Comparison between chromatin condensation and morphology from testis biopsy extracted and ejaculated spermatozoa and their relationship to ICSI outcome

A significant association between male subfertility, imperfect spermiation and abnormal nuclear condensation has been suggested. The DNA content of spermatozoa might be responsible for inducing alterations in sperm morphology. The final nuclear shape, which is species-specific, depends on chromatin condensation during spermatogenesis as well as a precise organization of DNA within the nucleus. Many reports have described the association between disturbances in sperm chromatin condensation, morphology and male infertility. Chromatin condensation is achieved by gradual substitution of lysinerich somatic histones by testis-specific histone and finally by protamine. In this study two groups of patients were compared: the first consisted of 63 patients who had undergone intracytoplasmic sperm injection (ICSI) with freshly ejaculated spermatozoa whereas the second included 47 patients assigned to ICSI with testes biopsy-extracted spermatozoa. In both groups chromatin condensation was assessed by aniline blue staining and morphology evaluated according to strict criteria. The condensed chromatin and morphology of spermatozoa were significantly (P < 0.0001) less in the second group compared to the first. However the fertilization, cleavage, implantation and pregnancy rates were almost the same in both investigated groups. There was no significant difference between the two groups with respect to ICSI outcome. The percentage of chromatin condensation (nuclear maturity) and morphologically-normal spermatozoa were significantly higher (P < 0.0001) in the ejaculated spermatozoa than in those from testis biopsy but the ICSI outcome (fertilization, cleavage, implantation and pregnancy rates) was the same. In view of these results the fertilization capability and the embryo quality obtained using testis biopsy extracted spermatozoa is not influenced by chromatin condensation and sperm morphology in testicular sperm extraction (TESE)-ICSI programmes. Therefore, it could be said that neither chromatin condensation nor morphology of testis extracted sperm could predict the fertilization, implantation and pregnancy rate in TESE-ICSI programmes.     

精子DNA碎片率与男性年龄、精子活动力和体外受精结局关系的研究

目的 探讨精子DNA碎片率(DNA fragmentation index,DFI)与男性年龄、精液检查指标、体外受精(in vitro fertilization,IVF)的受精率、优质胚胎率、周期妊娠率和胚胎着床率等关系.方法 随机选取本院生殖中心111例IVF患者,使用流式细胞仪行精子DFI测定,DFI值按不同界...     

Success of intracytoplasmic sperm injection in couples with male and/or female chromosome aberrations

This paper reports on results of intracytoplasmic sperm injection (ICSI) in patients in whom constitutional or secondary chromosome aberrations were detected in the male and/or female partner. Out of 434 couples treated by ICSI (590 cycles), 16 couples (3.7%) were affected by constitutional chromosome aberrations and 96 (22.1%) by secondary chromosome aberrations. Constitutional chromosome aberrations were found in eight male and eight female patients. Couples with the aberration in the male showed significantly lower fertilization, implantation and pregnancy rates (P < 0.05). The occurrence of female constitutional chromosome aberrations led to lower fertilization rates but implantation and pregnancy rates were similar to a control group; however, a higher abortion rate was noted. In the group with secondary chromosome aberrations, 22 males and 59 females carried an abnormality and in 15 couples, both partners. Compared to the remaining (unaffected) 322 couples, fertilization and embryo transfer rates were reduced but implantation rates and pregnancy rates were not different. In all couples where an abortion occurred, mainly parental autosomal aberrations were involved (six out of eight). Our retrospective analysis shows that an unexpectedly high number of infertile couples in an ICSI programme are affected by chromosome aberrations, which in turn may explain the reduced fertilization rates observed in this subgroup of patients.      

The use of intracytoplasmic sperm injection with electroejaculates from anejaculatory men

Electroejaculation has been successfully used for sperm procurement in anejaculatory men desiring fertility. However, electroejaculates typically have normal sperm numbers but poor motility, morphology, and functional deficiencies. Here we report the pregnancy outcome of a series of couples undergoing combined electroejaculation and in-vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI). In all, 13 couples underwent a total of 18 cycles. The aetiologies of anejaculation included history of retroperitoneal lymph node dissection for testicular cancers, spinal cord injury and psychogenic causes. ICSI was performed on 192 oocytes, resulting in a fertilization rate of 75.5%. A total of 15 embryo transfers were performed using a total of 51 embryos. Clinical pregnancy rate, as defined by positive fetal heart rate(s) using vaginal sonography, was 55.6% per retrieval; implantation rate was 33.3% per embryo. These rates appear to be similar to those obtained in standard IVF for non-male factor infertility, or ICSI for male factor infertility. The use of ICSI for electroejaculates undoubtedly provides these couples with the highest chance of pregnancy.      

