Distribution of spermatogenesis in the testicles of azoospermic men: the presence or absence of spermatids in the testes of men with germinal failure [published erratum appears in Hum Reprod 1998 Mar;13(3):780]

The aim of the study was to determine whether a prior diagnostic testicle biopsy can predict success or failure of testicular sperm extraction (TESE) with intracytoplasmic sperm injection (ICSI) in patients with non-obstructive azoospermia caused by testicular failure, and what is the minimum threshold of sperm production in the testis which must be surpassed for spermatozoa to reach the ejaculate. Forty- five patients with non-obstructive azoospermia caused by testicular failure underwent diagnostic testicle biopsy prior to a planned future TESE-ICSI procedure. The diagnostic testicle biopsy was analysed quantitatively, and correlated with the quantitative findings of spermatogenesis in patients with normal spermatogenesis, as well as with the results of subsequent attempts at TESE-ICSI. Men with non- obstructive azoospermia caused by germinal failure had a mean of 0-6 mature spermatids/seminiferous tubule seen on a diagnostic testicle biopsy, compared to 17-35 mature spermatids/tubule in men with normal spermatogenesis and obstructive azoospermia. These findings were the same for all types of testicular failure whether Sertoli cell only, maturation arrest, cryptorchidism, or post-chemotherapy azoospermia. Twenty-two of 26 men with mature spermatids found in the prior testis biopsy had successful retrieval of spermatozoa for ICSI, 12 of their partners became pregnant, and are either ongoing or delivered. The study suggests that 4-6 mature spermatids/tubule must be present in the testis biopsy for any spermatozoa to reach the ejaculate. More than half of azoospermic patients with germinal failure have minute foci of spermatogenesis which are insufficient to produce spermatozoa in the ejaculate. Prior diagnostic testicle biopsy analysed quantitatively (for the presence of mature spermatids) can predict subsequent success or failure with TESE-ICSI. Incomplete testicular failure may involve a sparse multi-focal distribution of spermatogenesis throughout the entire testicle, rather than a regional distribution. Therefore, it is possible that massive testicular sampling from many different regions of the testes may not be necessary for successful TESE-ICSI.      

Fertility with testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermic men

In non-obstructive azoospermia spermatozoa can usually onlybe isolated from the testicles, and thus the most promisingtreatment model is testicular sperm extraction (TESE). Hormoneconcentrations, testicular volume determinations and testicularbiopsy results are not uniform enough to select potential candidatesfor successful TESE and intracytoplasmic sperm injection (ICSI)approaches in advance. The aim of this study was to assess theefficacy of using ICSI with testicular spermatozoa in casesof non-obstructive azoospermia and to compare the inclusioncriteria and sperm existence in the testicles in sperm obtainableand non-obtainable groups. All men showed either complete orincomplete (n = 14) maturation arrest in spermatogenesis, severehypospermatogenesis (n = 10) or Sertoli cell-only syndrome (n= 5) in their testicular biopsies. Only 14 out of a total of29 men provided enough spermatozoa for the ICSI procedure, whileno spermatozoa were found in the testicular samples of the remaining15 men. Out of 123 oocytes obtained from 14 females, 101 wereinjected with the husbands' testicular sperm cells. Total fertilizationfailure was observed in three cases. Of 39 oocytes fertilized,38 cleaved. The fertilization and cleavage rates were 38.6 and97.4% respectively. The pregnancy rate was 20.7% per initiatedcycle. In the group from whom spermatozoa were obtainable, thepregnancy rate was 42.9% per initiated cycle and 54.5% per embryotransfer. A total of six pregnancies were achieved, of whichtwo Were twins and four were singletons. One singleton pregnancyresulted in abortion in the first trimester. There was no statisticaldifference concerning the serum follicle stimulating hormoneconcentration, testicular volume and biopsy results in groupsin which spermatozoa were obtainable or not. In conclusion,although the association of TESE with ICSI obtained pregnanciesfor some patients with non-obstructive azoospermia, furtherstudies are needed to determine the inclusion criteria for successfulTESE.     

Surgical sperm recovery for intracytoplasmic sperm injection: which method is to be preferred?

