Partners of men with Klinefelter syndromecan benefit from assisted reproductivetechnologies

Ulun Ulug, M.D.,a Faruk Bener, M.D.,a Mehmet Ali Akman, M.D.,a andMustafa Bahceci, M.D.a,b

Bahceci Women Health Care Center and German Hospital at Istanbul, Istanbul, Turkey

Objective: To report the birth of a healthy female infant from a father with nonmosaic Klinefelter syndrome(KS) and document the experience of men with KS undergoing assisted conception.

Design: Retrospective.

Setting: Private IVF center.

Patient(s): Twelve couples with male factor infertility due to Klinefelter syndrome undergoing assistedreproduction treatment.

Intervention(s): Controlled ovarian hyperstimulation, testicular sperm extraction, intracytoplasmic sperminjection (ICSI), round spermatid injection (ROSI), and preimplantation genetic diagnosis.

Main Outcome Measure(s): Testicular sperm retrieval rate, fertilization rate, and pregnancy outcome.

Result(s): There was a sufficient amount of motile sperm for injection into mature oocytes in 6 of the 11testicular biopsies (54.5%). Fertilization rates for ICSI and ROSI cases were 54.2% and 41.6%, respectively.The pregnancy rate per ET was 27.2%. None of the ROSI cases resulted in pregnancy. Two patients hadspontaneous abortions at 8 and 18 weeks of gestation, respectively. Only one patient delivered a healthyfemale baby after 36 weeks of an uneventful pregnancy.

Conclusion(s): Men with KS can benefit from assisted reproductive technologies, and the testicular spermretrieval rate among them is promising. Although sex chromosome aberrations among the embryos from menwith KS are not common, couples can be offered preimplantation genetic diagnosis before ET. (Fertil Steril�2003;80:903–6. ©2003 by American Society for Reproductive Medicine.)

Key Words: Klinefelter syndrome, ICSI, pregnancy

Klinefelter syndrome (KS) is one of themost common causes of male hypogonadismand is characterized by excessive X chromo-some. Supernumerary X chromosomes are gen-erated by meiotic nondisjunction, and the inci-dence of KS is 1 in 500–600 males (1). Theclinical features of this syndrome are typicallyassociated with postpubertal testicular failuresuch as hypoplastic testicules, gynecomastia,and sterility. Eighty-five percent of men withKS are nonmosaic and have nonobstructiveazoospermia because of the shortened germcell life and hyalinization of testicular tubules(2).

Several studies have focused on spermato-genesis in the testicles of men with nonmosaicKS (3, 4). In the era of assisted reproductivetechnologies (ART), testicular spermatozoa

can be recovered and used for achieving preg-nancy for men with KS. In this regard, healthybirths from those individuals were also re-ported.

In this study, we document our experiencewith a group of men diagnosed with KS whohad undergone assisted conception with testic-ular extracted spermatozoa. In addition, we re-port the birth of a healthy female infant from afather with nonmosaic KS after intracytoplas-mic sperm injection (ICSI).

MATERIALS AND METHODS

PatientsDuring the period from January 2000 to

August 2002, 12 patients diagnosed with KSunderwent assisted conception treatment out of

Received December 16,2002; revised andaccepted March 19, 2003.Reprint requests: MustafaBahceci, M.D., Azer IsMerkezi Kat 6 44/17, AbdiIpekci Cad. Nisantasi,80200 Istanbul, Turkey(FAX: 90-2122303990;E-mail: bahceci@hotmail.com).a Bahceci Women HealthCare Center and GermanHospital at Istanbul.b Yeditepe UniversitySchool of Medicine,Istanbul, Turkey.

FERTILITY AND STERILITY�VOL. 80, NO. 4, OCTOBER 2003Copyright ©2003 American Society for Reproductive MedicinePublished by Elsevier Inc.Printed on acid-free paper in U.S.A.

0015-0282/03/$30.00doi:10.1016/S0015-0282(03)01157-9

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3,714 cycles carried at the Istanbul German Hospital IVFunit. The diagnosis of KS was made according to cytogeneticevaluation of peripheral lymphocyte metaphases. As theprimary management for males with KS would not change,tissue cultures to confirm nonmosaicism were not requested.Physical examination of the external genitalia was per-formed on all males to rule out any coexisting anomaly.

All couples were thoroughly informed about testicularsperm extraction (TESE) procedure, pregnancy rate, and thefurther consequences of transmitting abnormal karyotype tooffspring. They were also informed about round spermatidinjection (ROSI) in case of failed testicular spermatozoaretrieval. Before undergoing testicular surgery, all males hada new sperm analysis according to World Health Organiza-tion criteria to confirm azoospermia. The committee forethics at the German Hospital in Istanbul approved thisstudy.

