Intracytoplasmic injection of round and elongated spermatids from azoospermic patients: results and review

Microinjection is established as the method of choice in the treatment of severe male factor infertility as well as in azoospermic patients. Recent studies have shown that fertilization and cleavage can be achieved by injection of ejaculated as well as testicular elongated spermatids into oocytes. Here we report on the two first pregnancies worldwide resulting from elongated spermatid injection from frozen-thawed testicular tissue. Four patients with complete Sertoli cell-only syndrome (SCOS) and two with spermatogenetic maturation arrest were included in our microinjection programme. Tissues from open testicular biopsies were cryopreserved until the time of follicle puncture. A total of 67 oocytes were harvested. In the two patients with maturation arrest, cryopreserved elongated spermatids were successfully injected, while in two of the other four SCOS patients only cryopreserved round spermatids were available to be injected into the oocytes. Out of 18 injected oocytes, 10 were fertilized in the first group, while nine out of 49 injected oocytes showed fertilization and cleavage in the second group. Two clinical pregnancies were achieved with elongated spermatids from frozen-thawed testicular tissue, while no pregnancy was established in the case of round spermatids. This study confirms that fertilization, cleavage and pregnancy can be successfully achieved in cases with spermatogenetic maturation arrest by injecting cryopreserved elongated spermatids into oocytes. The literature on pregnancies following spermatid injection, as well as the problems using this technique and possible risks, are discussed.     

Comparison of oocyte activation and Ca2+ oscillation-inducing abilities of round/elongated spermatids of mouse, hamster, rat, rabbit and human assessed by mouse oocyte activation assay

Oocyte activation and Ca2+ oscillation-inducing abilities of round spermatid (ROS) and elongated spermatid (ELS) of some rodents and human were assessed by their injection into mouse (B6D2F1) oocytes (mouse test). With mice (B6D2F1, ICR) and rat, ROS displayed no oocyte activation or Ca2+ oscillation-inducing abilities. Although ELS could induce activation at 87, 86 and 31% of injected oocytes respectively, most of the intracellular calcium concentration ([Ca2+]i) responses of ELS-injected oocytes did not show oscillation patterns; only several transient [Ca2+]i rises (transient pattern) were seen. Similarly, with hamster, rabbit and human, while ROS could induce oocyte activation efficiently (70, 71 and 52% respectively), most of the [Ca2+]i patterns of injected oocytes were transient patterns, and not oscillation patterns. When ROS nuclei only from these latter species were injected into mouse oocytes, most of the oocytes could not be activated. [Ca2+]i patterns of oocytes injected with immature sperm cells changed from transient pattern to oscillation pattern while the cells were maturing into spermatozoa. With hamster ROS, oocyte-activating factor was found to be distributed mainly in the cytoplasm. It was interesting that there is a dissociation between the timings of appearance of oocyte activation and that of Ca2+ oscillation of oocytes injected with developing immature sperm cells.     

Correlation of testicular pathology and sperm extraction in azoospermic men with ejaculated spermatids detected by immunofluorescent localization

Limiting testicular biopsy for intracytoplasmic sperm injection (ICSI) to those with a high chance of having testicular spermatozoa has not been possible because of the poor predictive value of current clinical and laboratory methods. In order to predict testicular pathology and sperm extraction, we characterised the semen of 28 men with azoospermia due to gonadal failure in terms of the presence of spermatids using an immunological method. The results were compared with the assessment of testicular biopsies by histology and the extraction of spermatozoa into culture medium. Washed cellular elements in the ejaculate were smeared on microscope slides and fixed in 100% methanol, before incubation with acrosome-specific monoclonal antibody (18.6), fluorescein isothiocyanate-labelled anti-mouse goat IgG, and examination by epifluorescent microscopy. Semen from men with oligozoospermia and obstructive azoospermia served as positive and negative controls, respectively. Twelve patients who had positive immunofluorescence (one or more spermatids present) had spermatozoa retrieved from their testes (five hypospermatogenesis, seven focal spermatogenesis), and 16 patients with negative immunofluorescence (spermatids absent) had apparent Sertoli cell-only syndrome (12) or maturation arrest histological pattern (four). However, four patients with apparent Sertoli cell-only syndrome had testicular spermatozoa present after extraction from the biopsy. Plasma follicle stimulating hormone concentration and testicular volume did not predict retrieval of seminal spermatids or testicular spermatozoa. We conclude that the immunofluorescent localization of one or more spermatids in the ejaculate can be used to predict the likelihood of obtaining testicular spermatozoa for ICSI. However, in some patients with Sertoli cell-only syndrome, spermatozoa could still be recovered in the absence of apparent seminal spermatids.      

Intracytoplasmic sperm injection: correlation of oocyte grade based on polar body, perivitelline space and cytoplasmic inclusions with fertilization rate and embryo quality

The extent to which the morphology of the oocyte at the light microscopy level is related to the results of intracytoplasmic sperm injection (ICSI) is controversial. In this study, after cumulus removal, oocytes were graded into four groups according to the status of the first polar body, size of the perivitelline space and the presence of cytoplasmic inclusions. Oocyte data from 65 consecutive patients were reviewed. The results showed that, for oocytes without cytoplasmic inclusions, the fertilization rate and embryo development beyond 2-cell stage were significantly lower (P < 0.01) in the oocytes at grade 1-2 (poor) than those in oocytes at grade 3-4 (good). Grade 4 oocytes without inclusions gave the highest proportion (66.7%) of good embryos with grading 1-2 (grade 1 best; P < 0.01). A higher proportion of grade 1-2 oocytes (44.7%; P < 0.05) was obtained from patients older than 35 years. More oocytes containing cytoplasmic inclusions were seen in patients diagnosed as having female factor infertility (24.9%; P < 0.01) and older than 35 years (26.5%; P < 0.05) compared to patients with male factor infertility and younger than 35 years. The fertilization rate and embryo development were not associated with the oestradiol concentration on the day of human chorionic gonadotrophin administration or the total number of oocytes retrieved. The results suggest that human oocyte grading based on the triple factors first polar body, size of perivitelline space and cytoplasmic inclusions is related significantly to fertilization rate and embryo quality after ICSI.      