精子DNA碎片与活性氧的关系研究及对IVF结局的影响

目的探讨不育男性的精子DNA损伤程度与精液常规分析各项指标及活性氧的关系,以及对常规体外受精-胚胎移植(IVF-ET)结局的影响。方法接受常规IVF受精的夫妇135例,男性患者作为研究对象,按照精子DNA损伤率将患者分为3组:正常组(精子DNA损伤率0%～15%)79例、轻度损伤组(精子DNA损伤率15%～30%)49例、重度损伤组(DNA损伤率>30%)7例。分析精子DNA损伤对精液常规指标(体积、浓度、活动力和形态)的影响,观察三组患者精浆中过氧化氢(H2O2)、过氧化氢酶(CTA)浓度的变化及三组间受精率、卵裂率、优胚率、种植率、临床妊娠率的差异性。结果重度损伤组的精子活力(a+b)明显低于正常组和轻度损伤组(P<0.05),重度损伤组及轻度损伤组的精子畸形率明显高于正常组(P<0.05)。重度损伤组的优胚率明显低于正常组和轻度损伤组(P<0.05),三组间受精率、卵裂率、植入率和临床妊娠率无统计学差异。重度损伤组精浆中的H2O2浓度明显高于正常组和轻度损伤组(P<0.05),而CTA浓度无统计学差异。结论精子DNA损伤与高H2O2水平有关。精子DNA损伤可导致精液的活动力下降,精子畸形率增高,降低优胚率,不利于IVF-ET的结局。     

Testicular sperm retrieval by percutaneous fine needle sperm aspiration compared with testicular sperm extraction by open biopsy in men with non-obstructive azoospermia

The efficiency of testicular sperm retrieval by testicular fine needle aspiration (TEFNA) was compared with open biopsy and testicular sperm extraction (TESE), in 37 rigorously selected patients with non- obstructive azoospermia. All patients underwent TEFNA and TESE consecutively. Thus, each patient served as his own control. The case was regarded as successful if at least one testicular spermatozoon was found allowing intracytoplasmic sperm injection (ICSI) of at least one oocyte. The mean age of the male patients was 32.7 years (range 24-47). Whereas by TEFNA spermatozoa enabling performance of ICSI were found in only four patients out of 37 (11%), open biopsy and TESE yielded spermatozoa in 16 cases (43%). The negative predictive value of high serum follicle stimulating hormone (FSH) concentrations (> or =10 IU/l) (predicting failure to find spermatozoa for ICSI) was low (38.4%). The positive predictive value (predicting the chance to find spermatozoa for ICSI) of normal-sized testicle was not different from that of small- sized (<15 ml) testicle (50%). Complications included one case of testicular bleeding following fine needle aspiration, treated locally, and two cases of extratunical haematomata following TESE requiring no intervention. In patients with non-obstructive azoospermia, TEFNA has a significantly lower yield compared to TESE. Performance of ICSI with testicular sperm in these cases resulted in satisfactory fertilization and high embryo transfer rates. The implantation and pregnancy rates per embryo transfer were 13 and 29% respectively. Neither serum FSH values nor testicular size were predictive of the chances to find spermatozoa for ICSI. Some complications may occur even following TEFNA.      

Effect of abnormal sperm head morphology on the outcome of intracytoplasmic sperm injection in humans

The present study was designed to determine the efficacy of intracytoplasmic sperm injection (ICSI) using spermatozoa with abnormal head morphology in 17 cases with total teratozoospermia. A total of 160 oocytes were retrieved and 144 metaphase II oocytes were injected. The fertilization and cleavage rates were 50.7 and 93.2% respectively. Fertilization failure occurred in two couples. A total of 54 embryos were transferred and pregnancy rates per initiated and per embryo transfer cycle were 17.6 and 20.0% respectively, while the clinical pregnancy rates per initiated and embryo transfer cycle were 11.8 and 13.3%. The implantation rate was 3.7% (2/54). Out of two pregnancies achieved, one resulted in abortion in the first trimester. The ongoing pregnancy rates per initiated and embryo transfer cycle were 5.88% (1/17) and 6.6% (1/15) respectively. Although the implantation and ongoing pregnancy rates are very low, ICSI seems to be the only treatment modality in cases where teratozoospermia was total with 100% abnormal head morphology.      

