Sperm retrieval by microdissection testicular sperm extraction and intracytoplasmic sperm injection outcomes in nonobstructive azoospermic patients with Klinefelter syndrome

Klinefelter syndrome is the most frequent chromosomal abnormality in patients with nonobstructive azoospermia. The development of advanced assisted reproductive techniques, such as testicular sperm extraction and intracytoplasmic sperm injection, has provided the possibility of biological fathering in nonobstructive azoospermic patients with Klinefelter syndrome. We aimed to evaluate our sperm retrieval rate by microdissection testicular sperm extraction and to analyse the intracytoplasmic sperm injection outcomes in these patients. Medical records of 110 nonobstructive azoospermic patients with Klinefelter syndrome were retrospectively reviewed. We found that the sperm retrieval rate by microdissection testicular sperm extraction is lower than published reports on other types of secretory azoospermia. The statistical analyses yielded that age, FSH and testosterone levels as predictive factors for successful sperm retrieval.     

显微切割睾丸活检在非梗阻性无精子症中的应用

一直以来,学者们认为非梗阻性无精子症因睾丸生精功能受损,导致精液中无精子,而无法生育自己的后代。但随着卵胞质内单精子注射技术的问世,近十几年来涌现出多种睾丸取精术(包括开放性睾丸活检、细针穿刺抽吸、显微切割睾丸活检等)。之后,大量研究表明非梗阻性无精子症患者睾丸中仍存有局部的生精灶,即使是Klinefelter综合征,也可成功取出精子。2010年欧洲泌尿外科学会(EAU)指南明确推荐非梗阻性无精子症采用开放性睾丸活检或显微切割睾丸活检取精。与开放性睾丸活检相比,显微切割睾丸活检的取精成功率高且并发症少,本文就其取精前预测指标、手术操作方法、取精成功率及术后并发症进行综述。     

男性不育症中染色体特殊核型

在男性不育症的遗传咨询门诊中，发现的染色体异常绝大多数为典型的４７，ＸＸＹＫｌｉｎｅｆｅｌｔｅｒ综合征，但我们也发现一些较为特殊的核型，其中多Ｘ及多Ｘ嵌合体的３例，Ｙ染色体结构异常及Ｙ染色体与常染色体易位２例；常染色体之间的平衡易位６例。本文讨论了染色体的异常与男性不育症之间的可能关系。     

Chromosomal abnormalities in 2 cases of testicular failure

This study investigated the underlying chromosomal abnormalities of testicular failure using molecular cytogenetic analysis. We report 2 cases of rare genetic anomalies that resulted in hypogonadism. The first patient presented with severe hypogonadism. Chromosome analysis revealed a mosaic 46,X,r(Y) (p11.3q11.23)/45,X karyotype, with a ring Y chromosome. A Y chromosome microdeletion assay showed a deletion in the azoospermia factor a region. The second patient presented with infertility and nonobstructive azoospermia. Cytogenetic and fluorescent in situ hybridization analysis revealed a 47,XY,+mar.ish i(15) (D15Z1++,SNRPN2,PML2) karyotype, with a small supernumerary chromosome derived from chromosome 15. These results emphasize the need for molecular cytogenetic evaluation in patients with testicular failure before using advanced reproductive techniques.     

Klinefelter综合征患者卵细胞胞质内单精子注射治疗和子代患染色体病的风险

Klinefelter综合征患者以47,XXY核型,睾丸精曲小管发育不良,无精子症和不育为特征。但极少数患者仍有 局部生精灶和少量精子发生。近年来由于辅助生育技术的发展,Klinefelter综合征患者经睾丸活检取精子和卵细胞 胞质内单精子注射(ICSI)治疗后已有30多个健康胎儿出生,但也有1例报告妊娠胚胎的核型为47,XXY而选择流 产。本文对近年来Klinefelter综合征患者的ICSI治疗的现状及子代患染色体病风险进行综述。     

Fertilit?t bei Patienten mit einem Klinefelter-Syndrom (47,XXY)

