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Success of Testicular Sperm Injection and Intracytoplasmic Sperm Injection in Men with Klinefelter Syndrome

Department of Urology (J.D.S., M.G., P.N.S.), The James Buchanan Brady Foundation and The Center for Reproductive Medicine and Infertility (G.D.P., L.L.V., M.G., Z.R., P.N.S.), The New York-Weill Cornell Medical Center, and the Population Council (M.G., P.N.S.), New York, New York 10021

Address all correspondence and requests for reprints to: Peter N. Schlegel, The James Buchanan Brady Foundation, Department of Urology, and The Center for Reproductive Medicine and Infertility, The New York-Weill Cornell Medical Center, and the Population Council, New York, New York 10021. E-mail: pnschleg{at}med.cornell.edu.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Purpose: The aim of this study was to report the successful fertility treatment of men with Klinefelter syndrome using testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI).

Methods: A total of 42 men with Klinefelter syndrome who underwent 54 TESE procedures were identified. Before TESE, patients with serum testosterone levels less than 15.6 nmol/liter were treated with an aromatase inhibitor. Sperm retrieval rates and results of ICSI, including fertilization and clinical pregnancy, were collected.

Results: Mean pretreatment FSH and testosterone levels were 33.2 IU/liter and 9.8 nmol/liter. During medical therapy, the mean testosterone level rose to 17.0 nmol/liter (P < 0.01). Spermatozoa were found during 39 microdissection TESE procedures, on the day before, or day of oocyte retrieval during a programmed in vitro fertilization cycle. The sperm retrieval rate was 72% (39 of 54) per TESE attempt, and 29 of the 42 different men (69%) had adequate sperm found for ICSI. Thirty-three in vitro fertilization cycles yielded embryos for transfer in the 39 (85%) cycles with sperm retrieved. Eighteen clinical pregnancies have resulted in 21 live births [18 of 39 (46%)]. All children had a normal karyotype.

Conclusion: TESE/ICSI is a successful intervention for the majority of patients with azoospermia and Klinefelter syndrome. Sperm retrieval and ICSI success in men with Klinefelter syndrome are comparable with other men with nonobstructive azoospermia treated at our center.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
MEN WITH KLINEFELTER syndrome have hypergonadotropic hypogonadism. This syndrome is diagnosed in 11% of azoospermic men and one in 500 male newborns overall (1, 2). The genotypic abnormality results from a meiotic nondisjunction event resulting in a 47,XXY genotype in most cases; however, up to 3% of men with Klinefelter’s syndrome are mosaic 46,XX/47,XXY (3). This disorder is the most frequent karyotypic abnormality reported in infertile men (4).

Mosaic patients occasionally have sperm in their ejaculate (5), but men with nonmosaic Klinefelter syndrome were considered sterile (2). These men with nonmosaic 47,XXY typically had extensive areas of Leydig cells and sclerotic tubules with only rare focal areas of spermatogenesis (6). With the advent of modern assisted reproductive techniques including testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI), isolated case reports demonstrated successful fertility treatment of men with nonmosaic Klinefelter syndrome (7). Until recently case reports of successful sperm retrieval and pregnancy for couples in whom the man has nonmosaic Klinefelter syndrome have been published (8, 9, 10, 11, 12, 13, 14, 15).

We retrospectively reviewed our experience in a large series of men with nonobstructive azoospermia treated with TESE and ICSI over an 8-yr period. Herein we present our experience in 42 patients with Klinefelter syndrome who underwent 54 attempted TESE procedures.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
A single surgeon’s (P.N.S.) experience with attempted treatment of men with nonobstructive azoospermia over an 8-yr period was retrospectively reviewed. Institutional board review of treatment was not required because data records were previously existent and identifiers were removed before publication. The records of 452 consecutive TESE procedures by one surgeon (P.N.S.) were analyzed, and all patients with a genetic diagnosis of Klinefelter syndrome were identified. All patients had peripheral karyotype identifying cells with a 47,XXY complement. At least 50 cells were cytogenetically analyzed for each patient. Forty-two patients were identified who underwent a total of 54 separate TESE procedures during this 8-yr time period.