Different methods for recovering epididymal or testicular spermatozoa have been described and each has its drawbacks and advantages. Percutaneous aspiration of the testis may be the method of choice in cases of irreparable obstructive azoospermia. Using a 21-gauge needle, spermatozoa may be recovered in 96 % of patients. More patients undergoing fine-needle aspiration experienced less pain than expected as compared with those undergoing open biopsy. Microsurgical epididymal sperm aspiration (MESA) is the preferred method in patients with an incomplete work-up because, if indicated, a vasoepididymostomy can be performed concomitantly with a full scrotal exploration. In azoospermic patients with testicular failure, the sperm recovery rate, i.e. the chance of finding at least one spermatozoon, is around 50% after multiple open biopsies. However, the fertilization rates after intracytoplasmic sperm injection (ICSI) are significantly lower than in men with normal spermatogenesis, and complete fertilization failure may occur more frequently. Although the combination of testicular sperm extraction (TESE) and ICSI may be the sole treatment available for infertility because of non-obstructive azoospermia, the overall success rate is limited and ongoing pregnancies are obtained in < or =20% of ICSI cycles. In patients with incomplete Sertoli cell-only syndrome, testicular damage may be limited by use of a selective microsurgical approach; less invasive methods such as fine-needle aspiration are not useful in these patients. Of 14 patients with primary testicular failure as proven by histopathology, only in one case (7.1%) were spermatozoa recovered by multiple aspirations, while in nine cases (64.3%) spermatozoa were recovered by open biopsy. Although the pregnancy rates reported after ICSI with frozen-thawed testicular spermatozoa from patients with primary testicular failure are relatively low, the recovery of testicular spermatozoa by open biopsy followed by cryopreservation may be the method of choice by which to prevent repeat surgery and pointless ovarian stimulation in the female partner.     

May-Grünwald-Giemsa stain for detection of spermatogenic cells in the ejaculate: a simple predictive parameter for successful testicular sperm retrieval.

BACKGROUND: Testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI) have become standard treatments for patients with non-obstructive azoospermia. A diagnostic testicular biopsy for histopathological examination is not always predictive of TESE outcome. Moreover, it is not without potential complications. The aim of this study was to determine the value of various clinical and laboratory parameters, particularly identification of seminal spermatids using May-Grünwald-Giemsa (MGG) stain in predicting TESE results. METHODS: A total of 100 patients with non-obstructive azoospermia was subjected to clinical examination, serum FSH measurement, identification of seminal spermatids and spermatocytes using MGG staining and TESE with multiple testicular sampling. Spermatozoa were retrieved from 49% of patients. Results of TESE were compared with previous parameters in addition to histopathology. RESULTS: Testicular histopathology was, in general, an inaccurate parameter, and identification of testicular spermatids by histology predicted successful TESE in only 74% of cases. Testicular volume and serum FSH concentration also had poor predictive values. Round spermatids were identified in the ejaculate of 83.7% of TESE-positive cases, and in 22% of TESE-negative cases. CONCLUSIONS: The detection of round spermatids in semen by MGG staining provides the greatest predictive value for successful testicular sperm retrieval, and also has the advantages of simplicity, low cost and availability.     

Successful testicular sperm extraction (TESE) in spite of high serum follicle stimulating hormone and azoospermia: correlation between testicular morphology, TESE results, semen analysis and serum hormone values in 103 infertile men

Spermatozoa recovered from testicular biopsies can be used through intracytoplasmic sperm injection (ICSI) to achieve a pregnancy. To assess the likelihood of successful testicular sperm extraction (TESE) in men suffering from severe oligo- or azoospermia, bilateral biopsy specimens were obtained. Following semi-thin sectioning, the morphology of testicular samples was graded according to a modified Johnsen score. TESE was performed in parallel to this histological examination. The number of isolated spermatozoa was assessed in a semiquantitative way. From 103 patients investigated, 64 (62.1%) showed azoospermia in a preceding semen analysis and 29 (28.2%) patients had sperm concentrations between 0.1 and 1 x 10(6)/ml. In 10 patients who had higher sperm counts, most spermatozoa were non-motile. Spermatozoa could be detected after TESE in the testicular tissue of 49 (77%) azoospermic men. When follicle stimulating hormone (FSH) concentration was normal, most patients had detectable spermatozoa after TESE. Nearly one-third of patients with mildly elevated FSH had no spermatozoa. Thirty-nine percent of patients in whom FSH was elevated to more than twice normal and 50% of patients with grossly elevated FSH had no detectable spermatozoa. In all, 82.8% of men with sperm concentrations between 0.1 and 1x10(6)/ml in their ejaculate showed spermatozoa in the tissue sample after TESE. Our data demonstrate that, contrary to previous recommendations, infertile men with azoospermia and high FSH values should be reconsidered for testicular biopsy, provided that tissue samples can be cryopreserved for later TESE/ICSI treatment.      

