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 (本篇榮獲台灣婦產科醫學會九十四年度最優秀論文獎)

2016-06-01

本篇論文已於2004年台灣婦產科醫學雜誌上發表，並獲得最佳論文獎                                          

       據統計，600個出生的男嬰中，就有一位是47XXY染色體異常(正常為46XY)，是最常出現的染色體異常疾病。這個疾病造成的原因是，父母生殖細胞在形成卵子或精子時，因性染色體（X，Y）沒有分離造成。2/3是母親的因素，1/3是父親的因素。因為多了一個X染色體的干擾，造成睪丸發育不全，製造精子困難。

      47XXY染色體造成的無精症在1998年以前被認為是無法生育的。在1998年美國康乃爾大學在新英格蘭雜誌上發表47XXY無精症患者利用睪丸切片取精懷孕成功的案例，成為全世界成功的首例。本院赴美學習此技術後，在2000年也第一次對這種病人治療而懷孕成功，成為台灣成功的第一例 ( 全球第七例 )。此後全台陸續多名47XXY無精症患者來到健安不孕症-試管嬰兒中心治療，成績斐然。 部分的經驗與結果，已集成論文發表於2004年出版的台灣婦產科醫學雜誌期刊上（Taiwanese J Obstet Gynecol 2004; 43 (2): 89-96），並獲得最佳論文獎。  論文摘要如下：

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

目的：本研究是評估對47XXY無精症患者 (Klinefelter’s綜合徵患者) 做睪丸切片取精後，取得精蟲的可能率及利用此精蟲作單一精蟲卵細胞質注射技術加上胚胎植入所導致懷孕的結果。

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 林芷謙，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 : 

Patients description：During the period from May 1998 through December 2001 14 consecutive azoospermic patients, aged from 21-39 years old, diagnosed after cytogenetic evaluation of peripheral blood lymphocytes as suffering from azoospermia non-mosaic 47,XXY (Klinefelter's syndrome), who sought infertility treatment in our fertility clinic, were enrolled into this study.

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

    Testicular biopsy was done on the day before oocyte retrieval or the similar day of oocyte retrieval. The method of performing open testicular biopsies for TESE were same as that described by Tournaye et al.(13). Briefly, all the patients received spinal anaesthesia and after hemiscrototomy the scrotal contents were inspected and small multifocal biopsies rather than one large biopsy were taken. If intraoperative search for fresh tissues in the wet preparation identified motile spermatozoa, testicular sampling was stopped. If no spermatozoa were observed, more biopsies were taken until the whole testicular mass has been randomly sampled. A careful haemostasis was performed by means of unipolar cautery and both testicular and scrotal incisions were closed by interrupted resorbable sutures.

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. 

Table I : Physical and serum hormones characteristics of the 14 azoospermia patients with non-mosaic KS
Patient No.		1	2	3	4	5	6	7	8	9	10	11	12	13	14	Mean± SD
Wife age		31	29	39	30	21	27	28	26	27	25	35	29	26	34	29 ±4.58
Husband  age		37	30	38	36	29	34	35	31	29	28	39	32	30	39	33 ±3.97
	Body hight	176	169	ND	173	170	180	176	169	ND	172	ND	175	175	170	173 ±3.54
	Body weight	86	64	ND	62	76	92	106	55	ND	91	ND	54	78	78	7 7 ±16.59
	LH	32.7	19.75	ND	18.6	18	12.32	28.6	28.5	18.9	21.6	16.1	34.1	46.84	ND	24.67± 9.77
	FSH	15.6	46	ND	40.1	65.9	29.72	32.7	30.2	49.7	13.2	36.05	29.2	83.6	ND	39.33±19.99
	Prolactin	10.4	13.54	ND	20.6	19.8	9.85	7.31	11.4	11.4	13.2	7.29	19.7	13.32	ND	13.15± 4.63
	Testosterone	2.24	3.18	ND	0.91	0.51	2.95	1.32	1.58	2.01	1.58	2.08	1.78	0.46	ND	1.72 ±0.85
	Gynaecomastia	+	-	-	-	+	+	-	-	+	+	-	+	-	+
*	Previous testis biopsy	+	+	-	-	-	-	-	-	-	+	+	+	-	+
	Orchepexy	-	-	-	-	-	-	-	-	-	+	-	+	-	-
	Sperm found by ESP	+	-	-	-	-	-	-	-	-	-	-	-	-	-
	Sperm found by TESE	+	-	+	+	+	+	-	+	+	+	-	-	-	-
	Clinical pregnancy	quads	-	Single	twins	-	-	-	quads	Single	triplets	-	-	-	-
	Fetal reduction	+	-	-	-	-	-	-	+	-	+	-	-	-	-
	Outcome of clinical pregnancy	ongoing twin preg.	-	Blight  ovum	ongoing twin preg.	-	-	-	1 boy 1 girl deliver	1 boy deliver	1 boy 1 girl deliver	-	-	-	-
*  It means that these patients had received testis biopsy previously in other medical center.

