Birth of healthy male twins after intracytoplasmicsperm injection of frozen-thawed testicularspermatozoa from a patient with nonmosaicKlinefelter syndrome

The birth of 10 healthy neonates has been reported after ICSI using spermatozoa from patients withnonmosaic Klinefelter syndrome. All infants had a normal karyotype. In the first reported pregnancy,frozen-thawed ejaculated spermatozoa were used. In all other cases, ICSI was performed by using testicularsperm. We report the first case, to our knowledge, of the use of frozen-thawed testicular sperm from anonmosaic 47,XXY man in ICSI that was followed by pregnancy and delivery of normal male twins. A26-year-old healthy man with normal phenotype presented to our center because of infertility. Physicalexamination revealed bilateral testicular atrophy (estimated testicular volume 6 mL), normal male hairdistribution, and no gynecomastia. Standard semen analyses (average volume 3.26 1.3 mL) showed nospermatozoa. Occasionally, few immotile sperm were visible in the pellet after centrifugation, most of whichhad poor morphology. Serum hormone analysis revealed an FSH level of 25.9 IU/L, an LH level of 11.8 IU/L,a T level of 12.2mg/L (low), and a prolactin level of 318 pmol/L (normal). Peripheral-blood chromosomeanalysis showed a 47,XXY karyotype in 40 cells.

Two biopsy samples were taken from the right testis during testicular sperm extraction (TESE) in 1997.Motile sperm were found and injected into 15 mature oocytes from the patient’s 24-year-old wife. Pregnancywas achieved and culminated with the birth of a normal boy (1). The remaining testicular tissue was frozenby diluting the extract in a 1:1 ratio with freezing medium and Test Yolk buffer (Irvine Scientific, Santa Ana,CA). The contents were sealed in freezing straws that were dropped into a nitrogen vapor chamber andstabilized at280°C for 20 minutes (cooling rate of210°C/min) before immersion in liquid nitrogen for spermstorage at2196°C.

The couple returned to our unit because they desired another pregnancy. The frozen sperm was quicklythawed to room temperature. The frozen-thawed sperm mixture containing cryoprotectant was diluted withinsemination medium and centrifuged at 300g for 7 minutes; the pellet was then processed for examinationin multiple droplets.

Eight MII oocytes were injected with immotile frozen-thawed spermatozoa; of these, four fertilized andcleaved. Forty-four hours after oocyte retrieval, one embryo developed to the four-cell stage with grade IImorphology. The remaining three embryos were at the two-cell stage; one each had I-II, II-III, and II-III grademorphology. (Grade I represents excellent morphology; grade IV represents poor morphology.) The ninthoocyte was injected with the only available motile spermatozoon. This oocyte fertilized and cleaved to thefour-cell stage and had grade I-II morphology 44 hours after oocyte retrieval.

The transfer of four embryos—three of the four embryos obtained after injection of immotile sperm andthe embryo obtained after injection of the motile spermatozoon—led to establishment of a twin pregnancy.Two sacs with appropriate fetal crown rump length and heart beats were visible on ultrasonography in theseventh week of gestation. However, the concepti were expelled 1 week later after heavy uterine bleeding.

In the next treatment cycle, five oocytes were injected with immotile frozen-thawed spermatozoa. Threeoocytes were fertilized; two of them cleaved and formed four-cell–stage embryos, each with grade II-IIImorphology. The sixth oocyte underwent ICSI with a motile spermatozoon; however, the next morning, threepronuclei were visible in that oocyte. The two normal embryos were transferred to the uterine cavity, but thepatient did not conceive.

The third treatment using frozen-thawed testicular sperm was initiated in December 1998. Seven of eightMII oocytes were injected with seven motile spermatozoa, yielding six fertilizations. The eighth oocyte wasinjected with an immotile spermatozoon; three pronuclei were visible. Of the six fertilized oocytes, fivereached the four-cell stage and one reached the two-cell stage on day 2. Three embryos had grade IImorphology, two had grade II-III morphology, and one had grade III morphology. Four embryos werereplaced, and a twin pregnancy was established.

Received December 29,1999; accepted March 31,2000.Reprint requests: RaphaelRon-El, M.D, IVF Unit,Assaf Harofeh MedicalCenter, Zerifin 70300, Israel(FAX: 972-89779003;E-mail: rronel@asaf.gov.health.il).

