Genetic risk of Klinefelter's syndrome in assisted reproductive technology

Abstract Aim The main cause of Klinefelter's syndrome (KS) has been believed to be XY sperm. Accordingly, in the intracytoplasmic sperm injection treatment of patients with KS, hereditary KS has been a concern. Therefore, this study attempted to estimate the risk before and after the assisted reproductive technology. Methods First, in order to validate the safety of the gametes of the patients with KS, a fluorescent in situ hybridization (FISH) analysis, following an original cell identification method using 1052 testicular gametes of 30 patients, was conducted. Second, in the resultant 45 babies, cytogenetic and physical–cognitive screening data were analyzed. In addition, a first attempt was conducted to investigate the origin of the extra X chromosome in 11 patients with KS by using 12 X‐chromosome short tandem repeat (STR) analysis in order to estimate the paternal contribution to KS. Results No sex chromosomally abnormal gamete was found in the FISH analysis and the babies were normal genetically, physically, and cognitively. In the STR, it was confirmed that most (7/11) of the patients with KS resulted from the fertilization of the XX oocytes, suggesting that a baby with KS that had been reported previously might not have resulted from XY sperm. Conclusion These results indicate that the risk of assisted reproductive technology for patients with KS is not as high as previously expected.

abnormality incidence increases in children who are born as a result of ICSI from patients with KS who produce sperm with XY disomy. [24][25][26][27][28][29] However, other reports 12 showed that only normal sperm with haploid X or Y were found in the testis of patients with KS. Accordingly, it is important to collect more precise data in order to determine the risk level of assisted reproductive technology (ART) by using the gamete of a patient with KS and in order to decide on how to deal with the clinical treatment of patients with KS.
Recently, the authors established criteria to distinguish testicular somatic and meiotic cells without fixation and any staining, 30 and accordingly, the precise cytogenetic analysis of these cells became possible. In this study, the criteria for a cytogenetic analysis (chromosome assay and fluorescent in situ hybridization [FISH]) were used so that the sperm and meiotic cells of patients with KS would be selectively isolated, instead of the previous method with a spermatogenic cell mixture. Furthermore, a follow-up review of a comparably large number (n=45) of newborn babies that were born from the ART treatment of patients with KS at the institute from 2000 to 2013 was performed in order to confirm that XY sperm were not selected for the ART treatment. Namely, the risk of hereditary KS was examined before and after the ART treatment. In addition, a first attempt of X-chromosome short tandem repeat (STR) analysis was conducted among patients and their parents in order to estimate XY sperm and XX oocyte contribution to the birth of patients with KS, as there is a possibility that KS resulting from XX oocyte fertilization might be more frequent.

| Patients
This study dealt with 280 men who had been diagnosed previously as having non-mosaic KS and had consented to receive microsurgical testicular sperm extraction (micro-TESE) treatment at the institute from 2000 to 2013.

| Biopsy of the testis tissues
Several different sites of each testis were biopsied under an operation microscope (micro-TESE). The biopsied testicular tissues were prepared in Dulbecco's phosphate-buffered saline (DPBS) containing 0.125% collagenase and 0.01% DNase in order to free the spermatogenic cells from the seminiferous tubules, as previously described. 30 The cells were used for cytogenetic analysis or clinical treatments (ICSI or round spermatid injection [ROSI]) after cryopreservation.

| Freezing and thawing of the sperm or spermatid
For the cryopreservation of the sperm, the testis tissue suspension was centrifuged and the pellets were transferred into a droplet of human tubal fluid (HTF) on a Petri dish with a glass pipette under a diverted microscope. Then, the cell suspension was mixed with a very small amount of a freezing medium (~2 μL of HTF with 10% serum protein substitute [SPS; Origio, Malov, Denmark] and 100 mmol/L sucrose) and placed on the tip of Cryotop (Kitazato Corp., Fuji, Japan) under an inverted microscope. The Cryotop was exposed to liquid nitrogen vapor for 2 minutes and stored in liquid nitrogen. For the thawing of the frozen cells, after maintaining the Cryotop in air for 5 seconds, it was dipped into a droplet that was covered with warm mineral oil (37°C) to suspend the cells. 31 The motile sperm were selected and used for ICSI.
For ROSI, the spermatids were selected from the testicular cell suspension under an inverted microscope and suspended in 0.15 mL of freezing medium (DPBS with 0.6 mol/L ethylene glycol, 0.125 mol/L sucrose, and antibiotics). The suspension was drawn in a 0.25 mL Cassou straw and cooled on ice (4°C). After the straw was maintained at −7°C in a cooling chamber of a programmable alcohol bath freezer for 20 minutes, it was cooled to −30°C at the rate of −0.3°C/min before being plunged into liquid nitrogen. Thawing was carried out by maintaining the straw in air for 5 seconds. The cell suspension then was diluted with HTF containing 10% SPS in a test tube to remove the cryoprotectant. 30

