Short-term in-vitro culture and cryopreservation of spermatogenic cells used for human in-vitro conception

Testicular cell suspensions were prepared from obstructive and non- obstructive azoospermic men and were cultured in vitro for 96 h as (i) mixed cell populations and (ii) isolated homogeneous populations of primary spermatocytes, round spermatids and elongating spermatids. The cells lost their viability gradually during the first 24 h period. By 72 h almost 90% of the cells were non-viable. Isolated pure fractions showed better viability at each time interval (P < 0.0005). Throughout the culture period primary spermatocytes, elongating spermatids and other non-spermatogenic cells showed no change in their morphology, but almost 22% of round spermatids showed growth of flagella. Most of the round spermatids developed their flagella during the first 4-8 h period of culture. Isolated pure round spermatids showed better flagellar growth compared with mixed cell suspensions (P < 0.0005). The spermatogenic cells were successfully cryopreserved. However, when mixed spermatogenic cell suspensions were cryopreserved, more cells lost their viability compared with when isolated pure fractions were cryopreserved (P < 0.0005).      

Evaluation of the fertilization potential of freshly isolated, in-vitro cultured and cryopreserved human spermatids by injection into hamster oocytes.

The clinical potential for fertilization was examined by using the human sperm-hamster oocyte assay system after microinjection of round (RS), elongating (ES) or elongated (EtedS) spermatids retrieved from obstructive and non-obstructive azoospermic patients. Freshly isolated, in-vitro cultured and cryopreserved spermatids were utilized. For each category of microinjected spermatids, we demonstrated that the more mature the injected spermatid, the higher the incidence of fertilization (for freshly isolated spermatids, P < 0.006 and P < 0.008, for in-vitro cultured spermatids, P < 0.007 and P < 0.007 and for cryopreserved spermatids, P < 0.006 and P < 0.007 for obstructive and non-obstructive azoospermic patients respectively). Short term in-vitro culture of the spermatogenic cells did not improve the incidence of fertilization. However, cryopreservation significantly decreased (P < 0.001) the incidence of fertilization when each corresponding spermatogenic cell stage was compared. The incidence of fertilization was not statistically different when corresponding stages of spermatogenic cells were compared from obstructive and non-obstructive patients.     

Ooplasmic injection of elongating spermatids for the treatment of non- obstructive azoospermia

We applied the technique of ooplasmic elongating spermatid injection to the treatment of non-obstructive azoospermia. Mature oocytes were injected with elongating spermatids isolated from testicular biopsy material obtained from 13 non-obstructed azoospermic men. Seventy-three oocytes were successfully injected with elongating spermatids and were then cultured for 36 h. At 13 h post-injection 68 oocytes were found to be activated and 52 of them were fertilized. Forty-one 2- to 4-cell stage embryos developed from normally fertilized oocytes were transferred. At least two embryos were transferred to each female partner. Two pregnancies were achieved. Elongating spermatid injection may have a role in the treatment of non-obstructive azoospermia.      

In-vitro maturation of round spermatids using co-culture on Vero cells.

In an attempt to determine whether co-culture could promote sperm maturation, three patients with non-obstructive azoospermia, two with maturation arrest at the level of primary spermatocytes and one patient with <1% tubules showing complete spermatogenesis, and one patient with total globozoospermia, gave consent to experimentally co-culture round spermatids retrieved from the testicle on Vero cell monolayers. In all azoospermic patients elongating spermatids could be obtained from round spermatids. In one case of maturation arrest, of 37 round spermatids co-cultured for up to 5 days, 30% developed flagella, 46% matured to elongating and 19% to elongated spermatids, with one mature spermatozoon also obtained (3%). In the same patient, primary cultures of three round spermatids with flagella enabled development of one further mature spermatozoon. In the case with total globozoospermia, of six round spermatids co-cultured for up to 5 days, one mature spermatozoon was obtained, with a flagellum and normal head morphology. These preliminary findings suggest that it may be possible to overcome the round spermatid block, and even the triggering of morphological abnormalities arising at the spermiogenic level, by in-vitro maturation under special environmental conditions.     