　

Conclusion :Almost all patients in our studied series had an elevated serum FSH level, low or normal level of serum testosterone and atrophic testis. Half of patients had mild gynecomastia. The clinical and hormonal pictures of our patients are similar to the characteristics of patients with non-mosaic KS reported by other groups (13, 15, 16, 17, 18, 19, 20, 21, 22).

Before TESE-ICSI, we carefully searched the presence of sperm in their ejaculate by ESP method (23) for azoospermic patients with non-mosaic KS in order to use ejaculated sperm as a first line treatment and it might avoid testicular biopsy for TESE. Both Ron-EI and Schlegel (29) could found spermatozoa in 35% and 20% of patients, respectively, with non-obstructive azoospermia by ESP and these spermatozoa could be used for ICSI, then, these patient could be avoid testicular biopsy. In Friedler's series included 12 patients with non-mosaic KS, two patients had dead spermatozoa in their ejaculate, but they did not point out whether these two patients from whom had spermatozoa been found in their ejaculate had spermatozoa been recovered or not by TESE (21). In both Reubinoffs and Tournaye's series, 4 out of 7 patients with non-mosaic KS had spermatozoa been found in their semen by ESP or scanning electron microscopy and three of these four patients from whom spermatozoa could be recovered by TESE (13, 17). In our series, one patient had a history of presence of dead spermatozoa in his ejaculate prepared by ESP and, then, from his biopsied testicular samples, motile spermatozoa could be retrieved and used for ICSI. To judge the value of past demonstration of spermatozoa in the ejaculate as a predictor of success of TEST, more data from a large series are needed.

In our 14 patients with non-mosaic KS, motile spermatozoa could be recovered by TESE from their biopsied testicular tissues. The retrieval rate was 57% (21). According the literature's survey by Frielder's, the average success rate of TESE in non-mosaic KS was 56%. Schlegel reported that they had 68% retrieval rate by microdissection method. The successful rate reported by Schlegel was the highest one in the literature (29).

In eight patients in whom motile spermatozoa were recovered, two patients had received testis biopsy previously for histological examination and searching the presence of spermatozoa in their biopsied tissues. One of these two patients also received orcheopexy for his undescending testis and testis biopsy simultaneously. The histological findings of testis sample of both patients showed no evidence of spermatogenesis and this finding implied that previous negative histological finding did not precluded the success of TESE in next attempt for patients with non-mosaic KS. In our series, six out of eight patients who did not received testis biopsy previously had spermatozoa been recovered in this attempt (6/8) compared to that only two out of six patients who had received testis biopsy previously had spermatozoa could be found (2/6). The difference between the two groups implied that previous testis biopsy might compromise the tiny germinal epithelium existed in the testis of patient with non-mosaic KS. This point was first time mentioned by us, as far as we knew. However, that previous attempt might decrease the successful rate of TESE in next time may needs more data from a larger series to verify it.

That the serum FSH level of the patients who had sperm recovered was ranged from 13.2 to 65.9 mIU/ml told us that high serum FSH is not a predictor of failed spermatozoa retrieval for the patient with non-mosaic KS. This point was also agreed by other authors (13, 16, 21).

Our fertilization and embryo cleavage rate (59% and 96% respectively) is similar to the results reported by Friedler et al. who achieved 66% fertilization rate and 98% embryo cleavage rate in their series (21). The spermatozoa retrieved from patients with non-mosaic KS also could induce same fertilization and embryo cleavage rate as spermatozoa retrieved from the non-obstructive azoospermia caused by other etiology (60).