| Cytological identification of the testicular cells
The authors already have established the criteria for identifying biopsied spermatogenic cells morphologically. The characteristics of the testicular cells were examined in detail under a differential interference microscope (10×40) and then their chromosomal constitution was determined by cytogenetic analysis to confirm whether the characteristics used and the meiotic phases correlated with each other. The mixture of the probes was applied to the slide under a cover slip and the nuclear and probe DNA were denatured simultaneously for 5 minutes at 75°C. The slide then was incubated in a chamber (Hybrite; Vysis) at 42°C for 120 minutes in order to allow hybridization, followed by counterstaining with 4,6-diamidino-2-pheniylindole (Vector Laboratories, Inc., Burlingame, CA, USA). 32

| Chromosome assay of the spermatogenic cells
For the chromosome analysis of the spermatogenic cells that were identified with the authors' morphological criteria, a chromosome assay with interspecific injection into mouse MII oocytes was used. 33 The meiotic cells were injected into the mouse oocytes 10 min after electrical stimulation (8 V/cm AC, 1000 KHz for 8 seconds, and 1200V/cm DC for 99 μseconds). Sperm or elongating sperm were injected without electric stimulation. After overnight incubation in the HTF containing 50 ng/mL vinblastine, the nucleus of the meiotic cells or injected sperm was allowed to form chromosomes in the mouse oocytes. Chromosome slides of the oocytes were prepared by the gradual fixation-air drying method. 34

| Microinjection of the testicular sperm or spermatid
Intracytoplasmic sperm injection was conducted with motile and morphologically normal sperm. Oocyte penetration by the spermatid was conducted with a comparably larger injection pipette than that used for conventional ICSI. In the cases of the STs and STs with a small flagellum, oocyte activation with electrical stimulation was required before injection, 30 but no oocyte activation was required for the elongating-elongated spermatids. After 5 days of incubation, the blastocysts were transferred into the uterus.

| X-chromosome short tandem repeat analysis
Using blood or oral mucous samples of 11 patients with KS and one or both of their parents with their consent, the origin of the extra X chromosome was determined with X-chromosome haplotype markers (STRs of 12 loci), according to the method of one particular study. 35  Electrophoresis was run on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) for the PCR products. The obtained data were analyzed with GeneMapper ID software (Thermo Fisher Scientific Inc., Waltham, MA, USA). All the steps described above were entrusted to Tohoku Chemical Company, Ltd., Tohoku, Japan.

| Screening of babies and children
Out of the 45 babies who were born by ICSI or spermatid injection treatment from the patients with KS in the institute, 29 underwent chromosomal analysis by using amniocentesis or peripheral blood samples before or after birth, respectively, along with the compulsory newborn or infant screening for physical and cognitive development in Japan. For the rest, only information in the screening was used to examine the possibility of KS.

| Ethical considerations
The clinical application of ROSI and genetic analysis with X-

| Morphological characteristics of the spermatogenic cells
In Figure 1A, typical images of the spermatogenic cells are shown. The elongating and elongated spermatids were easily identified, with deviated condensed nuclei and short flagella, respectively. It was comparably difficult to distinguish among the SG, early Pr-Scs, and STs. The STs were the smallest spermatogenic cells (6-8 μm in diameter; slightly smaller than erythrocytes). They were much smaller than the Pr-Scs (10-12 μm) and slightly smaller than the SG (8-10 μm). Two-to-three nucleoli were seen within the nuclei of the SG and Pr-Scs, but not in the STs. An area of the cytoplasm surrounding the nucleus was narrower in the STs than in the SG. Protruded active pseudopodia often were seen in the SG, 36 but not in the STs. Although an acrosomal vesicle or cap was considered to be strong evidence of the cell being a ST, such structures were found in <10% of the presumptive spermatids.

| Chromosome abnormality in the testicular cells of the patients with Klinefelter syndrome
In Figures 1B and C, chromosomal images of normal spermatogenic cells that were visualized by chromosomal assay and FISH analysis, respectively, are shown. When the chromosomes of the spermatogenic cells were induced to condense in the mouse oocytes, the SG had 46 of the dyad chromosomes, which are seen at the metaphase of somatic cell proliferation. The Pr-Scs had 23 of the tetrad chromosomes, in some of which the cross-overs were observed (see the arrow in Figure 1B). The STs had 23 monad chromosomes. Therefore, the chromosome assay showed that the authors' criteria for spermatogenic cell morphology allowed the cells to be identified correctly, and accordingly, the authors could apply the cells that were selected with the criteria for a FISH analysis of their interphase nuclei. In the SG, two blue spots of chromosome 18 and a green and orange spot of X and Y were found. In the Pr-Scs, each one spot of chromosome 18, X, and Y were visible, three spots in total. In the STs, a blue spot of chromosome 18 and either a green (X) or orange (Y) spot were visible, a total of two spots ( Figure 1C).

