本篇論文已於2004年台灣婦產科醫學雜誌上發表

對14位非嵌合型47XXY無精症患者（Klinefelter's綜合徵）進行睪丸切片取精並將取得的精蟲做單一精蟲卵細胞質注射的結果

方法：共有14位經血液染色體檢查為非嵌合型(純型) 47XXY男性不孕患者接受睪丸切片取精。這14位患者的配偶同時接受人工合成或尿液提煉的腦下垂體賀爾蒙注射，以刺激多粒卵泡成熟.。若於男性患者的睪丸組織中找到精蟲時，配偶也同時施行取卵手術。藉單一精蟲卵細胞質顯微注射的技術，將取得的精子注入卵子內，16小時後即可觀察精卵受精的情形。將有發育成胚胎的正常受精卵植入子宮內後，持續追蹤是否懷孕、懷孕情形及產下的胎兒是否正常。

結果：14位47XXY患者，其中8位在他們的睪丸組隻中可發現精蟲 (精蟲取得率為５７%)。這８位男性患者的太太都接受取卵手術與胚胎植入，有６位懷孕 (懷孕率為７５%) 。這６位有臨床懷孕的太太，其中三位目前都已成功生下健康的寶寶 (一個單胞胎，兩對雙胞胎)，另外兩位太太尚在懷孕中 (均為雙胞胎)，另外一位太太則不幸在懷孕三個月後流產。

結論：睪丸切片取精加上單一精蟲卵細胞質注射技術可為４７XXY不孕症患者帶來為人父的希望。本院睪丸切片取精找到精蟲的機率為５７%。綜合世界各國的文獻報告，睪丸取精的找到精蟲的機率也約為５６%。此外，本院能獲得這們高的臨床懷孕率(75%) 主要是因為患者的配偶平均年齡輕，且沒有其他不孕的問題。雖然截至目前為止，包括我們的病例，出生的小孩都是健康且染色體正常。但對於４７XXY患者經由這種生殖科技所孕育出的小孩，出現染色體異常的可能性還是不能忽略。一般可在懷孕初期的產檢時，以絨毛膜取樣或抽取羊水進行胎兒的染色體檢查，即可篩檢出正常與否。

OUTCOME OF INTRACYTOPLASMIC INJECTION OF SPERM OBTAINED BY TESTICULAR SPERM EXTRACTION FROM 14 MEN WITH 47XXY NONMOSAIC KLINEFELTER'S SYNDROME

  ¹ 江俊明， ¹ 林娟如，² 李良明

¹ Chun-Ming Chiang ,  ¹ Chuan-Ju Lin ,  ² Liang-Ming Lii

¹ 健安婦產科不孕症中心， ² 萬芳醫院泌尿科

¹ Jian-An OB/GYN Clinic & Fertility Center

² Department of Urology, Taipei Municipal Wann-Fang Hospital

Purpose: Our study was to evaluate the possibility of testicular sperm extraction (TESE) for the patients with non-mosaic Klinefelter's syndrome (KS) and the outcome of intracytoplasmic sperm injection (ICSI) using the extracted testicular sperm. 

Materials and Methods: 14 couples with primary infertility in which the men had secretory azoospermia and nonmosaic 47,XXY karyotype were included into this study. All the women in this study had no serious gynecological abnormalities. Open testis biopsies for sperm extraction were done one day before oocytes retrieval or simultaneously with oocytes retrieval. Sperm extracted from the biopsied tissues in wet preparation were used for ICSI. Outcome of ICSI using testicular spermatozoa were evaluated by fertilization rate, embryo cleavage rate, clinical pregnancy, ongoing pregnancy and birth of normal healthy baby. Before performing TESE - ICSI, all of these infertility couples were informed consent regarding the genetic risk of their potential offspring achieved by ICSI using testicular spermatozoa extracted from the husbands' testis. 

