Preimplantation Genetic Diagnosis (PGT) Results after ICSI in Patients with Klinefelter Syndrome: Should PGT be Performed?

Klinefelter syndrome (KS) is a disorder that causes infertility in men, is characterized by hypergonadotropic hypogonadism, azoospermia, and elevated FSH (Klinefelter et al., 1942) and is caused by a sex chromosome anomaly (Trisomy XXY) (Jacobs et al., 1959). 

Its incidence in infertile men (3-4%) and azoospermic cases (8-12%) is quite high compared to the normal population (0.1-0.2%) (Forti et al., 2010). While approximately 80% of KS cases have the classical type, that is, 47,XXY chromosome formation, the remaining approximately 20% have 48,XXYY, 48,XXXY, or 47,XXY/46,XY (mosaic) type chromosome anomalies. It is thought that the failure of chromosomes to separate (meiotic nondisjunction) during meiotic divisions during the gametogenesis stage that creates germ cells or mitotic division defects that occur in the early embryonic period (post-zygotic nondisjunction) cause this anomaly. It was determined that the extra X chromosome was inherited from the mother in 7 (63.6%) of the 11 KS cases in which DNA profiling was performed, and in 4 (36.4%) cases it was inherited from the father. These findings strengthened the possibility that most cases of KS may be caused by a division defect during oocyte formation (24,XX oocyte) rather than an abnormal sperm (24,XY sperm), as is believed (Miki et al., 2017). 

Although it is not exactly known how the excess on the X chromosome leads to azoospermia, it is suggested that it causes Leydig cell insufficiency and germ cell degeneration due to the disruption of apoptosis regulation in Sertoli cells (Aksglaede et al., 2006)

Although the presence of sperm in the ejaculate is observed in some cases, in most cases the only way to obtain sperm is through surgical procedures (TESE or micro-TESE). According to a meta-analysis based on data from more than 20 thousand KS cases, the sperm recovery rate after TESE/mic TESE is approximately 50%; Live birth rates after ICSI have been reported to be between 28-50%, although they vary depending on the age of the woman (Corona et al., 2017; 2019). Current information on the number of live births is limited in the literature, and according to the data shared in only 42 studies; A total of 1096 biochemical pregnancies and 569 live births were obtained at the end of these pregnancies (Corona et al., 2019). According to the results of these studies, sperm retrieval, pregnancy losses and live birth rates in cases with KS are parallel to azoospermia cases that do not have this chromosomal anomaly (Bakircioglu et al. 2011).

What are the Genetic Risks in Patients with KS?  

Considering that in individuals with KS, gamete cells are formed by germ mother cells with a 47,XXY chromosome structure, theoretically, approximately 50% of the gametes that will be formed will have a normal, that is, haploid chromosomal structure (23,X or 23,Y) and the other 50% It is expected to carry a sex chromosome anomaly (e.g. 24,XX or 24,XY). In parallel, it is predicted that there will be an increase in sex chromosome anomalies in the embryos of these cases. However, both the findings obtained from the babies born and the genetic test results performed on the embryos do not support that the gametogenesis stages occur as mentioned above. To date, only one case of 47,XXY has been reported, and no other sex chromosome anomaly or autosomal aneuploidy has been found among the babies born (Corona G et al. 2017).

Although the results of cases undergoing preimplantation genetic testing (PGT) are inconsistent, no increase in sex chromosome anomalies has been reported in general. In a study conducted in 2003 using the fluorescence in situ hybridization (FISH) method, it was found that the euploidy rate (54%) in embryos obtained in 32 cycles of 20 KS cases was lower than the control group (77.2%), and the frequency of sex chromosome anomalies was also higher in the control group. It was reported that it increased compared to (13.2% vs 3.1%). As a result of these findings, the authors emphasized the importance and necessity of preimplantation genetic diagnosis (PGT) in cases with KS (Staessen et al., 2003). However, the accuracy of the results is questionable in terms of the analysis technique used. According to a recent study using a sensitive technique such as next-generation sequencing (NGS), 64.4% of 45 embryos obtained from 18 KS patients whose spouses were 35 years of age or younger were observed to be euploid and did not carry any sex chromosome anomalies, and were compared with infertile cases in the same female age group. When compared, no statistical difference was found in euploidy rates (Tong et al., 2021). According to another study, XXY anomaly was found in only one of the 16 embryos obtained, and all other embryos were considered euploid (Bakırcıoğlu et al. 2003). This and similar studies show that there is no increased risk in the embryos of patients with KS that can be associated solely with this syndrome.

Why Are Sperm Chromosome Disorders Lower Than Expected in Patients with KS?

When all cases with and without PGT procedure are evaluated together, chromosomal anomalies in the born children and the embryos created are observed to be lower than theoretically expected. To understand the reasons, sperm cells and sperm mother cells need to be examined chromosomally. However, studies using sperm karyotyping or sperm in situ hybridization (FISH) methods are limited in number due to the very low number of sperm cells in these individuals. According to a study in which a total of 1052 testicular sperm cells belonging to 30 patients with KS were examined by the FISH method, no sex chromosome anomaly was found in any sperm and no KS was found in any of the children (45) born after ICSI (Miki et al., 2017). Most sperm FISH studies do not support the existence of an increased risk (Shi Q and Martin, 2001). These and similar findings suggested the existence of a slight mosaicism in the gonad cells, that is, the presence of euploid (46,XY) cell foci. In a study conducted in 2009 to examine the origin of sperm cells in cases with KS, the cases from which sperm were obtained by micro-TESE were examined histologically and genetically. 70-94% of Sertoli cells contained two XX (47,XXY) however, it was determined that all premeiotic spermatocytes in the tubules had a normal 46,XY chromosome structure (Sciurano et al., 2009). These findings show that the cell groups that form sperm are actually euploid cells (46,XY), therefore no increase in the risk of sex chromosome anomalies is expected in the resulting sperm cells. A possible mechanism has been suggested that a random loss of one of the X chromosomes (e.g. inactive X) during mitosis may cause diploid spermatogonia (46, Sciurano et al., 2009).

Current Clinical Approach to Patients with KS

Live birth rates after ICSI in cases with KS do not differ when compared to cases of severe male infertility without KS (Bakırcıoğlu et al. 2011). It does not seem possible, based on current data, that the sex chromosome anomaly in KS cases can be transmitted to other generations. Although the risk of sex chromosome and other chromosomal anomalies has not apparently increased, risks due to female age and the use of testicular sperm still exist. According to a very large series examining severe male infertility cases, it was noted that aneuploidy rates and mosaicism rates were higher in embryos obtained from patients whose partners were under 35 years of age and whose testicular sperm was used, compared to embryos obtained from women in the same age group and individuals with normal sperm count. These findings suggest that PGT-A may be indicated in cases of severe male infertility (Kahraman et al., 2020). For this reason, it should be aimed to make case-by-case evaluations and provide a personalized treatment plan, accompanied by comprehensive genetic counseling in which the presence of other accompanying indications will be evaluated.