Ovulation Induction ProtocolThe ovulation induction protocol for ovarian stimulation

to females began with pituitary desensitization by GnRHagonist (leuprolide acetate; Lucrin, Abbott, Aubonne, Swit-zerland) in the midluteal phase of the preceding menstrualperiod. Administration of gonadotropins (metrodin HP 75IU; Serono, Aubonne, Switzerland; or humegon 75 IU; Or-ganon, Oss, The Netherlands) was initiated when serum E2

level fell below 50 pg/mL. Gonadotropin dosages were tai-lored according to each individual ovarian response. When atleast two follicles reached 18 mm in diameter, hCG (Pregnyl10,000 IU, Organon) was administered IM.

Oocytes were retrieved 32–38 hours after hCG injectionand subjected to ICSI or ROSI. Embryos were transferredtranscervically 3 days after oocyte retrieval under ultrasoundguidance. Luteal phase was supported by 100 mg/day of P inoil IM.

Sperm Retrieval and PreparationThe TESE procedure, ICSI, and ROSI were performed as

described elsewhere (5, 6). Briefly, multiple-site testiculartissue was obtained by open biopsy under general anesthesia.After treatment with 20% human serum albumin and 20%Earle’s balanced salt solution (Sigma E2888, Sigma-Aldrich,Chemie GmbH, Stenheim, Germany) conditioned media,testiculer tissues were first observed under an inverted mi-croscope equipped with a Hoffman modulation contrast sys-tem. Magnifications of �200 and �400 were adjusted todetect the presence of spermatozoa or round spermatids.

The testicular tissue suspension was then centrifuged at600 g throughout a discontinuous Percoll (Sigma P-1644)gradient of three layers (50%, 70%, and 90%). The sperma-tids were mostly found in the 50% and 70% Percoll frac-tions, whereas spermatozoa were found in the 90% fraction.Round spermatids (ROS) at the Golgi cap phase and acro-some phase were observed and identified under a Zeiss135-M microscope equipped with a Hoffman modulation

contrast system. Spermatozoa injections were performedsmoothly. However, during injection of ROS, suction of theooplasm was rather vigorous, as this was thought to boostoocyte activation. The presence of two pronuclei (2PN)18–24 hours after ICSI or ROSI was considered fertilization.Pregnancy was defined as visualization of an intrauterinegestational sac with rising �hCG values.

RESULTSThe mean age of females and males were 30.41 � 4.3 and

33.4 � 2.9 years, respectively. The mean FSH, LH, and Tserum levels of males were 39.0 � 8.3 mIU/mL, 27.3 � 10.6mIU/mL, and 3.20 � 1.6 mg/L, respectively. All males hadbilateral reduced testicular volume (the mean testicular vol-ume for right and left testicles was 4.4 � 1.6 ml and 4.0 �1.6 ml, respectively). The characteristics of patients andoutcome of cycles are shown in Table 1. Embryo transfer(ET) was performed in 11 (91.6%) out of 12 cycles. Suffi-cient motile spermatozoa to be injected into mature oocyteswere found in 6 (54.5%) out of the 11 testicular biopsies.

In case number 9, spermatozoa used for ICSI were re-trieved from the ejaculate of the patient. ROSI and subse-quent ETs were done in 4 (36.3%) out of 11 ET cycles.Fertilization rates for ICSI and ROSI cases were 54.2% and41.6%, respectively. Overall implantation rate was 17.8%(5/28). Pregnancy was achieved in three patients (pregnancyrate per ET, 27.2%). None of the ROSI cases resulted inpregnancy.

In case number 4, preimplantation genetic diagnosis(PGD) was used on the 10 developing embryos. Fluores-cence in situ hybridization with probes for chromosomes X,Y, 21, 18, and 13 were used to detect aneuploidy. Twoembryos had multinucleated cells and were discarded. Theremaining eight embryos had diploid sets of chromosomesaccordingly assessed and were equally distributed for XXand XY.

In case number 2, transvaginal ultrasound demonstrated agestational sac without any embryonic pole and the patientspontaneously aborted at 8 weeks of gestation.

In case number 9, a trichorionic triplet pregnancy wasachieved; however, the patient aborted at 18 weeks of preg-nancy. During the course of the pregnancy, prolonged vag-inal bleeding due to subchorionic hematoma at the low-lyingfetus persisted (two female fetuses and one male fetus;external appearances were normal for the gestational age).The couple refused to have any further investigation of thefetuses after the abortion.

In case number 10, a healthy 2,750 g female baby wasdelivered by cesarean section after 36 weeks of an unevent-ful gestation. This couple opted to continue the pregnancywithout any prenatal diagnostic intervention. However, theman agreed to have a skin biopsy to confirm mosaicism, and26 metaphases with 47,XXY were detected in fibroblasts.

904 Ulug et al. IVF experience and Klinefelter syndrome Vol. 80, No. 4, October 2003

Chromosome analysis of peripheral blood cells from theneonate showed a normal 46,XX karyotype.

DISCUSSIONRecent advances in ART have allowed men with KS to

conceive. However, healthy deliveries after assisted concep-tion from those individuals are not abundant and are mostlypresented as case reports. This study describes several fea-tures of ART that can be used in combination for men withKS and reports three pregnancies and one successful deliv-ery.