Results: Physical examination of the 14 patients included in this series revealed varying clinical features. TESE was performed in all of the 14 patients with non-mosaic KS and resulted in retrieval of motile testicular spermatozoa in 8 cases (57%). ICSI using retrieved spermatozoa were performed for all the couples in which men had sperm extracted from their biopsied testicular tissues. The retrieved spermatozoa could induce normal fertilization and embryo development. All 8 wives in whom their husbands had spermatozoa been retrieved could undergo embryo transfer and 6 clinical pregnancies were achieved (75%). Outcome of the six pregnancies included birth of 5 healthy babies (1 singleton, 2 twins; 3 male and 2 female), 2 ongoing twins pregnancies (both gestational age beyond 5 months) and one blighted ovum at time this writing. 

Conclusion: The advent of TESE - ICSI had offered an opportunity for azoospermic KS patients to father their own genetic offspring. To our knowledge, this is the largest series reported to study the outcome of performing TESE - ICSI for patients with non-mosaic KS in Taiwan. According to our literature survey, we got same success rate of TESE (57%) comparing with other fertility centers in the world (56%). The testicular spermatozoa retrieved in non-mosaic KS had same ability to fertilize eggs and yielded normal embryo development comparing with spermatozoa obtained from normal man's ejaculate. The high pregnancy rate per embryo transfer (75%), achieved by these couples in which men has sperm been extracted, might be due to younger age and no serious gynecological disorder of their female partners. So far as we know, based on literature's review, the babies achieved after TESE - ICSI in the couples in which men with non-mosaic KS were normal karyotype and healthy. Because that there were increased abnormalities of sperm nuclei obtained from men with KS had been found by several researchers, preimplantation genetic diagnosis or prenatal diagnosis should be recommended to these couples in which men with KS who underwent TESE - ICSI. Although the potential risk of chromosomal abnormalities in their babies were informed before performing TESE - ICSI, all the couples with ongoing pregnancies in our series refused aminocentesis; they underwent careful ultrasound follow up and finally they got healthy babies. That the spermatozoa retrieved from the men with non-mosaic KS is the consequence of meiotic segregation in 47 XXY germ cell or 46XY germ cell is still debated now.

Introduction :Klinefelter's syndrome, initially described by Harry Klinefelter in 1942, is characterized by hypogonadism, small testis, azoospermia or oligospermia and gynaecomastia (1). In 1959, Jacobs and Strong found these patients had an additional X chromosome, total two X chromosome (2), explained by meiotic non-dysjunction during gamate meiosis (3). 90% affected patients is non-mosaic form (47,XXY chromosomal constitution) and 10% is mosaic form (46,XY / 47,XXY chromosomal constitution) (3).

Klinefelter's syndrome is the most frequent sex chromosomal abnormality, occurring in 0.1% general phenotypic male population (4). 3.1% in the infertile male population (5) and 11% in azoospermic men population (6). Klinefelter's syndrome with mosaic form sometimes presents with severe oligospermia and can father their own genetic offspring by combining oocyte retrieval / intracytoplasmic injection and embryo transfer technique. In contrast, men with non-mosaic KS usually are azoospermic and have been considered sterile. That the extra X chromosome causes germ cell loss during testicular development is seen in all male mammals including human being (7), so that virtually no sperm cells are present in the adult XXY testis (8, 9, 10). Nevertheless, rare small patches of spermatogenesis were found in the testis of non-mosaic 47,XXY males by Steinberger and Skakkebaek (11, 12) and occasionally, spermatozoa are present in the ejaculate (8, 9, 13).

Since pregnancy was achieved by using testicular sperm extraction (TESE) combined with intracytoplasmic injection (ICSI) for patients with abnormal seminiferous tubular function (14), many attempts were preformed for patients with non-mosaic azoospermic KS using TESE-ICSI technique, which offers these patients the opportunity to bear their own genetic offspring. According to our survey from the literature, at present, about 23 deliveries have been reported following ICSI using testicular spermatozoa extracted from testis of patient with azoospermia non-mosaic KS (15, 16, 17, 18, 19, 20, 21, 22).

The aim of this study was to evaluate the feasibility and outcome of ICSI using testicular spermatozoa surgically retrieved from azoospermic patients, in our series, with non-mosaic KS.