In the past 10 years, our knowledge about fertility chances of patients with Klinefelter syndrome (KS, 47,XXY) has changed considerably, especially when regarding the possibility of IVF ICSI treatment (in vitro fertilisation, intracytoplasmic sperm injection) with single testicular spermatozoa. Thus, it is important to take this knowledge into consideration when counselling Klinefelter patients.Germ cell degeneration in the testicles of Klinefelter patients due to their additional X chromosome is an important phenomenon in this disease which is not yet fully understood. When entering puberty, the testicular volume of KS patients increases for a short time with rising testosterone and inhibin B levels at the same time. These decrease, however, and FSH increases during puberty. This seems to indicate a critical point in time when spermatogenetic function of the testicles could still be existent. Thus, in early puberty there could possibly be a time slot when spermatozoa could be detected in the ejaculate or-if not-at least in the testicular tissue. These could be extracted by testicular sperm extraction, cryopreserved and used for intracytoplasmic sperm injection therapy later on. In the literature, a total of 133 births of children from Klinefelter fathers have been reported. This early specific procedure could lead to a better acceptance of their diagnosis and also offer the option of not being incurably infertile.     

Y-autosome translocation associated with male infertility: a case report

A 40-year-old man was referred to our hospital with a 12-year history of infertility. He was a well developed male weighing 78 kg with a height of 171 cm. Physical examinations revealed male habitus with normal adult pubic and axillary hair. The penis, epididymides, spermatic cords and prostate were normal. The right testis was about 15 ml in volume and left ne was approximately 12 ml, respectively. Repeated semen analyses showed azoospermia except for only one time when 4 immotile sperm were detected. The plasma levels of lactate hydrogenase, follicle stimulating hormone prolactin and testosterone were within normal limits. Chromosome analysis of peripheral lymphocytes revealed a balanced reciprocal translocation between the short arm of chromosome 12 and the long arm of the Y chromosome (46, X, t (Y ; 12) (q12 ; p13.3)). We performed microdissection testicular sperm extraction and retrieved 11 spermatozoa (10 progressive motile). Seminiferous epithelium showed maturation arrest at the stage of spermatid. Mean Johnsen's score count was 6. The etiology and clinical features of this rare disease were briefly discussed.     

A case report of successful testicular sperm extraction at 31 years and 41 years of age in a man with klinefelter syndrome

We report a case of Klinefelter syndrome with the chief complaint of male infertility. Normal pregnancy and delivery resulting from conventional testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI) was possible by a previous doctor when he was 31 years old. He was referred to our hospital for treatment of infertility when he was 41 years old. Abdominal ultrasonography demonstrated a low echoic region with a relatively uniform content in the left testis and a central low echoic small mass surrounded by a high echoic region in the right testis. In excised tissue of the left testis, only a single seminiferous tubule containing spermatozoa was found and sperm was successfully retrieved by microdissection TESE.     

A rare variant Klinefelter syndrome seen 40 years later: 47,X,del(Xq24),Y

Patients with Klinefelter syndrome (KS) show a typically 47,XXY karyotype; however, some variations have been observed, including 47,XX,der(Y), 46,XY/47,XXY, 48,XXXY, 48,XXYY, and mosaicism or structural sex chromosome abnormalities in some patients. In the literature, a rare KS variant, 47,X,del(Xq),Y karyotype, was reported in only a few cases prior to 1981. A 40-year-old man (IV-3) was referred to our department due to infertility. His phenotype did not differ from the classic KS phenotype. He had two siblings (1-male; 1-female). His brother (IV-5) had mental retardation and died one year earlier at age 32. Additionally, his sister (IV-2) also had a history of infertility due to her husband's azoospermia. His mother had a history of 12 miscarriages. Karyotype analysis revealed the 47,X,del(Xq24),Y karyotype, and no deletions were seen in the AZF and SRY regions. We thought this chromosomal abnormality in the patient might have resulted from X-autosome translocation in one of his parents since his mother had recurrent pregnancy loss and his sibling had mental retardation. However, we could not confirm it due to his parents were not alive. This study shows the first case of a long-arm X-chromosome deletion after a long period and reviews current knowledge concerning variant KS (deletion Xq).     