| Success rate of intracytoplasmic sperm injection and round spermatid injection
The success rates of the ICSI and ROSI treatment of patients with KS are shown in Table 2. Out of 280 patients, sperm with faint motility were recovered in 92 (32.9%) patients for ICSI. Spermatids, which are evidence of the completion of meiosis, were found in 33 (11.8%) patients for ROSI. In 155 (55.4%) patients, no spermatogenic cell was found. The incidence of pregnancy per treatment cycle, miscarriage,

| Physical and cognitive development of the babies of patients with Klinefelter syndrome
In the 29 babies who were cytogenetically analyzed, it was confirmed that they had a normal karyotype. The results of the newborn or infant screening for physical and cognitive development also showed that, in all 45 babies, no abnormality was found.

| Origin of the extra X chromosome in patients with Klinefelter syndrome
Examples of X-chromosomal STR DNA profiles are shown in Table 3.
Patient no. 09KY is a case in which both X chromosomes were inher-

| Genetic risk of the intracytoplasmic sperm injection treatment for patients with Klinefelter syndrome
The present study showed that 45 babies were successfully delivered by using oocyte penetration by sperm or spermatid from patients with KS from January 2000 to December 2013 at the institute and, among them, there was neither a case of chromosomal abnormality nor any case of physical or cognitive abnormality. The miscarriage rate (37.3%) in the treatment of patients with KS by using sperm and spermatid was not significantly higher, when compared with patients who do not have KS (20.1% of 134). 30 The results indicate the possibility that the genetic risk of the embryos that are produced in the treatment of patients with KS is not as high as previously believed.
This clinical result is consistent with the cytogenetic data of the FISH and chromosomal analysis in the gametes from patients with KS.
In the 25 patients with KS who were examined, no sex chromosome abnormality was found in 952 ST cells and 100 sperm ( Table 2). The mechanism to produce normal gametes in the testis of patients with KS is considered to be as follows. In patient no. 2, all the SG and Pr-SCs that were analyzed were XY in their sex chromosome constitution.
Therefore, there is no doubt that STs with X or Y chromosomes could be derived from the meiosis of sex chromosomally normal germ cells.
In the remaining four patients with KS with testicular mosaicism of XY  12 There is another possibility that the resultant abnormal daughter cells of XXY SG can become degenerative or apoptotic, 37 because in this study, only the spermatogenic cells that were alive with the intact plasma membrane and smooth round shape were selectively examined. This possibility seems to be a reason for an inconsistency of the present data with those of previous cytogenetic studies in patients with KS. Many previous FISH studies have reported that not only the sex chromosome abnormality rate, but also the rate of autosomal aneuploidies, 24 is higher in sperm from patients with KS than from infertile patients without KS. [24][25][26][27][28][29] In those studies, the testicular cell suspension was directly smeared onto a glass slide, treated with dithiothreitol, and hybridized with FISH probes. As after the successive treatment, the artificially swollen sperm heads were not allowed to be evaluated for their morphology, a tail was used to identify the sperm. KS are included in their data, the result seems to disagree with the present data. However, their data include some points that are hard to understand. First, the total aneuploidy rates did not differ among the embryos from IVF and ICSI with normal and suboptimal sperm groups.
Second, in the embryos of the suboptimal sperm group, aneuploidy increased in specific autosomes in addition to the sex chromosomes.
These incompatible phenomena seem to be explained by the possibility that patients with genetic backgrounds causing aneuploidy of a specific chromosome(s) are contained in the oligozoospermia group.
Therefore, their result might not necessarily be applicable to patients with KS, although close attention has to be paid to the genetic risk of ICSI treatment of patients with KS.

| Contribution of the XX oocyte in the production of Klinefelter syndrome
When the current study found that no XY aneuploidy could be observed in the gametes of patients with KS in the authors' cytogenetic analysis, it was hypothesized that the XY sperm did not contribute to the production of KS as much as the XX oocytes. In this X-chromosome STR analysis, the patients with maternal origin X chromosomes were comparably frequent (63.6%), suggesting that the contribution of the XX oocyte to the production of XXY embryos might be greater than by the XY sperm, although the sample number that was applied for the X-chromosomal STR DNA profiling is not large enough. Some studies have previously attempted to determine the origin of the extra X chromosome in patients with KS with X-chromosome restriction site polymorphism. [42][43][44] The maternal contribution to the production of KS was slightly greater in two studies (59% vs 41%) and was slightly lower in one study (42.8% vs 57.1%). In those studies, however, there were cases in which the X-chromosome origin was determined by the appearance or disappearance of a single band in a single allele, which might have resulted from mutation. In the X-chromosome STR analysis, the 12 X-chromosomal markers are clustered into four linkage groups, which consist of three alleles, and thus each set of three markers is handled as a haplotype for genotyping to avoid misjudgment.
The authors could find no previous study that applied X-chromosome STR with PCR to patients with KS. One study reported that the extra X chromosome is the result of meiotic non-disjunction 45 or possibly, as recently described, of the premature separation of sister chromatids, both paternally or maternally, because of an increased maternal age. 1,46 As the X-chromosome origin can affect the potency of spermatogenesis in patients with KS, the authors will collect further data by using this method.