Material and Method : 

All of these patients underwent complete history taking and physical examination on initial interview. Serum hormone analysis for LH, FSH, testosterone and prolactin were done on initial interview, too. All patients before undergoing testis biopsy for TESE needed to have received three times examination for ejaculate using the method named as extensive sperm preparation (ESP), described by Ron-EI, to search living spermatozoa, which occasionally could be found and used for ICSI (23).

Before undergoing TESE-ICSI, all patients were informed consent regarding the potentially genetic risks of their offspring yielded by testicular spermatozoa extracted from their testis. 

Testis biopsy, testicular sperm retrieval and preparation

The biopsied testicular samples were placed in a petric dish with pre-warmed HEPES-HTF / 3% SSS(Synthetic serum substitute) medium. Initially, the testicular samples were dispersed and stretched to isolate the individual seminiferous tubules by means of sterile glass slides. Subsequently, mechanical dispersal of the tubules was accomplished by mincing the extended tubules with a sterile scissors in pre-warmed HEPES-HTF / 3% SSS. Intraoperatively, a wet preparation of the suspension was observed under inverted microscopy at 200X and 400X power to identify spermatozoa. The testicular tissues were then processed to sperm retrieval as following. The dispersed and minced testicular tissues were mixed gently with pre-warmed HEPES-HTF / 3% SSS medium placed into Falcon 3002 tissue culture dish. The tissue suspension was centrifuged at 500g for 5 minutes after one hour incubation in 37℃, 5%CO₂ . The supernatant was further centrifuged up to 1800g for 5 minutes and the pellets were observed for the presence of spermatozoa. If the spermatozoa was observed, the pellet was resuspended in 100ml of HEPES-HTF / 3% SSS and incubated for ICSI use. If no spermatozoa was observed, the process for sperm retrieval was continued by adding erythrocyte buffer medium, which can induce erythrocyte lysis, or adding collangenase / DNase, which can digest the seminiferous tubular wall, into minced testis tissues which has been premixed with HEPES-HTF / 3% SSS in order to increased the chance to find spermatozoa (24, 25). When testicular spermatozoa were found enabling ICSI, excess tissues were cryopreserved and one small piece was sent to histological examination. 

Protocol of ovarian stimulation, oocyte retrieval and ICSI

For stimulating multiple oocytes growth for the female partners whose husband with non-mosaic KS were undertaken using ultra-short or long nasal spray ovarian stimulation protocol. In ultra-short protocol, the female partners received buserelin acetate (Hoechst Marion Rousell, Germany) 0.5mg injection subcutaneously from day 2 to day 4 of the menstrual cycle. Both human menopausal gonadotropin (HMG) and human urinary FSH (Follicular stimulating hormone) or recombinant FSH were administrated from day 3. The beginning dose of human gonadotropin given was depended on the serum LH, FSH level and follicular number of both ovaries on vaginal ultrasound finding on day 2 of the menstrual cycle. After day 9, the dose of gonadotropin administrated was adjusted according to the serum E2 level and follicular size. When three leading follicular sizes reached 18 mm and serum E2 level corresponded with 100-200pg/ml per follicle, HCG (Human Chorionic Gonadotropin) 10000IU was injected for final maturation of the oocytes.

In the long protocol, buserelin acetate (Suprecur, Hochst Marion Roussel, Germany) nasal spray 1200mcg per day, which was divided to four doses, commenced in the luteal phase of the cycle preceding to the IVF cycle. On the day 2 of menstrual cycle, serum E2 was measured to ensure that pituitary desensitization was achieved. When serum E2 level was below 30 pg/ml, the dose of buserelin acetate was switched to 600mcg/day and both HMG and urinary FSH or recombinant FSH were given to stimulate multiple oocytes growth. The criteria for administrating initial dose of HMG and FSH, adjustment of the dose after day 9 and the time of HCG injection were similar to ultra-short protocol.

Ultrasound guided transvaginal oocytes retrieval was performed in 34-36 hours after the administration of HCG under spinal anesthesia. ICSI was performed according to the procedure designed by Dr. Van Steirteghem et al.(26). 