To the couples in which men with non-mosaic KS, the journey of treating their infertility to get a baby was arduous and full of tear and blood, therefore, they seized any opportunity to get pregnancy. Although they was informed the high risk of multiple pregnancy, which could cause premature birth and subsequently increase the baby's morbidity and mortality, some of them still asked for being transfered more than three embryo to increase the chance of getting pregnancy. Totally, 54 embryos were transfered in fresh cycle and resulted in 16 gestational sac with visible beat (implantation rate 29%) and 6 clinical pregnancy (clinical pregnancy rate 75%). Among the six clinical pregnancies, four were multiple pregnancies beyond two (one was triplets and two were quads). All of the wives who had multiple pregnancy beyond two received fetal reduction to reduce their fetal number to two and all of them stood the operation well eventually.

That we could achieved high implantation (29%) and high clinical pregnancy rate (75%) might be due to three reasons. The first reason is that the wives of the patients in our series were young (mean 29±4.58) and no wives had gynecological disease associated with them, which could affect her reproductive ability, but one who had been suffered from endometriosis for many years. The second reason is that we forcefully and carefully aspirated and cleaned the mucus presented in the uterine cervix just before performing embryo transfer through uterine cervix. The third reason is that we performed embryo transfer under full bladder abdominal ultrasound guidance. Silber stated that neither the pathology, the source, the quantity, nor the quality of spermatozoa had an effect on fertilization or pregnancy rate and that could dramatic affect the embryo implantation, pregnancy and delivery rate was maternal age (30). In his report with 63 cases with non-obstructive azoospermia, these was a live delivery rate of 40% per ICSI-ET cycle when female age was below 37 years compared to live delivery rate of 11% when female age was beyond 37 years. In our series, we could get live delivery rate of 62.5% if two ongoing pregnancies were included. One woman who achieved clinical pregnancy by TESE-ICSI, in our series, suffered from abortion (blighted ovum) at 10 weeks of gestational age eventually and this event might be imputed to her higher age (39 y/o). Many authors mentioned that the detrimental effect of the cervical mucus presented in the uterine cervix during performing transcervial embryo transfer on embryo implantation rate (31, 32, 33, 34, 35). Mucus plugging of the catheter tip could be a cause of retained embryo, damage to the embryo (especially with assisted hatching) and improper embryo placement. Cervical mucus adhered to catheter tip was a carrier to bring bacteria into endometrium cavity, which caused the clinical pregnancy rate per ET cycle from 57% down to 29.6%, reported by Egbase (33). The technique of abdominal ultrasonography-guided embryo transfer under full bladder is getting popular because it let transfer the embryo atramatically and place the embryo in endometrium at optimal place. The advantage of using full bladder abdominal ultrasound guided embryo transfer has been appreciated by many authors (36, 37, 38).