Preoperative evaluation included a complete history and physical examination. Hormonal profiles including serum testosterone, estradiol, FSH, LH, and prolactin levels were obtained at least 6 months after cessation of any hormonal therapy. Patients with serum testosterone less than 15.6 nmol/liter or a testosterone to estradiol ratio less than 100 were treated with testolactone or anastrazole (16) for at least 2–3 months before surgery to enhance endogenous production of testosterone. Use of testolactone, then anastrazole, and subsequently testolactone was the initial oral treatment for patients with low testosterone levels during sequential periods from 1996 to 2004. Men who did not respond to initial oral aromatase inhibitor therapy with normal testosterone levels after 1 month were subsequently treated with human chorionic gonadotropin (hCG) injections. Treatment with hCG was initiated at a dose of 1500 IU twice a week and titrated upward until a response in serum testosterone was obtained or a maximum dose of 2500 IU three times a week was reached. For patients who were previously on other hormonal therapy (except exogenous testosterone) before referral to our center (e.g. clomiphene, recombinant FSH, etc.) with normal serum testosterone levels, previous treatment was maintained and testosterone, as well as estradiol, levels were monitored until attempted retrieval. All patients on exogenous testosterone had that treatment stopped for at least 6 months before TESE.

A diagnostic testis biopsy, when performed, was done at least 6 months before attempted TESE or during the TESE procedure. Biopsies were evaluated by a single individual experienced in the interpretation of such slides, and results were characterized based on the most advanced region of spermatogenesis present, as previously described (17). Five men who did not have a prior diagnostic biopsy had a single sample sent to pathology for analysis at the beginning of the TESE procedure. Twenty-five men had biopsies done before referral to our center, and an additional five biopsies were done at our center before TESE. This practice stopped when it became clear that diagnostic biopsy does not accurately predict the results of TESE.

TESE was performed by microdissection according to our previously described technique (18, 19). Briefly, the testis was widely opened in an equatorial plane and microdissection was carried out with examination of the morphology of seminiferous tubules using an operating microscope at x15–20 power magnification before the removal of tissue. Enlarged seminiferous tubules were selected, removed and the presence or absence of sperm evaluated in the operating room by an embryologist (19). Each sample (usually < 1 mg tissue/sample) was removed, mechanically cut, and dispersed in 0.1–0.3 cc simulated human tubal fluid buffered by HEPES and supplemented with 5% plasmanate in a microfuge tube. Specimens were passed several times through a 24-gauge angiocatheter to confirm disruption of tubules.

Each specimen was evaluated sequentially in the operating room under a phase-contrast microscope at x200 magnification. If intact spermatozoa were identified, then the procedure was terminated. If no sperm were seen, then continued microdissection of additional areas of testicular parenchyma was carried out and samples taken until sperm were found or all areas of both testes were examined. Care was taken during dissection to maintain the testicular vasculature and avoid removal of testicular interstitial tissue that contains Leydig cells. In 52 of 54 retrieval procedures, Leydig cells were predominantly present in visually identifiable hyperplastic nodules rather than as limited numbers of free interstitial cells. After dissection, closure of the tunica albuginea was effected with 5–0 polypropylene to mark the site of opening of the testis. Whenever possible, TESE attempts were performed on the day before oocyte retrieval in a programed in vitro fertilization cycle. When this was not logistically possible, TESE was done on the day of oocyte retrieval. Overnight incubation was provided in a similar simulated human tubal fluid medium containing plasmanate but without HEPES buffer.