Reproductive capacity of round spermatids compared with mature spermatozoa in a population of azoospermic men

The present study aims to evaluate the injection of testicular round spermatids from patients with complete failure of spermiogenesis compared with that of mature epididymal and testicular spermatozoa. Over a period of 8 months, 188 azoospermic patients were evaluated with a view to their inclusion in our intracytoplasmic sperm injection (ICSI) programme. All patients had had a previous testicular biopsy; 38 had pure obstructive azoospermia, while 150 had non-obstructive azoospermia. Mature spermatozoa were found in 93 patients, whereas spermatozoa were entirely absent, with a predominance of round spermatids in 87. In eight patients, spermatids could not be found and therefore their cycles were cancelled. There was an early appearance of the two pronuclei stage in the round spermatid group compared with the mature spermatozoa group of patients (10.2 and 16 h respectively). The fertilization rate was also significantly lower (P = 0.00001) in the round spermatid group. The numbers of embryos developed and of embryo transfers in the round spermatid injection group were significantly lower compared with the mature spermatozoa injection group (P = 0.05 and 0.0001 respectively). No pregnancies resulted from round spermatid injection, while 18 pregnancies were achieved from the injection of mature spermatozoa. In conclusion, injection of round spermatids from patients with complete failure of spermiogenesis resulted in a significantly lower fertilization rate and a higher developmental arrest compared with injection of mature spermatozoa. With no pregnancies achieved, one may question the unusual variability of reported success rates and stress the need for further research in order to improve the outcome of this novel technique.     

Chromosome analysis of epididymal and testicular sperm in azoospermic patients undergoing ICSI

BACKGROUND: Although ICSI provides a way of treating azoospermic men, concern has been raised about the potential risk for transmission of genetic abnormalities to the offspring. We quantified the incidence of chromosomal abnormalities in epididymal and testicular sperm retrieved from azoospermic patients undergoing ICSI. METHODS: Individual testicular sperm were collected from testicular biopsies with an ICSI pipette, and epididymal sperm were retrieved by microsurgical epididymal sperm aspiration. Samples were processed by fluorescent in-situ hybridization (FISH) for chromosomes 18, 21, X and Y and the results compared with those from normal ejaculated samples. RESULTS: The overall aneuploidy rate of 11.4% in men with non-obstructive azoospermia was significantly higher (P = 0.0001) than the 1.8% detected in epididymal sperm from men with obstructive azoospermia and also the 1.5% found in ejaculated sperm. No significant difference was found between the epididymal and ejaculated samples. When the chromosomal abnormalities were analysed, gonosomal disomy was the most recurrent abnormality in both obstructive and non-obstructive azoospermic patients, while autosomal disomy was the most frequent in ejaculated sperm. CONCLUSIONS: Sperm of non-obstructive azoospermic men had a higher incidence of chromosomal abnormalities, of which sex chromosome aneuploidy was the most predominant. Genetic counselling should be offered to all couples considering infertility treatment by ICSI with testicular sperm.     

Ultrastructural analysis of chromatin defects in testicular spermatids in azoospermic men submitted to TESE-ICSI.