Criteria predicting the absence of spermatozoa in the Sertoli cell-only syndrome can be used to improve success rates of sperm retrieval

In patients with non-obstructive azoospermia, testicular sperm extraction (TESE) is a method of choice to recover spermatozoa as a male therapeutic approach in intracytoplasmic sperm injection (ICSI) programmes. However, the efficacy of TESE in this indication is burdened by a frequent failure of sperm recovery, which renders useless both the invasive testicular intervention and ovarian stimulation of the patient's spouse. One of the most frequent pathological pictures characterizing complete absence of spermatozoa is germinal aplasia (Sertoli cell- only syndrome or SCOS). Two different histological patterns of SCOS have been already described during the past five decades. These two patterns can be characterized as the congenital (pure) and the secondary (mixed) forms. Both patterns, with different prognosis to retrieve spermatozoa by therapeutic testicular biopsy, are frequently confused when TESE is performed during ICSI programmes. Useful criteria to predict the absence of spermatozoa can be obtained by a definite recognition of the two typical histological patterns during the diagnostic testicular biopsy. The diagnosis of congenital or acquired SCOS can be refined by endocrine, chemical, immunohistochemical and molecular biology aids. Reduction of both sperm retrieval failure and unnecessary ovarian stimulation can be achieved by combination of these methods.     

Fertilization, pregnancy and embryo implantation rates after ICSI in cases of obstructive and non-obstructive azoospermia

The aetiology of azoospermia can be grossly divided into obstructive and non-obstructive causes. Although in both cases testicular spermatozoa can be used to treat male fertility, it is not well established whether success rates following intracytoplasmic sperm injection (ICSI) are comparable. Therefore, a retrospective analysis of fertilization, pregnancy and embryo implantation rates was performed following ICSI with testicular spermatozoa in obstructive or non-obstructive azoospermia. In total, 193 ICSI cycles were carried out with freshly retrieved testicular spermatozoa; in 139 cases of obstructive and 54 cases of non-obstructive azoospermia. The fertilization rate after ICSI with testicular spermatozoa in non-obstructive azoospermia was significantly lower than in obstructive azoospermia (67.8% versus 74.5%; P = 0.0167). Within the non-obstructive group, the fertilization rate in the group of maturation arrest (47.0%) was significantly lower than in case of Sertoli cell-only (SCO) syndrome (71.2%) or germ cell hypoplasia (79. 5%). Embryo quality on day 2 after ICSI was similar for all groups. Pregnancy rates per transfer between obstructive (36.8%) and non-obstructive groups (36.7%) were similar. In cases of maturation arrest the pregnancy rate per transfer was lowest (20.0%) although not significantly different from SCO syndrome or hypoplasia groups. Embryo implantation rates were not different between the obstructive (19.6%) and non-obstructive groups (25.8%), and were lowest in cases of germ cell hypoplasia (15.8%). This retrospective analysis shows that although fertilization rate after ICSI with testicular spermatozoa in non-obstructive azoospermia is significantly lower than in obstructive azoospermia, pregnancy and embryo implantation rates are similar.     

Highly sensitive quantitative telomerase assay of diagnostic testicular biopsy material predicts the presence of haploid spermatogenic cells in therapeutic testicular biopsy in men with Sertoli cell-only syndrome