Pituitary desensitization with GnRH agonists and ovulation induction with gonadotropins was performed for the female partners in accordance with previously described techniques (20). Spermatozoa were selected for microinjection based on morphology as previously described (21, 22). Spermatozoa were chosen for ICSI based on their motility (or motility after pentoxifylline stimulation), with the most morphologically normal motile sperm injected. Where all sperm had morphological defects (e.g. cytoplasmic droplets), sperm with fully developed tails and grossly normal heads were injected. The presence of fertilization was evaluated by examining oocytes 12–17 h after injection for the presence of distinct pronuclei and two polar bodies. Embryos were transferred 3 d after the microinjection procedure.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
Forty-two men underwent a total of 54 attempts at microsurgical sperm retrieval (Table 1). No patients had any postoperative complications. Thirty-nine men had classic 47,XXY Klinefelter’s syndrome. Three patients were mosaic 46, XX/47,XXY. Five patients had varicoceles repaired (at least 6 months) before TESE attempts. Five patients had been treated with exogenous testosterone for 2–14 yr (median 2 yr) before their fertility evaluation, and androgen therapy was stopped for at least 6 months before attempted TESE.

The mean age of men at the time of TESE was 32.8 yr (range, 24–52 yr). Mean partner age was 33.2 yr (range, 20–44 yr). Mean testicular volumes were 2.5 cc for each side (range 1–6 cc, SD 0.8). Mean FSH and testosterone pretreatment were 33.2 IU/liter (normal, 1–8 IU/liter) and 9.8 nmol/liter (normal, 8.2–27.2 nmol/liter), respectively. Pretreatment testosterone levels were obtained at least 6 months off any hormonal therapy. Thirteen men were treated with testolactone (50–100 mg orally twice a day) and hCG injections (1500 IU, twice a week), and 19 were treated with testolactone (50–100 mg orally twice a day) alone (see Table 4). Five were treated with anastrazole (1 mg/d), and one with anastrazole and hCG. Clomiphene citrate (25 mg/d) was used before TESE for three patients, and one patient received recombinant FSH injections before TESE. Six patients had normal testosterone levels and received no treatment before TESE attempt, and for another six patients, the pretreatment regimen was not known. Medical therapy (aromatase inhibitors, clomiphene, hCG, or FSH) to increase testosterone levels was continued until the time of TESE (median duration of treatment 4 months). On medical therapy, the mean serum testosterone of treated patients increased from 9.8 to 17.0 nmol/liter (P < 0.001).

Sperm were identified from 39 attempts at TESE, with at least twitching motility for 36 cases, and three cases had only nonmotile sperm identified. Pentoxifylline stimulation (3 mM incubation for 1 h) was used to identify viable spermatozoa from samples that did not initially have motility. Any spermatozoa that were retrieved but not used during the first ICSI attempt were cryopreserved. A repeat TESE procedure was performed only if no spermatozoa were viable after thawing. For TESE attempts performed on the day before oocyte retrieval, twitching motility was noted after overnight incubation in medium at 37 C. ICSI was performed for all cases in whom spermatozoa were identified intraoperatively. In only one case in whom no sperm was seen in the operating room, spermatozoa were later identified in the laboratory after additional processing of extracted tissue, including collagenase treatment (1% collagenase for 1 h at 37 C.) During 15 TESE procedures, no spermatozoa were identified, and further processing of tissue did not identify sperm. The overall sperm retrieval rate was 72% (39 of 54). Of the 42 men who underwent these procedures, 29 had testicular spermatozoa for ICSI after TESE (69%). Only one of the five men who had previously received testosterone replacement therapy had sperm retrieved with TESE (see Table 5).

At the time of initial evaluation, 86% (36 of 42) men had serum testosterone levels less than 15.6 nmol/liter and were started on medical therapy. The majority of men were treated with either testolactone alone (19) or testolactone with hCG (13). Mean testosterone levels increased significantly after therapy. Sperm retrieval rates for patients treated with these medications were 74 and 54%, respectively (see Table 4). All five men treated with anastrazole had sperm retrieved and all three men on clomiphene had successful TESE. Of the six men with no prior therapy, four had successful TESE procedures. Of note, these men had normal serum testosterone levels at the time of initial evaluation, potentially suggesting that they had better overall baseline gonadal function.