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The couple consistently refused our suggestion of preimplanta-tion genetic diagnosis. Subsequent amniocentesis revealed a normalkaryotype for both fetuses (both 46,XY). The pregnancy progresseduneventfully, and two healthy male infants were delivered bycesarean section; their birthweights were 3100 and 3380 g. Post-natal diagnosis by peripheral blood karyotypes (20 cell metaphasesfor each child) confirmed that both infants had normal chromo-somal constitution.

The recent reports of 10 normal infants born to fathers withnonmosaic Klinefelter syndrome after ICSI are encouraging andpresumably will lead to more such treatments. The presence ofspermatozoa in the ejaculate of patients with nonmosaic Klinefeltersyndrome is uncommon and may be inconsistent. In 7 of 13reported patients (2, 3), very few spermatozoa were seen in thepellet of their ejaculate, and motile spermatozoa were observed inthe ejaculate of only 1 of the 7 men. Therefore, it is logical toassume that TESE will be needed to obtain sperm from most or allpatients with nonmosaic Klinefelter syndrome. This consideration,along with the fact that most patients with nonmosaic Klinefeltersyndrome have small testicles, emphasizes the importance of use offrozen testicular tissue from the original testicular biopsy in ICSI.Freezing tissue enables the number of times that biopsy samplesmust be obtained from the testicles, with all the consequences of asurgical procedure.

It is interesting to compare the fertilizing capability of immotiletesticular thawed spermatozoa with that of motile sperm of thesame origin. In our case, of the 14 injected oocytes with immotilespermatozoa, 7 fertilized and 6 cleaved; in contrast, of the 8 oocytesinjected with motile spermatozoa, 7 fertilized and cleaved. How-ever, both sets of embryos led to the establishment of a pregnancy.

In cases in which TESE is part of treatment or when frozentesticular sperm is used, we transfer one additional embryo to thestandard number of embryos transferred to the uterus. The reasonfor doing so is that a patient with azoospermia can undergo alimited number of TESE procedures. In this case, we transferredfour embryos to the uterus rather than the usual three while takinginto account the increased risk of multiple pregnancy.

The couple had refused preimplantation genetic diagnosis, al-though we extensively discussed its importance with them. Theyagreed to prenatal diagnosis by amniocentesis in the 17th week ofgestation.

According to different published data, the incidence of hyper-ploidy of the sperm in patients with mosaic Klinefelter syndromevaries from 2.1% to 7.5% . A single published study (4) reported a2.7% incidence of hyperploidy in the ejaculated spermatozoa of apatient with nonmosaic Klinefelter syndrome. We provide thisinformation to couples in which the husband has the Klinefeltersyndrome. Although the information gathered so far does notindicate a high incidence of transmission of gonosomal hyperploidyin nonmosaic patients, preimplantation or prenatal genetic diagno-sis should continue to be suggested to patients before startingtreatment.

Raphael Ron-El, M.D.Arie Raziel, M.D.Dvora Strassburger, Ph.D.Morey Schachter, M.D.Orna Bern, Ph.D.Shevach Friedler, M.D.IVF and Infertility Unit, Assaf Harofeh Medical Center,

Tel Aviv University, Tel Aviv, Israel

References1. Ron-El R, Friedler S, Strassburger D, Komarovsky D, Schachter M,

Raziel A. Birth of a healthy neonate following the injection of testicularspermatozoa from a patient with Klinefelter’s syndrome. Hum Reprod1999;14:368–70.

2. Tournaye H, Camus M, Vandervorst M, Nagy Z, Jovis H, Van Stein-teghen A. Surgical sperm retrieval for intracytoplasmic sperm injection.Int J Androl 1997;20(Suppl 3):69–73.

3. Palermo GP, Schlegel PN, Sills ES, Veek LL, Zaninovic N, Menedes S,et al. Births after intracytoplasmic injection of sperm obtained by tes-ticular extraction from men with non-mosaic Klinefelter’s syndrome.N Engl J Med 1998;338:588–90.

4. Guttenbach M, Michelman HW, Hinney B., Engel W, Schmid M.Segregation of chromosomes into sperm nuclei in a man with 47,XXYKlinefelter’s karyotype: a FISH analysis. Hum Genet 1997;99:474–7.

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