The major concern regarding using the testicular spermatozoa from patient with KS for ICSI is the potential genetic risk in their offspring. Various studies of chromosomal abnormalities in ejaculated spermatozoa from patients with non-mosaic KS showed increased rate of sperm nuclei abnormalities, which varied from 3.7~9.8% (39, 40, 41, 42, 43, 44). The rate of sperm nuclei abnormalities in ejaculated spermatozoa from patient with non-mosaic KS varied from 7.75% to 23.63% (45, 46). While sex chromosomal hyperploidy was found at an incidence of 0.9-2.5% in the mosaic form (39, 40, 41, 42, 43, 44), it incidence in the non-mosaic form varied from 2.52~21.76% (45, 46, 47). The rate of abnormal sperm nuclei and sex chromosomal hyperploidy in normal population is around 0.6% and 0.4% respectively (47, 48). Since both successful pregnancies and birth by ICSI using testicular spermatozoa obtained from azoospermia patients with KS, several studies regarding the aneuploidy rate of these testicular spermatozoa had been published. They pointed out that the aneuploidy rate of retrieved spermatozoa from the testicular tissues of the patients with KS varied from 6.3~7.5% and that the rate of sex chromosomal hyperploidy also increased, too (49, 50, 51). Both the rates of abnormal sperm nuclei and sex chromosomal hyperploidy increased in patients with KS compared to that in normal population. However, all the babies had been delivered till now by ICSI using testicular spermatozoa from patient with non-mosaic KS, so far as we knew, were normal and healthy including our series. But, one is an exception which reported by Ron-EI; in his report, one woman whose husband with non-mosaic KS achieved triplets pregnancies using TESE-ICSI and karyotype analysis from chorionic villous sampling revealed a non-mosaic 47,XXY in one of three fetuses during 10th gestational week. This fetus was reduced on 14th gestational week finally (52). Although preimplantation genetic diagnosis had been recommended and may be indicated, but not all fertility center performing assisted reproductive technology have this facilities and technique. Furthermore, it has the several limitation of aneuploidy detection by this experimental technique. Actually, Munne' et al. reported an error rate of 5.4% for PGD by FISH of the common aneuploidies (53, 54). Therefore, at present, PGD cannot substitutes invasive prenatal diagnosis including chorionic villous sampling or aminocentesis for fetal karyotyping (17). In our series, we had recommended these couples which achieved pregnancy to receive aminocentesis for chromosomal karyotyping of their fetuses, but all of them refused to take prenatal diagnosis.  They were willing to take the risk of potential genetic anomalies of their fetus and did not take any action in case an abnormal sex chromosomal constitution was found in the fetus. Therefore, we monitored these fetuses by careful ultrasound and all these babies delivered in our series were normal and healthy eventually.

The spermatozoa produced by patient with non-mosaic KS is the consequence of meiotic segregation in 47,XXY germ cell or 46,XY germ cell still is a debated issue now. Some authors concluded that 47,XXY germ cell might enter and complete meiosis based on indirect evidence, which was the presence of 24XY or 24XX spermatozoa in ejaculate potentially issued from 47,XXY spermatozoa (39, 40, 45), and on the basis of distorted sex ratio of spermatozoa examined (46, 55). On the other hand, some authors concluded that only 46,XY spermatogonia could enter and complete meiosis based on observing that only 46,XY pachytene primary spermatocyte was found in testis cell obtained from patients with non-mosaic KS by histology, electron microscopy or FISH (19, 20, 57). The latter authors thought the 46,XY cell certainly existed in the testis of patients with non-mosaic KS who had sperm been recovered by TESE or found in ejaculate although peripheral blood lymphocyte karyotype revealed pure type of 47,XXY (germinal mosaicisms concept). Hunt et al. and Morz et al. discovered that the two X reactivation occurred during the period of testis formation which occurred in early fetal age. That two X reactivation in the presence of SRY gene expression in pre-Sertoli cell, which took place in the period of testis formation could cause loss of mitotic primodian XXY spermatogonia by studying murine Klinefelter model (58, 59).  They stated that exceptional germ cell survived in the postnatal XXY testis were resulted from rare nondisjunction events of proliferating XXY germ cells, and this events gave rise to clones of XY cells and concluded that exclusively all the spermatozoa found in XXY non-mosaic was yielded by XY spermatogonia. Now that spermatozoa obtained from patients with 47,XXY is exclusively yielding by 46,XY spermatogonia, why both the rate of abnormal sperm nuclei and sex chromosomal hyperploidy are increased in these patients. Morz et al. thought that was the result of compromized testicular environment which took place in heterogenetic etiology causing azoospermia or severe oligospermia including KS (7).

In conclusion, to our best knowledge, this is the largest series to study the feasibility and the outcome of performing TESE/ICSI for patients with non-mosaic KS in Taiwan. Our result pointed out that testicular spermatozoa could be retrieved form patients with non-mosaic KS and by means of ICSI technique, the spermatozoa could induce normal fertilization, normal embryo cleavage and birth of a healthy baby. Furthermore, our outcome encourages and reassures physician and their patients with KS to purse the use of their own sperm which being treated infertility. Although, so far as we know by literature's survey and our own findings that the babies yielded by testicular spermatozoa obtained from patients with non-mosaic KS were favorable, it is still needed to do detailed counselling regarding the potential genetic risk of the babies yielded by their own sperm.

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