Six patients in our series underwent multiple TESE/ICSI cycles. One patient underwent five TESE procedures, all of which successfully identified sperm. Five other patients underwent two procedures each. The overall success rate per sperm retrieval attempt for these five men was six of 10 (60%), with one man having no sperm identified during either of two sequential TESE procedures.

ICSI and pregnancy results

A mean of 7.7 meiosis phase II ova were injected (range 3–22) and the mean number of ova fertilized was 4.6 ovum (range 1–15). Two hundred eighty-five ova were injected and 172 were successfully fertilized (60%). Eighteen ICSI cases resulted in clinical pregnancy, with 21 babies born thus far, 11 boys and 10 girls. One pregnancy is ongoing. All children had normal peripheral karyotypes confirmed after birth.

Histological analysis of testes

Histological analysis of diagnostic testicular biopsies performed simultaneous to or before sperm retrieval revealed 34 attempted TESE cases with Sertoli cell-only pattern, seven with Leydig cells-only seen, three with focal hypospermatogenesis, and one with maturation arrest. No patient with mosaic Klinefelter syndrome had hypospermatogenesis. The histological findings for the other seven attempts were not known. Sperm retrieval rates per attempt at TESE, relative to diagnostic biopsy histology are displayed in Table 2.

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
Successful TESE-ICSI for men with nonmosaic Klinefelter syndrome with delivery of healthy children was first reported in 1997 (22). Since that time, several groups have published case reports (7, 8, 9, 11) and small series (10, 13, 18, 23) of successful pregnancy outcomes combining these techniques. The treatment of men with Klinefelter syndrome has been thought to be limited by the small testicular volume, extensive tubular sclerosis, and high FSH typically found for these men. Herein we report our experience in a consecutive series of men with predominantly nonmosaic Klinefelter syndrome who underwent attempted simultaneous TESE in a programmed in vitro fertilization cycle. We included three cases of mosaic Klinefelter syndrome in this series because the success rate was no better (one of three with sperm retrieved in mosaic cases.)

A comparison of the sperm retrieval success rates reported in this study with those of prior series of attempted TESE procedures in patients with Klinefelter syndrome is presented in Table 3 (10, 11, 15). Other groups operated on men with similar ages and slightly lower mean serum FSH levels, with 40–48% success in retrieving testicular spermatozoa. The overall clinical pregnancy rates reported varied from 33 to 50% in small series. Our data suggest a slightly higher success rate in retrieving sperm with 72% of TESE attempts (or 69% of men) having sperm found. Once sperm were identified, 56% of ICSI attempts resulted in pregnancy.

The mean age of patients did not differ between those with sperm found and those with no sperm found, and serum testosterone levels did not differ between these groups either. Most of our patients received preoperative stimulation with an aromatase inhibitor or a combination of hCG and aromatase inhibitor to enhance intratesticular (endogenous) production of testosterone. Although four of six men who were not treated with an aromatase inhibitor had sperm retrieved by TESE, these men had normal testosterone levels at initial evaluation and may have had better overall testicular function. The lower retrieval rate for men who required hCG and testolactone (or hCG and anastrazole) because they did not respond to the aromatase inhibitor alone (Table 4) suggests that androgen-producing capacity of the testes may be related to spermatogenic function. Our observation that the sperm retrieval rate appeared to be lower (20%) in men who previously received exogenous androgens may argue against the routine treatment of Klinefelter patients with testosterone therapy at the time of diagnosis. We have also seen two patients with Klinefelter syndrome who were managed with long-term testosterone replacement before attempted TESE at another institution who could not have sperm found with TESE. Such treatment may suppress the hypothalamic-pituitary-testis axis, impairing FSH secretion and causing decreased intratesticular androgen levels that could impair spermatogenesis. Return of spermatogenesis after testosterone pretreatment may require a washout and recovery period of substantive duration (years.) The failure to find sperm in men after testosterone suppression could suggest a permanent adverse effect of testosterone in men with Klinefelter syndrome after long-term therapy (Table 5). The concern for maintenance of fertility potential in young men with Klinefelter syndrome must be balanced with potential benefits of testosterone replacement, including the possible psychosocial benefits of early androgen replacement therapy in these patients.