ICSI in cases of sperm DNA damage: beneficial effect of oral antioxidant treatment

BACKGROUND: Most studies examining the use of ICSI for cases of elevated sperm DNA fragmentation report poor pregnancy and implantation rates. ICSI with testicular sperm samples has recently been suggested for these cases. Here we test a less invasive approach based on oral antioxidant treatment prior to ICSI with ejaculated spermatozoa. METHODS: Thirty-eight men with an elevated (> or =15%) percentage of DNA-fragmented spermatozoa in the ejaculate were treated with antioxidants (1 g vitamin C and 1 g vitamin E daily) for 2 months after one failed ICSI attempt. In 29 (76%) of these cases this treatment led to a decrease in the percentage of DNA-fragmented spermatozoa, and a second ICSI attempt was performed. Outcomes of the two attempts were compared. RESULTS: No differences in fertilization and cleavage rates or in embryo morphology were found between the ICSI attempts performed before and after the antioxidant treatment. However, a marked improvement of clinical pregnancy (48.2% versus 6.9%) and implantation (19.6% versus 2.2%) rates was observed after the antioxidant treatment as compared with the pretreatment ICSI outcomes. CONCLUSIONS: Oral antioxidant treatment appears to improve ICSI outcomes in those patiens with sperm DNA damage, in whom this treatment reduces the percentage of damaged spermatozoa.     

人精子膜功能完整性与精液参数的关系

目的探讨精子膜功能完整性与精液参数的关系。方法收集503例精液样本,其中对照组149例,不育组354例。根据WHO标准分析精子密度、活率、活力、精液白细胞浓度、精子形态和精子尾部低渗膨胀(HOS)率。结果不育组精子尾部低渗膨胀率显著低于对照组(P<0.05)。与对照组相比,HOS正常不育组和HOS异常不育组形态正常精子率、密度、活力、活率均显著降低(P<0.05),头部异常精子率、精液白细胞浓度显著增高(P<0.05);此外,HOS正常不育组的尾部异常精子率显著高于对照组,其精子活力、活率均显著高于HOS异常不育组(P<0.05)。精子HOS与精子密度、活力、活率呈显著正相关,与精液白细胞浓度呈显著负相关(P<0.05)。结论精子膜功能可能与精液参数密切相关,精子膜功能是评价男性生育力的重要指标。     

A comparative analysis of embryo implantation potential in patients with severe teratozoospermia undergoing in-vitro fertilization with a high insemination concentration or intracytoplasmic sperm injection

The objective of this study was to assess fertilization, implantationand pregnancy rates in infertile patients with severe teratozoospermia[P (poor prognosis) pattern sperm morphology assessed by strictcriteria] treated by in-vitro fertilization (IVF) using a highinsemination concentration (HIC), or by intracytoplasmic sperminjection (ICSI). This was a retrospective cohort study performedin an academic tertiary institution. The outcome of 115 consecutiveICSI cycles was compared to that of a similar number of cyclesof IVF with HIC performed during a similar time frame and matchedby woman's age and basal serum (cycle day 3) follicle stimulatinghormone concentrations. The inclusion criteria were sperm morphology4% normal forms (P pattern) and 1 x106 total motile spermatozoaper ejaculate. The diploid fertilization rate in the HIC-IVFgroup was 86% and in the ICSI group 68% (P < 0.05). Importantly,an equal number of embryos was transferred to both groups ofpatients. The morphological quality of the embryos (proportionof transfers having superior morphology embryo scores) was significantlybetter in the ICSI group than in the patients receiving HIC-IVF.Although there was a clear trend for better implantation andpregnancy rates in the ICSI group, these differences were notstatistically significant We conclude that, although HIC-IVFresulted in a higher fertilization rate than ICSI in patientswith severe teratozoospermia, ICSI produced a significantlyhigher proportion of morphologically superior embryos with atendency towards a higher implantation potential. Therefore,teratozoospermic patients having adequate numbers of motilespermatozoa should be offered ICSI as an alternative to modified(HIC) IVF treatment.     

