Other abnormalities detected were triploidy (n = 1), haploidy (n = 1), Figure Sex chromosome abnormalities detected in embryos of one non-mosaic Klinefelter patient a, nucleus of a blastomere showing Klinefelter chromosomal constitution 47,XXY (Note the two blue signals representing the X chromosome and a yellow signal for the Y chromosome.) b, blastomere obtained from an embryo detected as monosomy X trisomy 13 (n = 1) and monosomy 13 (n = 1) Five embryos of one patient were screened for only sex chromosomes (due to a possible transmission of X-linked disease) and all of them were found to be normal for these chromosomes Overall, for 28 embryos, FISH results gave an abnormality rate of 17.8% (5/28) for sex chromosomes Among the embryos biopsied, eight of the 33 embryos were 349 Articles - Preimplantation genetic diagnosis in Klinefelter s syndrome - S Kahraman et al found to be have a group II pronuclear morphology with blurred cytoplasmic halo and irregularly scattered unclear PNBs which has been defined as a poor prognosis group in a previous study (Kahraman et al., 2002) Five of these embryos efficiently reached the 7–8 cell stage and were biopsied on day However, they showed retarded or arrested growth afterwards and were found to be abnormal after FISH analysis Discussion Despite numerous sperm FISH studies reported previously (Bernardini et al., 1997, 2000; Martin et al., 2000; Calogero et al., 2001, 2003; Vicari et al., 2003), genetic screening of sperm cells before ICSI does not always give a definitive answer concerning the extent to which abnormal spermatozoa may contribute to the chromosomal structure of a fertilized zygote There is controversy about the incidence of aneuploidy in sperm cells in fertile and infertile males as well as in ejaculated and testicular spermatozoa No difference has been reported in the incidence of sex chromosome aneuploidy when comparing sperm cells from infertile with fertile males (Miharu et al., 1994; Guttenbach et al., 1997) However, recent studies have shown that compared with the fertile group, a relatively higher rate of sex chromosome abnormalities exists among infertile couples (Aran et al., 1999; Pang et al., 1999; Pfeffer et al., 1999) Similarly, for the source of spermatozoa used in ICSI, one of the groups reported a slightly high post-meiotic aneuploidy rate which was not statistically significant (Martin et al., 2000) However, Huang et al (1999) reported a high percentage of aneuploidy In a more recent study, an increased incidence of chromosomal anomalies has been found in the testicular spermatozoa of men with non-obstructive azoospermia, in which it was implied that increased sex chromosome aneuploidy is due to meiotic errors (Levron et al., 2001) For non-mosaic patients, however, the rate of chromosomal abnormality in the testicular spermatozoa used for ICSI is not well established and cytogenetic and FISH results for the testicular spermatozoa analysed by several groups are conflicting However a recently published study (Morel et al., 2003) has indicated that there is an increased incidence of autosomal aneuploidies for chromosomes 13, 18 and 21 in the spermatozoa from patients with Klinefelter’s syndrome compared to the general population Thus, the offspring of patients with Klinefelter’s syndrome born by ICSI may be at higher risk of autosomal aneuploidy Another recently published study analysed the meiotic segregation not only of the sex chromosomes but also of autosomal chromosomes and 21 in the spermatozoa of a non-mosaic Klinefelter patient (Hennebicq et al., 2001) They found an increased frequency of 24,XY and 24,XX spermatozoa in the patient compared with controls 350 It has been suggested that some 47,XXY germ cells are able to go through meiosis and produce spermatozoa with hyperhaploidy (Levron et al., 2000) On the other hand, other reports indicate that in 47,XXY males, the abnormal cells are unable to enter meiosis Although a variable number of premeiotic cells have an exclusively XXY chromosomal constitution, all pachytenes analysed were exclusively XY (Blanco et al., 2001) Theoretically, the application of ICSI with sperm cells containing abnormal sex chromosome constitution would produce embryos that may fail to be implanted or produce a progeny with sex chromosome abnormalities In a recent study performed by Macas et al (2001) male-derived genomes of ICSI and IVF-3 PN zygotes were analysed for disomy of chromosomes X, Y and 18 and a higher incidence of numerical chromosomal abnormalities was observed in the ICSI group This may imply that the selection of chromosomally abnormal spermatozoa may in fact increase chromosomal abnormalities in the fertilized zygote However, studies reporting chromosomal analysis of preimplantation embryos of Klinefelter patients are too small in number to draw any conclusion about a possible risk of chromosomal abnormalities in the preimplantation stage First, Staessen et al (1996) reported three cases in which a total of five embryos were biopsied for PGD, four embryos were found to be normal for the chromosomes analysed and transferred to three patients, resulting in one biochemical pregnancy