Embryo transfer, luteal support and embryo cryopreservation

The state of fertilization of oocytes was assessed 16-20 hours after the injection by looking for the presence of pronuclei. Fertilization was achieved normal when two pronuclei were present. Grading criteria of embryo quality was according to the standard described by Lucinda L. Veek (27).

Embryos with best quality were transferred through uterine cervix on day 3 after oocyte retrieval under full bladder abdominal ultrasound guidance using Labotech or Wallace embryo transfer catheter. Sometimes, we transferred more than four best quality embryos (if available) but not beyond six embryos, after being asked for by some couples who were afraid that the quality of frozen embryo was in inferior to the fresh embryo and transfer in frozen cycle might decrease the pregnancy rate. Of course, all couples in which women transferred more than four embryos were informed the possibility of getting multiple pregnancies. All of them consented to fetal reduction if multiple pregnancies more than triplets were achieved after TESE-ICSI. Surplus embryos were frozen using slow cooling method (28). For luteal support, micronized progesterone (Utrogestan, Piette, Belgium) 200 mg tid/day were administrated into vagina from the day of oocyte retrieval and continued for the following 16 days, then serum-bHCG were measured to assessment of a pregnancy. All pregnant women received an additional dose of micronized progesterone 600mg/day until 10 gestational weeks were reached. Clinical pregnancy means sonographic demonstration of a gestational sac with visible fetal beat.

Results :Physical and serum hormones characteristics of the azoospermic patient with non-mosaic KS in our series are presented in table I. The mean age of the male patients is 33±3.97 (range 28-39 years). The mean age of the female partners is 29±4.58 (range 21-39 years). The mean serum FSH (Follicular stimulating hormone) level of male patients was elevated, which ranged from 13.2 to 83.6 mIU/ml (mean 39.33±19.99). Their serum testosterone level, which ranged from 0.46 to 3.18 ng/ml (mean 1.72±0.85), was below normal or near normal. Half of our male patients had mild gynecomastia. All patients with non-mosaic KS in our series had small testicles of 2-4 ml in volume. Six of them had received testis biopsy before this attempts for histological examination and searching the presence of spermatozoa in biopsied tissues in other medical centers; two among these six patients had an undescending testis and received orcheopexy and testis biopsy simultaneously. During the period of this study, we had two patients with non-mosaic KS from whom sperm could be found in their ejaculate by ESP method. Sperm found from one of these two patients was dead and could not be used for ICSI but another could avoid testis biopsy because motile sperms were found in their ejaculate by ESP method, therefore, this case was not enrolled into this series.

Mature motile spermatozoa were found in the wet preparation of biopsied testicular tissues from eight patients in our studied series. The retrieved rate was 57%. Two patients who had spermatozoa retrieved had received testis biopsy before this attempt; one of these two patients had received orcheopexy simultaneously in addition to testis biopsy. In all of these six patients, no spermatozoa were found in previous biopsied testis tissues, which was described by patients themselves. In one patient who had a dead spermatozoa been found in ejaculate by extensive sperm preparation, spermatozoa also could be retrieved by TESE from his biopsied testicular sample. All patients in this series, histological findings of testicular tissues biopsied showed tubal hyalinization, no evidence of spermatogenesis, and relative hyperplasia of the Legding cells.

Totally, 118 mature oocytes retrieved from the female partners whose husbands had spermatozoa been found in biopsied testicular samples were injected (ICSI) and induced 70 pronuclei; the fertilization rate was 59%. 67 pronuclei changed into cleaved embryos (cleavage rate 96%). 62 cleaved embryos were transferred and resulted in 29 gestational sac with visible beat (implantation rate 29%). All 8 wives whose husbands had spermatozoa been retrieved could undergo embryo transfer and achieved 6 clinical pregnancies (clinical pregnancy rate 75%) including 2 singleton, one twin, one triplets and two quads. Multiple pregnancies more than two were reduced to twins at 10th gestational age after informed the risk of premature birth accompanied with multiple gestation. Outcome of the six clinical pregnancies included birth of five healthy babies (1 singleton and 2 twins; 3 male and 2 female), 2 ongoing twins pregnancies (both gestational age were beyond 5 gestational months) and one blighted ovum at time this writing.