The role of a telomerase assay in the recognition of Sertoli cell-only syndrome with testicular foci of haploid cells was evaluated. Men with Sertoli cell-only syndrome (n = 23) were given a new diagnostic testicular biopsy. Part of the biopsy was stained and the remainder was processed for the quantitative telomerase assay. After 3-13 months, a therapeutic testicular biopsy was performed. This material was minced and then examined using confocal laser scanning microscopy and fluorescent in-situ hybridization. Histology of diagnostic testicular biopsy material confirmed the diagnosis of Sertoli cell-only syndrome in all the participants. All seven men with a telomerase assay value in their diagnostic testicular biopsy of >42 total product generated (TPG) U/microg protein had haploid cells (i.e. spermatozoa and/or spermatids) in their therapeutic testicular biopsy. Among participants with telomerase assay values <42 TPG U/microg protein, only one man had haploid cells in his therapeutic testicular biopsy. Thus, telomerase assay values >42 TPG U/microg protein in the diagnostic biopsy identified 87.5% of the Sertoli cell-only syndrome men with haploid cells in their therapeutic testicular biopsy. Significantly higher values of the telomerase assay were found in men with testicular foci of haploid cells than in men without these foci. The use of a quantitative telomerase assay biopsy appears to be important for identifying those men with Sertoli cell-only syndrome who have foci of haploid cells and can be candidates for assisted reproduction techniques.     

Testicular sperm extraction: impact of testicular histology on outcome, number of biopsies to be performed and optimal time for repetition

Testicular sperm extraction (TESE) may not always be successful in patients with non-obstructive azoospermia, as they only have minute foci of active spermatogenesis from which a tiny number of spermatozoa can be extracted. The aim of this study was to find the percentile incidence of successful TESE in non-obstructive azoospermia patients in relation to various histopathological patterns and the number of performed biopsies, and to determine the optimal time needed for repetition. A total of 216 patients underwent bilateral testicular biopsy taking a single piece from each testis for sperm retrieval and pathological evaluation. In another 100 patients, the same procedure was done but taking multiple samples (maximum four samples/testis). Spermatozoa were successfully retrieved from 37.5 and 49% of patients who supplied single and multiple samples respectively. TESE was significantly higher when multiple samples were taken in all histopathological groups except for Sertoli cell-only syndrome, tubular sclerosis and Klinefelter's pattern. Twenty-seven patients underwent repeated TESE for ICSI between 1 and 24 months from the first procedure; all of them had easy sperm retrieval during the first procedure. Although sperm retrieval was successful in 75 and 94.7% of patients who underwent the second attempt, before and after 3 months respectively, a second TESE was usually more difficult and necessitated multiple sampling.     

Human fertilization with round and elongated spermatids

Human spermatids from ejaculate and testicular tissue have been utilized for evaluating human fertilization by intracytoplasmic sperm injection (ICSI) and, where possible, compared with spermatozoa utilizing sibling oocytes. Round and elongated spermatids obtained from ejaculates were either prepared through Percoll gradients or isolated and washed individually using subzonal insemination needles (SUZI; 10- 14 microm internal diameter). Seminiferous tubules obtained after biopsy were placed into HEPES-buffered Earle's medium and dissected using 21-gauge needles. Spermatogenic cells and spermatozoa were isolated and washed individually using SUZI needles. Spermatozoa were subsequently injected into the ooplasm using 5 microm (internal diameter) ICSI needles, whereas 8-9 microm (internal diameter) needles were used for spermatid injection. Only metaphase II oocytes (n = 207) were injected: 64 with round spermatids, 92 with elongated spermatids and 51 with spermatozoa; the fertilization rate was 30, 24 and 67% respectively. There was a significant (P < 0.001) increase in the fertilization rate using spermatozoa compared with spermatids. The fertilization rate was not different between round and elongated spermatids, although the fertilization rates for round and elongated spermatids in the ejaculate were 33 and 18% respectively, compared with 22 and 38% respectively when testicular spermatids were utilized. In three patients sibling oocytes were used to compare round and elongated spermatids found in the ejaculate with spermatozoa extracted from seminiferous tubules. The fertilization rate was 24% for spermatids and 79% for testicular spermatozoa. This result suggests that, should only spermatids be available in the ejaculate, a testicular biopsy in the hope of obtaining testicular spermatozoa would be worth while.      