Genetic investigations on causes of male infertility in Western Saudi Arabia

In recent years, genetic studies have yielded great progress in elucidating causes of male infertility. This investigation aims to identify frequent genetic abnormalities, that is, sex chromosome aneuploidies and Y‐chromosome microdeletions among infertile men in Western Saudi Arabia. From a population of infertile patients, 88 male patients with either azoospermia or severe oligozoospermia (sperm concentration <5 million/ml) were selected. In addition to a thorough clinical workup, karyotypes and Y‐chromosomal microdeletions were investigated. Among those 88 infertile patients, we detected six patients with Klinefelter syndrome, two with 47 XYY syndrome and two with Y‐chromosome microdeletions AZFb,c. While the prevalence of sex chromosome aneuploidies was in the range of globally investigated populations, the microdeletions appeared to be less frequent in Western Saudi Arabia compared to other regions of the world. All genetically abnormal cases showed sperm concentration <1 million/ml, and hence, this appears to be the threshold for warranting genetic investigations in Western Saudi Arabia. Since Klinefelter and 47 XYY syndromes were only discovered late in life, upon an infertility investigation, sex chromosome aneuploidies due to their many‐fold comorbidities require earlier medical attention. A neonatal screening programme is suggested for detection of these aneuploidies in Saudi Arabia for the general health benefit of these patients.     

Genetic causes and management of male infertility

Infertility affects approximately 15% of couples. With infertility such a common problem in a generally healthy age group, complete evaluation is needed of both men and women. Infertility work up for men includes a semen analysis, the results of which suggest various supplemental studies, including karyotype. Karyotype is indicated when a patient has findings on history or physical exam concerning for chromosomal abnormalities, azoospermia, or severe oligospermia (count <5 million/mL). The most common chromosomal numerical abnormality found on karyotype is Klinefelter syndrome which is classified as redundant sex chromosomes, with the most common chromosomal arrangement being 47, XXY. If a patient is found to have a chromosomal abnormality such as Klinefelter’s, there is still a chance of fertility using testicular sperm extraction and in-vitro fertilization.     

Klinefelter syndrome and fertility—Impact of X‐chromosomal inheritance on spermatogenesis

With the use of testicular sperm extraction (TESE), spermatozoa can be retrieved in about 30%‐50% of men with Klinefelter syndrome (KS). The reason for the absence or presence of spermatozoa in half of the men with KS remains unknown. Therefore, the search for an objective marker for a positive prediction in finding spermatozoa is of significant clinical value to avoid unnecessary testicular biopsies in males with (mostly) low testicular volume and impaired testosterone. The objective of this study was to determine whether paternal or maternal inheritance of the additional X‐chromosome can predict the absence or presence of spermatogenesis in men with KS. Men with KS who have had a testicular biopsy for diagnostic fertility workup TESE were eligible for inclusion. Buccal swabs from nine KS patients and parents (trios) were taken to compare X‐chromosomal inheritance to determine the parental origin of both X‐chromosomes in the males with KS. Spermatozoa were found in TESE biopsies 8 of 35 (23%) patients after performing a unilateral or bilateral TESE. Different levels of spermatogenesis (from the only presence of spermatogonia, up to maturation arrest or hypospermatogenesis) appeared to be present in 19 of 35 (54%) men, meaning that the presence of spermatogenesis not always yields mature spermatozoa. From the nine KS‐trios that were genetically analysed for X‐chromosomal inheritance origin, no evidence of a correlation between the maternal or paternal origin of the additional X‐chromosome and the presence of spermatogenesis was found. In conclusion, the maternal or paternal origin of the additional X‐chromosome in men with KS does not predict the presence or absence of spermatogenesis.     

Surgical recovery of sperm in non-obstructive azoospermia

The development of intracytoplasmic sperm injection (ICSI) opened a new era in the field of assisted reproduction and revolutionized the assisted reproductive technology protocols for couples with male factor infertility. Fertilisation and pregnancies can be achieved with spermatozoa recovered not only from the ejaculate but also from the seminiferous tubules. The most common methods for retrieving testicular sperm in non-obstructive azoospermia (NOA) are testicular sperm aspiration (TESA: needle/fine needle aspiration) and open testicular biopsy (testicular sperm extraction: TESE). The optimal technique for sperm extraction should be minimally invasive and avoid destruction of testicular function, without compromising the chance to retrieve adequate numbers of spermatozoa to perform ICSI. Microdissection TESE (micro-TESE), performed with an operative microscope, is widely considered to be the best method for sperm retrieval in NOA, as larger and opaque tubules, presumably with active spermatogenesis, can be directly identified, resulting in higher spermatozoa retrieval rates with minimal tissue loss and low postoperative complications. Micro-TESE, in combination with ICSI, is applicable in all cases of NOA, including Klinefelter syndrome (KS). The outcomes of surgical sperm retrieval, primarily in NOA patients with elevated serum follicle-stimulating hormone (FSH) (NOA including KS patients), are reviewed along with the phenotypic features. The predictive factors for surgical sperm retrieval and outcomes of treatment were analysed. Finally, the short- and long-term complications in micro-TESE in both 46XY males with NOA and KS patients are considered.     