BACKGROUND: Testicular sperm extraction (TESE) combined with intracytoplasmic sperm injection (ICSI) is offered to treat obstructive and non-obstructive azoospermia, but factors that influence the outcome of ICSI are not well defined. METHODS AND RESULTS: The percentage of elongated spermatids with normal chromatin condensation in azoospermic patients submitted for TESE-ICSI was determined. The quantitative analysis could be applied to nine of 19 biopsies classified as incomplete late maturation arrest (LMA) and compared with 10 biopsies with normal spermatogenesis. The percentage of elongated spermatids with normal chromatin was lower in LMA than in normal histology (mean 4.4%, range 0-20, and mean 52.9%, range 40-70 respectively; P = 0.0001). The percentage of elongated spermatids with normal chromatin was negatively correlated with the serum concentration of FSH (r = -0.86, P < 0.0001) and the number of degenerated germ cells per 100 Sertoli cells nuclei (r = -0.68; P < 0.0001), while it was positively correlated with the number of elongating spermatids per 100 Sertoli cell nuclei (r = 0.81; P < 0.0001). The percentage of elongated spermatids with normal chromatin was not correlated with the rate of oocyte fertilization, while the delivery rate/cycle was higher in cases with normal histology compared with cases of LMA. CONCLUSIONS: These preliminary data suggest that an altered chromatin condensation is a ubiquitous defect in spermatids of non-obstructed azoospermic men submitted for TESE-ICSI.     

Diagnostic epididymal and testicular sperm recovery and genetic aspects in azoospermic men.

Various procedures for sperm recovery in azoospermic men have been described, from open testicular biopsy to simple needle aspiration from the epididymis and the testis. Fifty-one obstructive and 86 non-obstructive azoospermic men were treated to compare the recovery of spermatozoa obtained by percutaneous aspiration from the epididymis (PESA) and aspiration/extraction from the testis (TESA, TESE) with histopathology. If TESA failed, the work up proceeded with TESE. All patients were karyotyped. Spermatozoa were recovered by PESA or TESA in all obstructive men (51/51 patients). In 22 out of 86 patients with non-obstructive azoospermia, testicular spermatozoa could be successfully recovered by TESA. In five additional patients TESE was successful in recovering spermatozoa where TESA had failed. In 43 patients, neither TESA nor TESE was successful. Sixteen patients chose not to proceed with TESE. Seven out of 86 patients had an abnormal karyotype in the non-obstructive group (8%), none in the obstructive group. In the non-obstructive patient group testicular histopathology showed hypospermatogenesis, incomplete maturation arrest and germ cell aplasia with focal spermatogenesis in cases where spermatozoa were recovered and complete germ cell aplasia, complete maturation arrest and fibrosis in cases where no spermatozoa were found. Spermatozoa were recovered by PESA or TESA from all patients with obstructive azoospermia and from approximately 40% of patients with non-obstructive azoospermia by TESA or TESE. Retrieval of viable spermatozoa in the infertility work-up was highly predictable for sperm recovery in subsequent ICSI cycles. TESA performed under local anaesthesia seems almost as effective as more invasive procedures in recovering testicular spermatozoa, both in obstructive and non-obstructive azoospermic men.     

Developmental potential of human spermatogenic cells co-cultured with Sertoli cells.

BACKGROUND: Development of an in-vitro culture system capable of supporting human early germ cell differentiation would be important for treatment of azoospermic patients. METHODS: Sertoli cells, spermatogonia and spermatocytes were isolated from testicular biopsies of 61 non-obstructive azoospermic patients, and co-cultured using Vero cell conditioned medium only or supplemented with recombinant (r)FSH or rFSH plus testosterone. Germ cell purity was checked by fluorescent in-situ hybridization (FISH) analysis. RESULTS: Best results were achieved with both hormones, which elicited 6.9% of meiosis index and 22.7% of differentiation into normal late spermatids after 2-3 weeks of culture. In-vitro matured spermatids were microinjected into oocytes to study their developmental potential. Round spermatids elicited 37.5% of fertilization and 28.6% blastocyst rates. Abnormal elongating and elongated spermatids enabled 8.3 and 27.3% fertilization rates respectively, but none achieved the blastocyst stage. Normal elongating and elongated spermatids elicited 30.5% fertilization and 42.9% of blastocyst rates. FISH analysis showed sex chromosome anomalies in all embryos, except in the case of morulae from normal late spermatids. CONCLUSIONS: Results suggest that meiosis and spermiogenesis can be resumed in vitro, with normal differentiated spermatids showing a low fertilization potential but regular rates of blastocyst formation. However, most of the embryos did not reach the morula stage and showed major sex chromosome abnormalities.     

