Klinefelter’s syndrome (KS) is the most frequent
sex chromosomal abnormality and is usually pathologically
characterized by spermatogenic dysfunction
Pericentric inversion of chromosome 9 is one of the
frequent chromosomal rearrangements, considered
a normal variant with a prevalence of 1-3% (
Here, we report a case of KS, in which fluorescence in-situ hybridization (FISH) was used to analyze the aneuploidy rate of the ejaculated sperm of the patient, while identifying an inversion in chromosome 9 in his wife. For the first time, intracytoplasmic sperm injection (ICSI) was performed to treat the patient, who fathered a twin pregnancy successfully.
A married couple (a 24-year-old man and a 22-year-old woman) were treated at our center for a diagnosis of primary infertility. A physical examination found the following in the man: height, 178 cm; span height, 178 cm; sitting height, 98 cm; weight, 79 kg; a history of mumps; normal secondary sexual characteristics; and a negative response to anti-sperm antibody.
Results of an external genitalia examination
were normal for pubic hair, penis length (8 cm),
bilateral epididymitis, vas deferens, and prostate,
but no antheridiogen varicose veins and bilateral
testes of a small size (~5 ml). A semen examination revealed severe asthenozoospermia, of which the
sperm showed a volume of 0.8 ml and a density
of 0.7×106/ ml (World Health Organization standard,
2010). His peripheral blood karyotype was
non-mosaic 47, XXY after we analyzed 200 peripheral
lymphocytes. The karyotyping results of
100 oral exfoliated cells were also analyzed and
the diagnosis was confirmed. No AZF deletion
was found after multiplex PCR analysis. Based
on a previously published method (
His wife had a menstrual cycle of 4/37 and mild dysmenorrhea,
normal vulva and vagina, smooth cervix
without masts or lacerations, a normal-sized posterior
uterus with medium texture, no active tenderness; and
normal bilateral attachments with no lesions or tenderness.
The basal endocrine index was as follows:
FSH, 6.8 mIU/mL; LH, 5.2 mIU/mL; E2, 19.6 pg/mL;
thyroid hormone (T1), 17.7 ng/dL; PRL, 20.7 ng/mL;
testo, 0.94 ng/mL and P, 0.41 ng/mL. Multiple immature
follicles were observed in both of ovaries by
transvaginal ultrasound (TVS) (
Karyotype examination of 200 peripheral blood
cells showed pericentric inversion of one chromosome
9, with the breakpoint in the short arm at 9p11
and in the distal region of the long arm at 9q21 (
The clinical details of the couple. A. Karyotype of the husband (47, XXY). B. The sperm were analyzed by FISH (×1000). C. The result indicated the husband without AZF deletion after multiplex PCR analysis. D. karyotype of the wife [46, XX, inv(9) (p11q21), and E. TVS found multiple immature follicles in her both of ovaries
Before the couple agreed to ‘assisted reproductive treatment, they received detailed genetic counseling and chose the ICSI treatment. Furthermore, the patient and his wife rejected a preimplantation genetic diagnosis, but were willing to undergo second trimester prenatal diagnoses. The wife received a short controlled ovarian hyperstimulation (COH), and the protocol was as follows: 1. at menstrual day 3, an intramuscular injection of Decapeptyl (gonadotropin- releasing hormone agonist, GnRHa) at a dose of 0.65 mg was carried for down-regulation; 2. from day 5, Gonal-F (recombinant FSH, rFSH) was continually used at a dose of 150 IU/d to promote ovulation; 3. at day 9, the dose of Gonal-F was reduced to 75 IU/d, at the same time, Luveris (recombinant LH: rLH) was injected at a dose of 75 IU/d; 4. serial ultrasound examinations and the evaluation of serum E2, LH, and P levels were used to monitor the follicular maturation. Pregnyl (hCG) was employed for ovulation induction at a dose of 10000 IU IM when at least two follicles achieved a mean diameter of 18 mm; 5. oocyte aspiration was performed at 35th hour after hCG administration. Controlled ovarian stimulation, followed by TVS-guided follicle aspiration, was performed and resulted in the recovery of 11 mature MII stage eggs.
On the same day as the oocyte aspiration, 0.5 mL of semen was obtained from the patient . After centrifugation, the pellet of the semen was mixed with 0.1 mL of culture medium, and sperm was acquired to carry out the ICSI procedure.
Eleven mature MII stage eggs were selected and fertilized by ICSI. At 72 hours post-egg acquisition, two of eleven 8-cell embryos were randomly selected for embryo transfer, and the other 9 embryos were frozen. A twin pregnancy was established and at 18 weeks, amniotic fluid cells were obtained through transabdominal amniotic fluid puncture to perform prenatal diagnosis. The results showed that the karyotypes of the two embryos were 46, XY and 46, XY, inv(9) (p11q21), respectively. Twin boys were delivered at 34 weeks of gestation because of PROM and admitted to the neonatal intensive care unit (NICU). After 7 days, they were discharged. To date, the twin brothers have developed normally with no signs of impaired nervous system function or cognition.
The gold standard of genetic diagnosis for
KS remains karyotyping of metaphase spreads
from cultured peripheral blood lymphocytes,
yet karyotype analysis of other additional tissues
(buccal smear, skin, etc.) could provide
more precise diagnosis (
Before the development of ICSI, patients with
KS could only acquire offspring by either adoption
or artificial insemination with another’s sperm.
However, in some cases, sperm could still be occasionally
found in the patient’s semen or through
a testicular biopsy. Hence, development of testicular
sperm retrieval technology can improve the
chance that KS patients can father their own biological
Before ICSI, it is necessary for patients to receive
adequate genetic counseling. Analysis of
the chromosome aneuploidy rate of the patient’s
ejaculated sperm can provide direct evidence
of the need for genetic counseling, for which
FISH is the most rapid and accurate method
Chromosome 9 has the highest degree of morphological
In summary, the application of FISH in the investigation of the sperm aneuploidy rate can facilitate evaluating the risk of patients with KS and generating genetically normal offspring. For those patients that still can provide sperm, ICSI treatment can be performed after receiving adequate genetic counseling and obtaining informed consent. Further, ICSI can be performed even when the female partner has an inversion of chromosome 9. Lastly, a follow-up study will be conducted on the patient’s offspring.