不同来源精子对ICSI助孕结局的影响

研究不同来源的精子对于ICSI辅助生殖助孕结局的影响。对本中心115例卵胞浆内单精子显微注射（ICSI）治疗周期进行回顾性分析,比较分别使用射出精液来源精子以及睾丸、附睾穿刺来源精子行ICSI后,受精率、卵裂率、优质胚胎率以及妊娠率、种植率等各项指标。精子来源的不同造成了受精率的显著差异,一旦受精完成,随后胚胎的发育及种植将更多的依赖于患者的年龄。     

Potential adverse effect of sperm DNA damage on embryo quality after ICSI

BACKGROUND: Sperm DNA damage is prevalent amongst infertile men and has been shown to strongly impact adversely natural reproduction, intrauterine insemination-assisted reproduction and to a lesser degree IVF/ICSI fertilization. The objective of this study was to examine further the relationship between sperm DNA denaturation (DD) and reproductive outcomes after ICSI. METHODS: We evaluated infertile couples (n = 60) undergoing IVF/ICSI at a single centre. Sperm DD was assessed by flow cytometry analysis of Acridine Orange-treated sperm and expressed as the percentage of sperm with DD. Couples were sub-grouped according to sperm DD results: group 1: 0-15%; group 2: >15-30%; group 3: >30%. RESULTS: There were no differences between the three groups with regard to maternal age, sperm parameters, oocyte maturation, fertilization or pregnancy rates. Group 3 had a significantly higher rate of multinucleation among the embryo cohorts compared to either groups 1 or 2 (20% versus 10% and 8% respectively, P = 0.04). There was a statistically insignificant trend toward an increased spontaneous pregnancy loss rate in group 3 (P =0.50). CONCLUSION: Although we did not observe significant relationships between sperm DNA damage and either fertilization or pregnancy rates, the potential adverse effect of sperm DNA damage on embryo quality and spontaneous pregnancy loss is concerning.     

High implantation and pregnancy rates with testicular sperm extraction and intracytoplasmic sperm injection in obstructive and non-obstructive azoospermia

Thirty-two infertile couples with obstructive and non-obstructiveazoospermia were included in this study. Testicular sperm extraction(TESE) was performed in 16 obstructive azoospermic cases wheremicrosurgical sperm aspiration (MESA) or percutaneous spermaspiration (PESA) were impossible because of totally destroyedepididymis and 16 non-obstructive azoospermia cases with severespermatogenetic defect where the testicles were the only sourceof sperm cells. A total of 288 oocytes was obtained from 32females and 84% were injected. The fertilization rates (FR)with 2 pronuclei (PN) and cleavage rate were 50.8 and 68.2%respectively. A total of 15 pregnancies was achieved (53% perembryo transfer), nine from the obstructive and six from thenon-obstructive group. Four pregnancies resulted in clinicalabortion (26.6%). The ongoing pregnancy rate was 39.2% per embryotransfer (ET) and 343% per started cycle. A high implantationrate was also achieved (26.6% in non-obstructive and 30% inobstructive azoospermia group). Using testicular spermatozoain combination with ICSI in both obstructive and non-obstructiveazoospermic groups, high implantation and pregnancy rates canbe achieved.     

Low pregnancy rate is achieved in patients treated with intracytoplasmic sperm injection due to previous low or failed fertilization in in-vitro fertilization

The main indications for intracytoplasmic sperm injection (ICSI) are severe male factor and fertilization failure or a low fertilization rate in previous in-vitro fertilization (IVF) treatments. The fertilization and pregnancy rates after ICSI, however, are seldom reported separately for these two different indications. The aim of this study was to compare the treatment outcome and pregnancy rate after ICSI between 65 patients with previous failed fertilization or a low fertilization rate without male factor, and 219 patients with a primary male factor. From the 2726 oocytes collected, 2087 (77%) were micro-injected and 1355 (65%) achieved normal fertilization. The oocyte fertilization rate was similar in the group with previous failed fertilization or a low fertilization rate and the group with a male factor (65 and 65% respectively), as was the cleavage rate of normally fertilized oocytes (92 and 94% respectively). Despite the similar fertilization and cleavage rates and the similar number and morphological quality of embryos transferred in both groups, the pregnancy rate was significantly lower (P < 0.05) in the group with previous failed fertilization or a low fertilization rate than in the group with a male factor (19.6 versus 33.5% respectively; 95% confidence intervals for the difference, 2-26%). The implantation rate was also lower (P = 0.01) in patients with previous failed fertilization or a low fertilization rate (9.6%) than in the group with a male factor (19.5%). We conclude that patients with previous failed fertilization or a low fertilization rate in standard IVF without male factor have a significantly smaller chance of becoming pregnant after subsequent ICSI than patients with a primary male factor. This poor outcome probably reflects intrinsic oocyte defects not bypassed by ICSI.      