Flow cytometry of human semen: a preliminary study of a non-invasive method for the detection of spermatogenetic defects

BACKGROUND: The pathway of spermatogenesis involves the conversion of diploid stem cells (spermatogonia) to tetraploid primary spermatocytes, followed by meiosis and two cell divisions, first forming diploid secondary spermatocytes and then haploid round spermatids. Differentiation of round spermatids results in spermatozoa containing condensed chromatin. It has long been known that semen from patients with non-obstructive azoospermia or oligospermia contains small numbers of immature germinal cells. In this article, a flow cytometric procedure is described for assessing defects in spermatogenesis by identifying the ploidy of those immature cells. METHODS: Cells in semen samples from 44 infertile patients and 14 controls were stained with propidium iodide, which displays red fluorescence when intercalated between bases in double-stranded DNA. The resulting cell suspension was examined by quantitative flow cytometry, with excitation by laser light (488 nm) and red fluorescence recorded on a logarithmic scale to allow easy differentiation between intensities of tetraploid, diploid and haploid round spermatids, and spermatozoa containing condensed chromatin. RESULTS: The flow cytometric method differentiated between cases of 'Sertoli cell-only' syndrome (complete absence of tetraploid and haploid cells) and cases where spermatogenesis was blocked in meiosis or in spermiogenesis. Flow cytometric histograms from semen samples from normozoospermic, oligozoospermic and azoospermic patients fell into patterns that correlated well with the results obtained from testis histology findings. CONCLUSIONS: The method described may serve as a simple, non-invasive and reliable assay to help clinicians counsel patients with severe male infertility before referring them for testicular surgery to locate spermatozoa for ICSI.     

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.     

Multiple pregnancies obtained by testicular spermatid injection in combination with intracytoplasmic sperm injection

Recent studies have shown that the injection of spermatid cells into the human oocyte can result in normal fertilization, embryo development and even delivery of live, healthy offspring. In our study, 23 azoospermic cases with severe spermatogenetic defects in their testicular biopsy are presented. The serum follicle stimulating hormone (FSH) concentrations and histopathological results of these males have been documented and compared in terms of fertilization and embryo development. The mean FSH value of the azoospermic males was 15.8 +/- 2.3 mIU/l, ranging from 1.6 to 39 mIU/l. Elongated spermatids were used in three cases only, as these more mature forms were mostly present in the testicular sample. In the remaining 20 cases, only round spermatids were found for use in intracytoplasmic sperm injection (ICSI). The fertilization rate with two pronuclei was 31.3%. The fertilization rate was found to be as high as 71% in three patients in the elongating and elongated spermatids group and as low as 25.6% in the round spermatid group. A few immature, non-motile spermatozoa were seen in only two cases from the elongated spermatid group. However, in the remaining cases, no spermatozoa were observed. The number of pronuclear (PN) arrest was quite high when only round spermatids were used (36.1%). Total fertilization failure was observed in two cases from the round spermatid group with Sertoli cell only and germ cell aplasia. A total of three pregnancies was achieved in 23 cases (13.0%), two from the elongated spermatid group and one from the round spermatid group. One biochemical pregnancy with a round spermatid resulted in an early spontaneous abortion and surprisingly, the remaining pregnancies were achieved with elongated spermatids resulting in multiple pregnancies. One twin and one triplet pregnancy were established following four embryo transfers in each patient. The twin pregnancy resulted in a live birth with two healthy babies; unfortunately, the triplet pregnancy ended in an abortion at 11 weeks. The use of testicular spermatids in the treatment of non-obstructive azoospermia may give hope by offering a novel treatment model. In cases with very severe spermatogenetic defect, even multiple pregnancies can be achieved with elongated spermatid cells by yielding a high implantation rate. However, the efficiency of round spermatids in achieving fertilization and pregnancy was disappointing.      