Clinical analysis of patients with azoospermia factor deletions by microdissection testicular sperm extraction

Microdeletions of the azoospermia factor (AZF) locus on the Y chromosome have been implicated as a major genetic component of idiopathic male infertility, and the incidence of AZF deletions has been reported to be 15-20% in men with non-obstructive azoospermia (NOA). Numerous studies have described AZF deletion rates in patients with azoospermia; however, a clinical comparison of azoospermic patients with AZF deletion and those with no deletion has not been reported well. A new technique for testicular sperm extraction, microdissection testicular sperm extraction (TESE), has been used widely on NOA patients. Although testicular spermatozoa are reliably detected and retrieved from NOA patients by microdissection TESE, sperm retrieval rates for patients with AZF deletions are not well known. Therefore, characteristics of NOA patients with AZF deletion were investigated. Six of 60 patients (10%) who underwent microdissection TESE were found to have AZF deletions by genomic polymerase chain reaction. Testicular data, outcome of sperm retrieval and endocrinological profiles, were compared between patients with AZF deletions (n = 6) and those with no deletions (n = 54). Testicular size, varicocele rates and testicular histology were similar between the groups. Significant differences were not detected in the endocrinological profiles. Sperm retrieval rates were not significantly different between the groups. In conclusion, AZF deletions do not appear to confer specific characteristics to NOA patients.     

Primary male infertility in Kuwait: a cytogenetic and molecular study of 289 infertile Kuwaiti patients

Infertility is one of the major public health problems, affecting 15% of couples who attempt pregnancy; in 50% of these, the male partner is responsible. Chromosomal abnormalities and Y microdeletions in the azoospermia factor (AZF) region are known to be associated with spermatogenetic failure. In the present study, 289 patients with primary male infertility because of spermatogenetic failure were studied in order to highlight the molecular background of male infertility in Kuwait, and to avoid the possibility of transmission of any microdeletions/chromosomal aberrations to offspring via intracytoplasmic sperm injection (ICSI). Of the 289 infertile men, 23 patients (8%) had chromosomal aberration in the form of Klinefelter syndrome/variant (16/23; 69.6%), XYY syndrome (3/23; 13%), XX male syndrome (2/23; 8.7%), 45,X/46X, i(Yp)(1/23; 4.4%) and 45,XY, t(9;22) (1/23;4.4%). Y-chromosome microdeletion in the AZFb and AZFc regions were detected in 7/266 cases (2.6%). Testicular biopsy was carried out in 31 azoospermic patients, of whom five men had Sertoli-cell only syndrome, while 26 patients had spermatogenic arrest. In conclusion, this study showed that the frequency of both chromosomal anomalies and Y microdeletions were found in 10.4% of the infertile men. The potential risk of transmitting these genetic disorders to offspring provides a rationale for screening infertile men prior to ICSI.     

Outcome of testicular sperm extraction in nonmosaic Klinefelter syndrome patients: what is the best approach?

Klinefelter syndrome (KS) is the most common chromosomal disorder associated with male hypogonadism and infertility. Parenthood can be achieved in men with KS by intracytoplasmic sperm injection (ICSI) using testicular spermatozoon. The aim of this study was to evaluate surgical sperm retrieval (SSR) rate in patients with KS and to investigate the approach associated with the highest SSR. This is a retrospective study where all medical records of patients with KS who underwent SSR for ICSI, in our centre in the past 14 years, were reviewed. Forty‐three patients were included in this study. Twenty‐three underwent conventional testicular sperm extraction (TESE), while 20 patients underwent microsurgical TESE (Micro‐TESE). The SSR was significantly higher in the Micro‐TESE group when compared with the TESE group (30% versus 0% respectively). In the Micro‐TESE group, hormonal stimulation was given to 16 patients, while no treatment was given to four patients. SSR was only successful in hormonally treated patients (6/16). When the type of hormone stimulation was evaluated, SSR was higher in patients receiving aromatase inhibitors (27.8%). SSR in patients with KS is significantly higher when using hormonal stimulation by aromatase inhibitors followed by microsurgical testicular sperm extraction.     