Several concerns can be addressed to men with KS un-dergoing assisted conception treatment. The first one is theretrieval rate of motile spermatozoa from the testicular ex-tractions. In this regard, prognostic values of the clinical andlaboratory parameters were assessed for the prediction ofspermatogenesis in patients with KS and testicular volume, Tlevels, and hCG tests were found to be important predictivefactors (7). Thus, the efficiency of TESE in men with non-mosaic KS was evaluated in several studies and the testicularspermatozoa retrieval rate was shown to be comparable withthe general population of patients with nonobstructiveazoospermia (8–10). Adequate testicular spermatozoa forICSI were detected in 42%–70.4% of TESE procedures (2,11).

Moreover, some studies reported significant rates of sper-matozoa retrieval from the ejaculate of men with nonmosaicKS (8, 12) and the birth of healthy twins using ejaculatoryspermatozoa from a man with nonmosaic KS was also re-ported (13).

The second concern is the fertilization potential of thetesticular retrieved spermatozoa from men with nonmosaicKS. Friedler et al. (2) reported a 66% fertilization rate fromfresh testicular spermatozoa and 58% from cryopreserved-thawed testicular spermatozoa, respectively. In our series,the fertilization rate was 55.4% from testicular retrievedspermatozoa. It is therefore apparent that the majority ofspermatozoa retrieved from males with KS are effective inachieving fertilization and subsequent embryo development.

In cases of failed testicular spermatozoa retrieval in menwith KS, ROS can be used for assisted fertilization. In onestudy, ROS from 24 men with nonmosaic KS were evaluatedand 93% of them were shown to be haploid (14). In ourgroup, the oocytes of four patients were subjected to ROSI,and 41.6% of them were fertilized and developed embryos tothe stage of transfer. However, we can conclude that ROSIfor men with KS is not efficacious in terms of achievingpregnancy.

The third concern is the probability of transmitting ab-normal sex chromosome material to offspring. Testiculargerm cells in KS can have different chromosomal aberra-tions, and it was proposed that the proportion of spermatozoawith hyperhaploid chromosomes was relatively increased(15). However, Kruse et al. (16) found hyperhaploid spermin only 7.5% of retrieved spermatozoa from men with KS.Similarly, Levron et al. (17) demonstrated that most of thetesticular sperm from KS patients had a normal pattern of sexchromosome segregation.

Furthermore, it was also shown that 47,XXY spermato-gonia can undergo meiosis and complete the formation ofcytogenetically normal spermatozoa (18). On the other hand,

T A B L E 1

A breakout of outcome of patients with KS undergoing assisted conception treatment.

CaseFemaleage (y)

Maleage (y)

Male FSH(mIU/mL)

Male LH(mIU/mL)

Male T(mg/L)

Sperma-tozoaa

Roundcell

Oocytesretrieved

MIIoocytes 2PN ETs Pregnancy Outcome

1 35 36 41 39 4.2 � � 15 13 6 4 �2 33 33 55 51 3.9 � 4 4 2 1 Singleton Missed3 28 34 28 19 0.8 � � 18 15 7 4 �4 37 34 32 24 5.5 � 26 22 13 4b �5 36 35 48 22 0.6 � 16 12 4 1 �6 32 35 28 16.9 3.3 � � 7 6 1 1 �7 31 34 40 29 4 � 19 14 6 4 �8 26 26 43 14 1.7 � � Cancelled9 28 32 37 35 6.1 �c 21 17 14 3 Triplets Abortus

10 29 37 33 19 2.7 � 7 5 2 2 Singleton Lived birth11 27 35 43 28 3.1 � � 4 2 1 1 �12 23 30 41 31.5 2.6 � 25 20 10 3 �

a Testicular sperm extraction was used to retrieve spermatozoa.b Preimplantation genetic diagnosis was performed on transferred embryos.c Ejaculatory spermatozoa were used.d 46,XX.

Ulug. IVF experience and Klinefelter syndrome. Fertil Steril 2003.

FERTILITY & STERILITY� 905

prenatal and postnatal diagnosis of 47,XXY fetuses was alsodescribed from men with KS (2, 19). PGD can be recom-mended to men with KS undergoing ART for the selection ofnormal diploid embryos. Reubinoff et al. (20) reported thebirth of a healthy neonate from a man with nonmosaic KSafter PGD. In their case, excessive X chromosome wasdetected in one out of the two developed embryos. In anotherstudy, five embryos underwent biopsy for PGD and none ofthem was found to have abnormal sex chromosomes (21). Inour series, PGD was used in 10 embryos in one individualand none of them was found to carry any sex chromosomalaberrations.

The drawback of this study is the lack in the confirmationof nonmosaicism by tissue culture among 11 men with KS.On the other hand, both clinical and laboratory characteris-tics of nonmosaic KS were apparent in our subjects. Theultimate aim of this study is to document the IVF experiencewith such a group of patients. Furthermore, regardless ofmosaicism, which accounts for more than half of the cases,testicular sperm retrieval has been accomplished and a27.2% pregnancy rate was achieved in that group. Our re-sults with the evidence discussed above suggest that menwith KS can benefit from ART treatment.

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