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.      

一种利用吖啶橙染色快速鉴定重力密度梯度沉降法中生精细胞类型的方法

目的建立一种重力梯度沉降法分离粗线期精母细胞,圆形及长形精子细胞的快速精确的镜检方法。方法利用重力密度梯度沉降法分离,将收集到的每管细胞分别放入96孔板中,每孔加入一定浓度的吖啶橙染液,于荧光显微镜下观察。结果通过分析对应荧光通道下3种生精细胞的细胞核/细胞质染色结果,可快速精确的分辨出粗线期精母细胞,圆形及长形精子细胞。结论建立了一种在重力密度梯度沉降法分离生精细胞过程中快速镜检检测粗线期精母细胞,圆形及长形精子细胞的方法。     

Reproductive capacity of spermatozoa from men with testicular failure.

Controversial reports have been published about the influence of sperm source and of the underlying testicular pathology on success rates of intracytoplasmic sperm injection (ICSI). In this controlled study, ICSI treatment cycles with testicular spermatozoa from men with obstructive and non-obstructive azoospermia were compared with ICSI ejaculated sperm cycles with semen parameters < or = 5 x 10(6)/ml and < or = 10% progressive motility. The control cases were matched for female age, rank of trial, female basal follicle-stimulating hormone serum concentrations and close proximity to the study group's procedure. The fertilization, cleavage, pregnancy and abortion rates were similar in matched groups irrespective of the type of azoospermia. However, the implantation rate in the non-obstructive azoospermic patient group was significantly lower than that in the matched ejaculated sperm group (13.4% versus 26%, P = 0.05). On the other hand, no impairment of the implantation rate was observed in the obstructive azoospermic patient group. These data show that testicular pathology has a negative impact on reproductive performance of testicular spermatozoa, resulting in a decreased implantation potential without any apparent effect on fertilization and early preimplantation development.     

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.     

Microsurgical TESE and the distribution of spermatogenesis in non-obstructive azoospermia

We wished to map the distribution of spermatogenesis in different regions of the testis in 58 men with non-obstructive azoospermia, and to develop a rational microsurgical strategy for the testicular sperm extraction (TESE) procedure. One goal was to maximize the chances for retrieving spermatozoa from such men, to minimize tissue loss and pain, and to preserve the chance for successful future procedures. Another goal was to expand upon the previously reported quantitative histological analysis of testicular tissue in 45 azoospermic men undergoing conventional TESE, this time using microsurgical as well as histological mapping. Tubular fullness observed at microsurgery and the presence of spermatozoa in the TESE specimen was compared with the quantitative histological analysis of spermatogenesis. Thus, our conclusions about the distribution of spermatogenesis are based on our experience with TESE in 103 consecutive cases of non-obstructive azoospermia. It was confirmed that men with non-obstructive azoospermia caused by germinal failure have a mean of 0 to 3 mature spermatids per seminiferous tubule in contrast to 17-35 mature spermatids per tubule in men with normal spermatogenesis and obstructive azoospermia. The former represented the threshold of quantitative spermatogenesis which must be exceeded in order for spermatozoa to 'spill over' into the ejaculate. Both testicular 'mapping' by multiple biopsy (n = 15) and microsurgical removal of contiguous strips of testicular tissue (n = 43) revealed a diffuse, rather than regional, quantitative distribution of spermatogenesis. A microsurgical approach resulted in the minimal amount of tissue loss and minimal-to-no pain (compared with the original 45 cases already reported). By this means it is often possible to immediately locate the few tubules with spermatogenesis at microsurgery, under local anaesthesia. But even in cases where greater amounts of tissue must be removed in order to find spermatozoa, the microsurgical TESE procedure prevents secondary testicular damage by protecting blood supply and preventing pain and atrophy from increased testicular pressure. Thus, future attempts at TESE-ICSI need not be compromised.     

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.