First polar body morphology before ICSI is not related to embryo quality or pregnancy rate

BACKGROUND: The aim of this study was to analyse the relationship between the first polar body (1st PB) morphology and the fertilization rate, cleavage rate, embryo quality, pregnancy and implantation rate. METHODS: This was a retrospective study on 167 consecutive cycles undergoing assisted reproduction with ICSI. The 1st PB morphology was evaluated at the moment of ICSI in the 596 injected oocytes and it was coded as intact or fragmented. The fertilization rate, cleavage rate, embryo quality (three grades), pregnancy rate, implantation rate and the time elapsed between oocyte retrieval and ICSI were evaluated. The 1st PB morphology was checked twice (denudation and ICSI) in a random sample of 180 oocytes in order to verify the effect of the in vitro culture. RESULTS: No significant relationship was found between the 1st PB morphology and the fertilization rate (P=0.703), cleavage rate (P=0.055), embryo quality (P=0.673), pregnancy rate (P=0.201) and implantation rate (P=0.511). A significant positive relationship (P=0.006) was found between the frequency of the 1st PB fragmentation and the time elapsed between denudation and ICSI. The pregnancy rate was significantly higher (P=0.008) when oocytes were injected between 5 and 7 h after retrieval rather than earlier or later. CONCLUSIONS: Our data suggest that the embryo quality, pregnancy rate and implantation rate are not related to the 1st PB fragmentation. The time which elapses between the oocyte retrieval and ICSI should be maintained at approximately 6 h in order to obtain optimal results.     

Efficient treatment of infertility due to sperm DNA damage by ICSI with testicular spermatozoa

BACKGROUND: Sperm DNA damage (fragmentation) is a recently discovered cause of male infertility for which no efficient treatment has yet been found. Previous findings have suggested that clinically relevant sperm DNA damage may occur at the post-testicular level. This study was undertaken to assess the clinical usefulness of ICSI with testicular spermatozoa in this indication. METHODS: The percentage of spermatozoa with fragmented DNA, assessed by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling assay, and ICSI outcomes were compared in two sequential attempts performed, respectively, with ejaculated and testicular spermatozoa in 18 men with increased sperm DNA fragmentation. RESULTS: The incidence of DNA fragmentation was markedly lower in testicular spermatozoa as compared with ejaculated spermatozoa. No differences in fertilization and cleavage rates and in embryo morphological grade were found between the ICSI attempts performed with ejaculated and with testicular spermatozoa. However, eight ongoing clinical pregnancies (four singleton and four twin) were achieved by ICSI with testicular spermatozoa (44.4% pregnancy rate; 20.7% implantation rate), whereas ICSI with ejaculated spermatozoa led to only one pregnancy which was spontaneously aborted. CONCLUSIONS: These data show that ICSI with testicular spermatozoa provides the first efficient assisted reproduction treatment option for men with high levels of sperm DNA damage.     

卵胞浆内单精子注射在男性与非男性不育患者中的应用

目的比较卵胞浆内单精子注射（ICSI）在男性不育与非男性不育患者中的助孕结局。方法回顾性分析2009年1月至2010年12月在我院接受ICSI治疗109个周期,将其分组：A组：男性不育73周期,包括射精组43个周期,手术抽吸精组30个周期,B组：非男性不育36周期,均为射精组。结果①A组：手术抽吸精组中女方年龄、平均获卵数与B组比较结果有统计学意义,P〈0.05;②射精组与手术抽吸精组：两组的正常受精率、卵裂率、优胚率、种植率、妊娠率比较,结果无统计学差异,P〉0.05;③B组中移植（ET）胚胎数、种植率、妊娠率、流产率与A组相比较,结果有统计学差异,P〈0.05;与正常受精率、卵裂率、优胚率,结果比较无统计学差异,P〉0.05。结论①ICSI仍是治疗男性因素的首要指证;②取精方式对妊娠率无影响;③对非男性因素,能获得较高的受精率,有临床治疗价值。     