Morphometric and cytogenetic characteristics of testicular germ cells and Sertoli cell secretory function in men with non-mosaic Klinefelter's syndrome

BACKGROUND: Klinefelter's syndrome is the most frequent chromosomal abnormality in infertile men. In this study, the chromosomes of round spermatids and spermatogonia/primary spermatocytes from men with non-mosaic Klinefelter's syndrome were examined, together with the Sertoli cell secretory function and sperm morphometry. METHODS: Twenty-four men with non-mosaic Klinefelter's syndrome and nine men with obstructive azoospermia underwent therapeutic testicular biopsy. When spermatozoa in the final filtrate were present, they were processed for sperm morphometry or ICSI. Sperm morphometry was evaluated by the maximal length and width of the sperm head, the length of the midpiece and the ratio of the acrosomal region to the total surface area of the head. When round spermatids were present, they were processed for fluorescent in-situ hybridization (FISH). FISH was also applied to fragments of seminiferous tubules. Sertoli cell secretory function was measured by the amount of androgen binding protein (ABP) secreted in vitro. RESULTS: More than 93% of the evaluated round spermatids were normal. The proportions of 24,XY and of 24,XX round spermatids to the total number were significantly larger in men with Klinefelter's syndrome than in obstructed azoospermic men. Men with Klinefelter's syndrome who had spermatozoa in their testicular tissue (n = 12) were positive for both 46,XY and 47,XXY spermatogonia in their seminiferous tubules. In contrast, men with Klinefelter's syndrome without spermatozoa in their testicular tissue (n = 12) were positive for 47,XXY spermatogonia but negative for 46,XY spermatogonia in their seminiferous tubules. ABP profiles were significantly smaller in men with Klinefelter's syndrome who were negative for spermatozoa compared with men who were positive. Four pregnancies were achieved and five healthy babies were born. CONCLUSIONS: This study suggests that few 46,XXY spermatogonia undergo meiosis with an XX pairing and a Y univalent type of pairing. Hyperhaploid round spermatids (24,XY and 24,XX) may be produced by meiosis of 47,XXY spermatogonia. Men with Klinefelter's syndrome who are negative for testicular spermatozoa have a greater degree of Sertoli cell secretory dysfunction compared with men with Klinefelter's syndrome who are positive for spermatozoa. There are several defects in sperm morphometry with functional significance in men with Klinefelter's syndrome.     

Are there any predictive factors for successful testicular sperm recovery in azoospermic patients?

Recovery of testicular spermatozoa from azoospermic patients with testicular failure followed by intracytoplasmic sperm injection (ICSI) is a recent advance in the treatment of male infertility. This study aimed at investigating which parameter(s) may predict successful testicular sperm recovery. We reviewed 395 testicular sperm recovery procedures and analysed the most frequently available parameters for clinical decision-making in azoospermic patients: (i) presence of at least one single spermatozoon in at least one preliminary semen analysis; (ii) maximum testicular volume; (iii) serum follicle stimulating hormone (FSH); and (iv) presence of spermatozoa in the histology of a randomly-taken testicular biopsy. Sensitivity, specificity, positive and negative predictive value, positive and negative likelihood ratio and accuracy were calculated for the above index parameters in different clinically relevant subgroups using receiver operating characteristic (ROC) curves whenever possible. Spermatozoa were always successfully recovered in patients with normal testicular histological findings (n = 173) or hypospermatogenesis (n = 16) but not in some patients with tubular sclerosis (seven out of 18), Sertoli cell-only pattern (55 out of 112) or maturation arrest (39 out of 76). Histopathology was the best test for predicting successful sperm recovery in the whole population (sensitivity: 86%, specificity: 93%, accuracy: 0.87). In patients with secretory azoospermia, histopathology was again the most accurate parameter (accuracy: 0.74), especially in patients showing Sertoli cell-only pattern (accuracy: 0.83) but not in patients showing maturation arrest (accuracy: 0.55). In patients with serum FSH concentrations > 12 IU/l and maximum testicular volume < 15 ml, histopathology was not found to be accurate. Semen analysis, maximum testicular volume and serum FSH were not highly predictive in all subgroups studied. Our analysis shows that no strong predictors for successful testicular sperm recovery are available except for testicular histopathology.      

Testicular sperm extraction in a patient with metachronous bilateral testicular cancer.