They also analysed the cells of the remaining embryo and concluded that it was also normal for sex chromosomes In another study, Reubinoff et al (1998) reported an application of PGD on three embryos in two couples with Klinefelter’s syndrome Transfer of one normal embryo for chromosomes 18, X and Y resulted in one karyotypically normal offspring However, in the other two embryos chaotic sex choromosomal distribution was observed More recently Bielanska et al (2000) reported the chromosome analysis of 10 spare embryos obtained from a couple in which the male partner was diagnosed as mosaic Klinefelter (46,XY/47,XXY) Seven out of 10 embryos were found to be abnormal for the chromosomes analysed (18, X and Y); two being mosaic with most cells detected as normal and seven were chaotic mosaic Interestingly none of the embryos analysed showed a uniform XXX1818 or XXY1818 chromosomal pattern In the present study, PGD was performed on 33 blastomeres in eight couples Although the number of analysed embryos was limited, a rate of 39.1% (9/23) abnormality was detected for chromosomes 13, 18, 21, X and Y in 23 embryos Strikingly, five of the abnormalities detected in fact contained sex chromosome abnormalities, producing an overall sex chromosome abnormality rate of 17.8% (5/28) among the blastomeres analysed In one case with ROSI, out of three embryos analysed, one presented a complex aneuploidy, one had an apoptotic nucleus and one showed normal chromosomal constitution for chromosomes 13, 18, 21, X and Y No pregnancy was established in this case after transfer of the latter embryo Although female factors can not be eliminated, this point deserves additional attention since it is likely that the problem of meiotic segregation in spermatozoa of Klinefelter patients may persist during preimplantation development according to these results Other blastomeres of abnormal embryos were re-analysed for the rate of mosaicism and only one embryo initially diagnosed as monosomy X was rediagnosed as monosomy X and monosomy 13, thus now showing a mosaic pattern The remaining abnormal embryos that were re-analysed confirmed the same chromosomal abnormality as in the initial diagnosis Therefore the rate of mosaicism was found to be 10.1% A recent study in which PGD was performed on embryos from testicular spermatozoa of non-obstructive azoospermia cases Articles - Preimplantation genetic diagnosis in Klinefelter s syndrome - S Kahraman et al reported a higher percentage of mosaicism compared with embryos of severe oligoasthenoteratozoospermia patients The authors argued that this result may be due to suboptimal sperm centriole function (Silber et al., 2003) It would be very interesting to analyse whether presence of an additional chromosome or a malfunctioning centriole gives similar results or additive effects with respect to embryo development and chromosomal mosaicism since all Klinefelter patients in this study were azoospermic In this study, unfortunately none of the abnormal embryos were further processed for detection of mosaicism therefore the rate of mosaicism cannot be determined Compared with sperm FISH results of mosaic Klinefelter cases, a lower rate of chromosomal abnormalities was detected in newborns possibly due to two mechanisms: firstly, abnormal spermatozoa may be selected and eliminated (most but not all) through apoptosis and/or some other unknown mechanism(s) As Blanco et al (2001) suggested, abnormal cells resulting from non-disjunction can be arrested at meiosis II and this would explain the lower spermatozoa with sex chromosomal abnormalities found with respect to the figures observed in post-reductional germ cells The second possibility would be that fertilization can in fact occur but embryos with chromosomal abnormalities are eliminated (most but not all) at the pre- or post-implantation stage According to current literature, both mechanisms are in fact likely to occur, however embryos having both types of sex chromosome abnormalities detected in this study (47,XXY and 45,XO) can implant in the uterus and are compatible with life In this study, a high rate of abnormalities of chromosomes other than the sex chromosomes was also detected Furthermore most of the abnormalities were monosomic for a given chromosome Therefore according to these results, PGD and genetic counselling should be considered for couples where the male partner has been diagnosed as having Klinefelter’s syndrome and patients should also be informed about the possible risk of autosomal and gonosomal chromosome abnormalities for developing embryos This study, together with others, in which PGD was performed on embryos developed from spermatozoa obtained in non-mosaic Klinefelter cases, will increase knowledge of the possible risk of chromosomal abnormality in embryos of those patients Further and more detailed studies are needed to clarify the exact role of spermatozoa from Klinefelter’s patients in an embryo’s overall chromosomal abnormality References Aran B, Blanco J, Vidal F et al 1999 Screening for abnormalities of chromosomes X, Y, and 18 and for diploidy in 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