Fertility of ejaculated and testicular megalohead spermatozoa with intracytoplasmic sperm injection

In this study the fertility and outcome of intracytoplasmic sperm injection (ICSI) using megalohead spermatozoa from the ejaculates and testicles was evaluated. Seventeen males with megalohead and pinhead sperm forms in their ejaculate were studied in 22 cycles. A high number of sperm heads without tails and abundant round spermatid forms were commonly observed. Round-headed spermatozoa were seldom accompanied by these severely abnormal spermatozoa. The majority of megalohead spermatozoa were observed to have multiple tails, were predominant in the sample, and were used for ICSI. Ejaculated megalohead spermatozoa were used for ICSI in 15 cycles, while testicular spermatozoa were used in seven cycles where there were no vital spermatozoa or spermatozoa of low vitality in the ejaculate. The same abnormal morphology was observed in the testicles as in the ejaculated spermatozoa in the same males. Mean (+/- SD) low motility 4.7 +/- 5.6% and sperm count (3.8 +/- 4.19 x 10(6)) were common findings in these severely teratozoospermic patients. A low fertilization rate (43.2%) was achieved by using megalohead sperm forms (group I, n = 17) in comparison with the control group (60.2%) which had zero normal sperm morphology according to strict criteria (group II, n = 30) (P <0.01). Furthermore, a low pregnancy rate (9.1%) was obtained in the megalohead sperm group in comparison with the control group (40%) (P <0.05). Low fertilization and pregnancy rates may be due to a high incidence of chromosomal abnormalities from severely defective spermatozoa in the ejaculate. Couples should be counselled and warned about possible low fertilization and pregnancy rates with ICSI when only pinhead and megalohead forms with a high number of sperm heads without tails are present in the ejaculate.     

The impact of endometriosis in couples undergoing intracytoplasmic sperm injection because of male infertility

To assess the impact of endometriosis on intracytoplasmic sperm injection (ICSI) outcome, we have retrospectively evaluated 980 ICSI cycles, comparing the results of women with and without endometriosis. A total of 101 cycles was identified in which various degrees of endometriosis were involved, and in the remaining 879 cycles, male infertility was the only cause of infertility. Ejaculated spermatozoa were microinjected in all cycles. There was a significant reduction (P = 0.004) in the number of oocytes retrieved from women with endometriosis as compared to those without endometriosis. However, there were no significant differences in either fertilization or pregnancy and implantation rates between women with or without endometriosis. We conclude that the presence of endometriosis in patients undergoing ICSI because of severe male infertility does not affect fertilization, pregnancy and implantation rates, although significantly fewer oocytes are retrieved from patients with endometriosis.      

Pregnancy with frozen-thawed and fresh testicular biopsy after motile and immotile sperm microinjection, using the mechanical touch technique to assess viability

BACKGROUND: There are few reports of pregnancy using immotile sperm, and none using a purely mechanical assessment of viability. METHODS: In this pilot study, we retrospectively analysed 66 cycles in 61 patients with determinant male factor, recording rates of fertilization, implantation, normal pregnancy and take-home babies achieved with ICSI. Sperm selection was based on morphologically normal appearance under the inverted microscope. Viability of immotile spermatozoa was assessed by the mechanical touch technique to observe tail flexibility and tail shape recovery. RESULTS: Of 17 ICSI cycles using frozen-thawed testicular sperm, six microinjected with immotile and 11 with motile sperm, we achieved fertilization rates of 65.7 and 74.3%, respectively, and five pregnancies (two and three, respectively). Of 49 ICSI cycles using fresh testicular sperm, 10 microinjected with immotile and 39 with motile sperm, we achieved fertilization rates of 73.4 and 64.4%, respectively, and 12 pregnancies (three and nine, respectively). CONCLUSIONS: Immotile (fresh and frozen-thawed) testicular sperm of normal morphological appearance can be used to achieve clinical pregnancy with ICSI. Our results strongly suggest that immotile sperm viability can be assessed by the mechanical touch technique.