A new indication for testicular tissue cryopreservation is demonstrated in a patient with metachronous bilateral testicular tumours and azoospermia. At the age of 18 (1982) the patient underwent left orchidectomy and radical retroperitoneal lymphadenectomy for a testicular teratoma (pT1N0M0). Semen samples were not cryopreserved because of absence of motile spermatozoa after thawing. Seventeen years after the primary testicular cancer, a seminoma of the contralateral right testis was diagnosed (pT1N0M0). Since the patient was azoospermic, no semen samples could be cryopreserved. However, spermatozoa were detected in testicular biopsy material of the right testis and were cryopreserved for ICSI. Since all spermatozoa were dead after thawing, testicular sperm extraction (TESE) was performed in the remaining tissue samples at the time of ICSI treatment. Only spermatids could be extracted from frozen-thawed samples due to the inhomogeneous distribution of spermatogenic activity in the testicular tissue. Although one oocyte was fertilized with these spermatids, a clinical pregnancy was not achieved. Despite the disappointing results of ICSI in the couple presented here, this case report demonstrates that cryopreservation of testicular tissue and TESE should be considered in patients with bilateral testicular tumours and azoospermia, if frozen semen samples are not available.     

A prospective study of multiple needle biopsies versus a single open biopsy for testicular sperm extraction in men with non-obstructive azoospermia

Little is known about the efficacy and the factors affecting the outcome of fine needle aspiration biopsy of the testis for sperm retrieval in azoospermic men with defective spermatogenesis. A prospective study was designed to compare the efficacy of needle and open (window) testicular biopsies for testicular epididymal sperm extraction (TESE) in 35 consecutive men with azoospermia due to defective spermatogenesis undergoing testicular biopsy for intracytoplasmic injection of oocytes. Each of the consecutive 35 patients underwent TESE using a 19 gauge butterfly needle followed by a window (1-1.5 cm-sized incision) testicular biopsy in the same procedure. The extraction of spermatozoa into culture medium was compared with the assessment of testicular biopsies by histology, the mode of biopsy (needle or open biopsy) and the amount of tissue retrieved by either method. Testicular spermatozoa were retrieved in 22 (63%) who had an open testicular biopsy compared with five (14%) patients who had multiple needle biopsies, respectively; the difference was statistically significant. Open testicular biopsy retrieves more testicular tissue than needle biopsy. Needle testicular biopsy retrieved testicular spermatozoa in 50% of those with hypospermatogenesis, 10% with focal spermatogenesis and in no patients with maturation arrest or Sertoli cell-only pattern. In contrast, sperm retrieval was successful in 100%, 90% and 66% of those with respective histologies using open testicular biopsy. Other than bruising, for which they required no analgesia, none of the patients suffered any obvious complications associated with traditional testicular biopsy. We conclude that open testicular biopsy is more effective than needle biopsy for the retrieval of testicular spermatozoa in azoospermic men with defective spermatogenesis. The difference observed may be related to the amount of testicular tissue retrieved and to the influence of testicular histology.      

Testicular sperm extraction in azoospermic men submitted to bilateral orchidopexy

BACKGROUND: This study was carried out to evaluate whether bilateral orchidopexy represents a poor or good prognostic factor in azoospermic men undergoing testicular sperm extraction (TESE). METHODS: One hundred and seven presumed non-obstructive azoospermia (NOA) patients, according to conventional clinical parameters (volume of testis, FSH, clinical history) were submitted to testicular biopsy with TESE. Thirty men (28%) had a history of bilateral orchidopexy for cryptorchidism. RESULTS: Normal spermatogenesis or mild hypospermatogenesis was diagnosed in 12/30 ex-cryptorchid patients and in 7/77 presumed NOA patients (P = 0.0004). Conversely, pure Sertoli cell-only syndrome or complete maturation arrest was found in 10/30 ex-cryptorchid patients and in 48/77 presumed NOA patients (P = 0.0094). In 53/107 patients (49.5%), TESE allowed a positive sperm retrieval. At least one spermatozoon was observed in 22/30 ( approximately 73%) ex-cryptorchid patients and in 31/77 ( approximately 40%) presumed NOA patients (P = 0.0026). A large number of spermatozoa (equivalent to an obstructive pathology) were retrieved in 13/30 ex-cryptorchid and in 10/77 presumed NOA patients (P = 0.001). A history of bilateral orchidopexy in presumed NOA patients correlates positively for the chance of retrieving testicular spermatozoa (odds ratio 3.8; 95% confidence interval 1.41-10.21; P = 0.008). CONCLUSIONS: Although bilateral cryptorchidism is usually considered a testicular secretive dysfunction, TESE permits retrieval of a large number of spermatozoa in almost 40% of cases. Our data suggest the existence of congenital or acquired obstructive anomalies of the seminal ducts in azoospermic orchidopexed men.     