For couples with nonobstructive azoospermia including Klinefelter syndrome, it is possible that TESE will fail to retrieve sperm. Although our experience suggests a better pregnancy rate when sperm are freshly retrieved, the possibility that ovarian stimulation could occur without sperm for ICSI must be considered. Couples should be counseled before treatment regarding this possibility and the potential use of donor spermatozoa discussed.

Friedler et al. (10) also reported on the success of cryopreserved sperm from men with Klinefelter’s syndrome who underwent TESE. They found that cryopreserved sperm produced comparable results with freshly obtained sperm in these men. This observation is important for couples who desire multiple pregnancies with a male partner with Klinefelter syndrome who could be spared TESE procedures with every cycle.

We have not observed similar pregnancy success for couples using cryopreserved sperm despite the fact that the couples who underwent ICSI with cryopreserved sperm had previously achieved pregnancies using freshly retrieved spermatozoa. This observation may occur because we find rare spermatozoa in a proportion of men that are not treatable with other sperm retrieval approaches. These rare, weak sperm may not survive freeze-thaw as effectively. If nonviable sperm are present after freeze-thaw, it is worthwhile to have men followed up at least 6 months after prior testicular surgery to all adequate recovery before consideration of a repeat attempt at simultaneous fresh sperm retrieval (23).

The testicular pathology in Klinefelter syndrome is postulated to be progressive with the gradual disappearance of viable germ cells from the tubules with time (24). We do not have experience with sequential diagnostic biopsies in men with Klinefelter syndrome, and we have been able to repeat TESE successfully in the majority of patients. Our observation that the age of men who had successful sperm retrieval was no different from that of men who failed to have sperm identified with TESE suggests that the decline in spermatogenesis (if such decline actually exists) for men with Klinefelter syndrome may not be clinically relevant to their treatment. Young men with Klinefelter syndrome who have sperm in the ejaculate should certainly be counseled to cryopreserve semen samples for later ICSI. If a progressive decline in spermatogenesis occurs in Klinefelter syndrome, it is possible that such impairment of testicular function may be related to elevated estrogens and could be reversible with aromatase inhibition.

Despite very limited testicular volume, extensive tubular sclerosis, and markedly elevated FSH levels, sperm retrieval with microdissection TESE is possible for 72% of men with nonmosaic Klinefelter syndrome. High sperm retrieval rates were observed despite the presence of Sertoli cell-only pattern on diagnostic biopsy and previous failures of TESE using random multibiopsy approaches. The use of microdissection TESE for men with nonmosaic Klinefelter syndrome had a high sperm retrieval rate, comparable with that seen for other men with nonobstructive azoospermia. Once sperm were obtained, ICSI resulted in pregnancy in 46% of cycles.

	Footnotes

 
First Published Online August 30, 2005

Abbreviations: hCG, Human chorionic gonadotropin; ICSI, intracytoplasmic sperm injection; TESE, testicular sperm extraction.

Received November 29, 2004.

Accepted August 18, 2005.

	References

Top Abstract Introduction Patients and Methods Results Discussion References

 

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This Article Abstract Full Text (PDF) A correction has been published Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Schiff, J. D. Articles by Schlegel, P. N. Search for Related Content PubMed PubMed Citation Articles by Schiff, J. D. Articles by Schlegel, P. N. Related Collections Male Endocrinology