Interchromosomal effect in sperm of males with translocations: report of 6 cases and review of the literature

Somatic chromosomal abnormalities are frequently found in infertile men, particularly in those with low sperm count and/or seeking intracytoplasmic sperm injection. These abnormalities mostly consist of numerical sex chromosome abnormalities and translocations (Robertsonian or reciprocal). In this study, we searched for the occurrence of non-disjunction of chromosomes not involved in translocations during meiosis, phenomenon called interchromosomal effect (ICE) and first described by Lejeune (1965). Ejaculate samples of two patients carrying a Robertsonian translocation and four a reciprocal translocation patients and four controls (men with a 46,XY karyotype and normal sperm parameters) were studied in dual FISH 7-9, dual FISH 13-21 and triple FISH X-Y-18. A statistically significant increase of disomy X, Y and XY (P = 0.009, P = 0.004, P < 0.001) was found in the Robertsonian der(13;14)(q10;q10) carrier but not in the der(14;21)(q10;q10) carrier compared with controls. Among reciprocal translocation carriers, a significant increase of disomy 21 (P = 0.033) was observed in a sole patient with a t(9;22)(q21;q11.2). The increase of meiotic non-disjunction for chromosome 21 and sex chromosomes is a recurrent event found in other studies. According to our results and published data, the ICE on some specific chromosomes is likely in men carrier of a translocation, although it cannot be excluded that the aneuploidy is related to the oligoasthenoteratozoospermia usually present in these men. Moreover, this phenomenon showed interindividual variations which cannot be predicted. The risk of aneuploidy in sperm of males used for ICSI need to be evaluated. It could be superadded to that of meiotic segregation of the translocation to give a more precise and personalized risk assessment of aneuploidy in the offspring of those men.     

非嵌合型克氏综合征显微睾丸取精术后临床妊娠1例报告及文献复习

目的 探讨非嵌合型克氏综合征患者生育遗传学意义后代的可行性.方法 1例就诊于华中科技大学同济医学院附属同济医院的男性患者,年龄29岁,多次精液分析提示无精子,查体双侧睾丸体积小,质硬,约2 ml.性激素水平:卵泡刺激素37.34 IU/L,黄体生成素15.69 IU/L,明显升高;睾酮2.13 ng/ml,稍微偏低.查染色体核型为47,XXY,Y染色体微缺失正常,确诊为非嵌合型克氏综合征.行显微睾丸取精术,同时配合女方取卵后行卵细胞胞质内单精子注射(intracytoplasmic sperm injection, ICSI),随访1个月,观察患者术后并发症及其配偶妊娠情况.结果 显微睾丸取精术中找到少量活动精子,次日女方取卵后行ICSI,3 d后移植2枚新鲜胚胎.术后患者未出现阴囊血肿、感染等并发症,术后随访1个月患者睾酮水平较术前偏低,但性功能未受到影响.女方移植后2周查血HCG,为801 mIU/ml;移植后4周超声提示宫内早孕,单胎,胚胎存活.结论 非嵌合型克氏综合征患者可通过显微睾丸取精术获取精子,同时配合ICSI技术,生育自己遗传学意义的后代.     

A familial analysis of two brothers with azoospermia caused by maternal 46,Y,t(X; 1) (q28; q21) chromosomal abnormality

Chromosomal abnormality is a primary genetic factor that lead to azoospermia and male infertility. Here, we report the cases of two brothers with primary infertility, whose chromosomes displayed a balanced translocation, and their karyotypes were 46,Y, t(X; 1) (q28; q21). Both presented an azoospermia phenotype without abnormal clinical symptoms. Their mother's karyotype was 46,X, t(X; 1) (q28; q21), and their father's chromosome karyotype was 46,XY. No abnormal changes were noted in the copy number of chromosome fragments in the whole genome. This study is the first to report showing that 46,Y, t(X; 1) (q28; q21) chromosomal abnormalities are associated with azoospermia.