Extended sperm preparation: an alternative to testicular sperm extraction in non-obstructive azoospermia

Testicular sperm retrieval for the treatment of non-obstructive azoospermia requires the execution of an invasive procedure, with all its possible attending complications and subsequent long-term effects. This study suggests a new non-invasive approach for collection of spermatozoa in these patients: the extended sperm preparation (ESP). ESP consists of conducting a thorough microscopic search through many droplets of ejaculate sediment. ESP was performed for 49 patients; in 17 patients (35%), spermatozoa were found and subsequently used in intracytoplasmic sperm injection (ICSI). Of these preparations, five yielded fewer motile spermatozoa than the number of corresponding oocytes available, and in one patient only non-motile spermatozoa were recovered. The remaining 32 ESP-negative patients underwent testicular sperm extraction (TESE) from testicular biopsy. Spermatozoa were found in 16 of 32 biopsies (50%) and subsequently used in ICSI. Fertilization and cleavage rates were comparable in both ESP and TESE groups, yielding four clinical pregnancies in each group (27 and 29% respectively). Embryo morphology was defined as excellent in significantly more cases in the ESP group than the TESE group, and implantation rate appeared somewhat higher in the ESP group (16%) than the TESE group (13%). The ESP technique yields results similar to TESE, and can be applied in cases of non-obstructive azoospermia as a prerequisite modality enabling us to avoid testicular biopsy in 35% of cases.      

Frequency of hyper-, hypohaploidy and diploidy in ejaculate, epididymal and testicular germ cells of infertile patients

The hypothesis that sperm aneuploidy and diploidy increase as a function of spermatogenesis impairment was addressed. Ejaculated semen samples from a series of men (n = 22) with very low total normal motile count (1 x 10(6)) was analysed in terms of sperm aneuploidy and diploidy by in-situ hybridization and compared with controls (n = 10). Germ cell aneuploidy was also analysed in an additional series of infertile patients presenting unexplained infertility (n = 3), congenital absence of the vas deferens (CAVD) (n = 6) and non-obstructive azoospermia (n = 3) undergoing IVF, microsurgical epididymal sperm aspiration (MESA)/ICSI and testicular sperm extraction (TESE)/ICSI cycles respectively. In-situ hybridization for chromosomes 1, 17, X and Y was performed on ejaculate, epididymal and testicular spermatozoa. Significantly higher sperm aneuploidy and diploidy rates where found (for the four chromosomes analysed) in spermatozoa from oligoasthenoteratozoospermia (OAT) over controls (18 versus 2.28% and 2.8 versus 0.13% respectively; P < 0.001). Testicular germ cells had even higher rates of sperm aneuploidy and diploidy. However, in this group it was difficult to determine whether the cells analysed were dysmorphic spermatozoa or spermatids. The data warrant further investigation on the cytogenetic abnormalities found in most germ cells identified in testicular tissue biopsies of azoospermic patients.     

Establishment of predictive variables associated with testicular sperm retrieval in men with non-obstructive azoospermia

Although testicular biopsy for sperm extraction is a procedure with a potential for complications, sperm retrieval is successful in 30-70% of patients with non-obstructive azoospermia. In order to predict the probability of retrieving at least one testicular spermatozoon we conducted a prospective study of a set of variables in 40 patients with non-obstructive azoospermia. Using the receiver operating characteristic curves, we determined the probability estimates of testicular volume, plasma follicle stimulating hormone (FSH) concentration, Johnsen score and visualization of testicular spermatids in discriminating between patients with successful and failed testicular sperm extraction. Visualization of testicular spermatids provided the best estimate of success of testicular sperm extraction. Of the factors studied using logistic-regression analysis (age, maternal and paternal age at birth, body mass index, luteinizing hormone, testosterone, FSH, testicular volume, the presence of testicular spermatids and Johnsen score), only the presence of spermatids and Johnsen score were independent variables able to predict the success of testicular sperm extraction. The visualization of the presence of spermatids gave a correct prediction of 77% and Johnsen score of 71%. The diagnostic model derived from these independent predictors when validated in 40 patients using the Jackknife technique gave a correct overall prediction of 87%. The probability of successful testicular sperm extraction in patients with non-obstructive azoospermia could be objectively predicted on the basis of simple histopathological criteria represented by the visualization of testicular spermatids and Johnsen score.     

Testicular fine needle aspiration: the alternative method for sperm retrieval in non-obstructive azoospermia.

The objective of this prospective open study was to determine the feasibility of obtaining mature spermatozoa for intracytoplasmic sperm injection (ICSI) by testicular fine needle aspiration (TEFNA) in men diagnosed with non-obstructive azoospermia. TEFNA consisted of a mean of 15 punctures and aspirations in each testis, using 23 gauge butterfly needles, connected to a 20 ml syringe with an aspiration handle. Patients (n = 85) underwent 111 TEFNA cycles. Mature testicular spermatozoa were recovered in 65 (58.5%) cycles from 50 (58.8%) patients. The sperm recovery rate by testicular histology was 14 out of 29 (48.3%) in patients with Sertoli cell-only, 13 out of 28 (46.4%) in patients with maturation arrest, 19 out of 20 (95%) in patients with hypospermatogenesis, four out of six (66.6%) in patients with tubular hyalinization due to non-mosaic Klinefelter's syndrome. No spermatozoa were found in two cases with post-irradiation fibrosis. ICSI was performed in all 65 cycles. In 58 cycles in which only the husbands' spermatozoa were used, 406 mature oocytes were injected, and 154 (37.9%) were normally fertilized. Of the 143 embryos that developed (92.8%), 119 were transferred in 42 cycles resulting in 18 clinical pregnancies (42. 8%), with 31 gestational sacs, providing an implantation rate of 26%. One abortion of a singleton pregnancy occurred (5.6%). No major side-effects, such as haematoma or infection were recorded. In conclusion, we have found TEFNA to be efficient, easy to learn, safe and well tolerated by all patients. In our opinion, TEFNA should be considered the first choice whenever sperm recovery is attempted in patients with non-obstructive azoospermia.     

Testicular sperm extraction: comprehensive analysis with simultaneously performed histology in 1418 biopsies from 766 subfertile men

The introduction of intracytoplasmic sperm injection (ICSI) has revolutionized treatment of male-factor infertility. Even with a single spermatozoon a pregnancy can be achieved. In cases of azoospermia due to obstruction or highly impaired spermatogenesis, spermatozoa can be retrieved directly from testicular tissue recovered by testicular biopsy followed by sperm extraction. The predictive value of histology from semi-thin sections of testicular biopsies was assessed in relation to testicular sperm extraction (TESE) results, using 1418 biopsy samples from 766 subfertile men which were evaluated simultaneously using a modified Johnsen score and an ordinal classification system for spermatozoa in TESE samples. In 655 men bilateral samples were available. Based on histological findings and TESE results, the quality of spermatogenesis in the right testes was significantly better than that in the left testes. There was a difference between the two sides in 35.7% of all patients for histology and 32.7% for TESE results. When best results from either testis were used for analysis, 76.9% of all men revealed spermatozoa in TESE preparations, although during histological evaluation of semi-thin sections only 64% of all men had shown mature spermatids. In a core group of 250 azoospermic men without anamnestic hints to obstruction and most likely to benefit from ICSI, TESE was successful in 62.8% men. Subdivision of this group dependent on follicle stimulating hormone (FSH) serum concentrations revealed that even in cases of increased FSH concentration, between 39.1 and 64.7% of men showed mature spermatids in their TESE samples. A subset of 70 azoospermic men from the main sample with symptoms and history suggestive of an obstruction and considered as positive controls showed a positive TESE result in all patients. The histology had failed to predict this in 2.9% of all cases. Nevertheless, in five men an early stage of testicular tumour (carcinoma in situ = CIS) was detected. Two of these males suffered from bilateral CIS. This reflects a prevalence of 0.7% testicular malignancy in the group of patients without a history of excurrent duct obstruction. The data demonstrate that a trial TESE with histology based on the semi-thin sectioning technique is a powerful diagnostic and therapeutic procedure, which justifies the invasive nature of sperm retrieval for ICSI. In addition, the results stress the importance of bilateral biopsies to gain optimal diagnostic and therapeutic results.