Light and electron microscopic analysis of human testicular spermatozoa and spermatids from frozen and thawed testicular biopsies.

The morphological changes caused by freezing and thawing human testicular spermatozoa have been assessed here. Retrieval of testicular biopsies was carried out on six patients with obstructive azoospermia preparatory to intracytoplasmic sperm injection (ICSI). Light microscope analysis was carried out on testicular cells and ultrastructural analysis was carried out on spermatozoa and different spermatid stages before and after the freezing procedure. Upon examination under light microscopy, all germ cells presented increased vacuolization in their cytoplasm and shrinkage or swelling of the nuclei and cytoplasmic membranes. These altered structures were accentuated in the spermatocyte I cell which often presented disrupted membranes. The ultrastructural findings under transmission electron microscopy demonstrated that after freezing and thawing the major types of cryoinjury were the swelling and rupture of inner and outer acrosomal and plasma membranes. The acrosome material often appeared as dispersed material or as condensed spots or was even lost. Such damage was observed mainly at the spermatozoa and late spermatid stages. We conclude that the freezing and thawing of testicular biopsies causes similar morphological damage to testicular spermatozoa and frozen-thawed ejaculated spermatozoa. It is still unclear whether these changes in testicular spermatozoa after freezing and thawing may compromise its use in the ICSI procedure.     

Fertilization and early embryolgoy: Human oocyte activation following intracytoplasmic injection: the role of the sperm cell

The aim of this study was to investigate whether the human spermatozoonparticipates in the activation of human oocytes following intracytoplasmicsperm injection (ICSI) and if so, by what mechanism. In thefirst series of experiments, we randomized human oocytes whichhad remained unfertilized after in-vitro fertilization (IVF)or ICSI, for intracytoplasmic injection with live spermatozoa,spermatozoa presumed to be dead and no spermatozoa. Secondly,unfertilized human oocytes and freshly ovulated mouse oocyteswere randomized for intracytoplasmic and sub-zonal injectionwith human sperm cytosolic fraction (CF) before and after heattreatment. We found that oocyte injection with initially motilespermatozoa induces human oocyte activation at a significantlyhigher rate than injection with dead spermatozoa (61 versus0%; P < 0.001) or injection without a spermatozoon (61 versus14%; P < 0.001). Intracytoplasmic injection of CF activatedboth human and mouse oocytes at the same rate as sperm injectionof human oocytes (activation rates of 70 and 65% respectively).This effect was greatly reduced by heat treatment of the CF.From these experiments we conclude firstly that the human spermatozooninjected intracytoplasmically contributes to human oocyte activationand secondly that the spermatozoon releases into the oocytea heat-sensitive, intracellularly active factor, which is notspecies-specific.     

Sperm-induced oocyte activation in the rhesus monkey: nuclear and cytoplasmic changes following intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) has increased the potential of the assisted reproductive technologies to propagate mammalian species and has provided an opportunity for research into cell cycle control and the mechanisms involved in sperm-induced oocyte activation. We have investigated the efficacy of ICSI in the rhesus monkey, the mechanism of fertilization following sperm injection and the cytoskeletal rearrangement that occurs upon oocyte activation. These studies were conducted on mature, and to a lesser extent, immature oocytes. Ejaculated spermatozoa, washed, capacitated and activated before immobilization, were injected into oocytes using conventional ICSI methodology. Sperm injection into mature oocytes induced oocyte activation (19/22; 86%) and pronuclear formation. In contrast, sham- injected oocytes did not activate readily (2/16; 13%). To localize oocyte activation factor(s), spermatozoa were separated mechanically into heads and tails which were then injected individually into mature oocytes. Activation occurred in 87% (20/23) of oocytes receiving heads. After tail injection, a single microtubule aster was nucleated and one pronucleus (PN) was seen in four of 21 oocytes. Intracytoplasmic injection of sperm extract (SE) resulted in oocyte activation at a significantly higher rate than occurred following sham injection (76 versus 13%). Sperm-induced oocyte activation was also evaluated in immature metaphase (MI) oocytes; activation occurred in 46% (12/26) of cases; however, only 8% of the activated oocytes exhibited 2 PN. Finally, beta-tubulin localization in untreated and taxol-treated oocytes was established as a marker for cytoplasmic changes associated with oocyte activation. These results are consistent with the hypothesis that spermatozoa contain an oocyte activating factor(s) which is primarily localized in the sperm head. Moreover, an activation response is limited to mature oocytes and is accompanied by cytoskeletal changes analogous to those seen following conventional fertilization.      

Fertilization and early embryology: Intracytoplasmic sperm injection does not overcome an oocyte defect in previous fertilization failure with conventional in-vitro fertilization and normal spermatozoa

A cohort comprising a total of 447 oocyte aspirations due tomale factors (n±258) or to previous fertilization failureby IVF in the presence of normal sperm parameters (n±189)was studied. We found a significantly reduced implantation andpregnancy rate per transfer in the group with previously failedIVF attempts compared to the male factor group (P <0.001).No differences were found in age, number of oocytes retrieved,number of embryos transferred or quality of embryos scored atthe time of transfer. However, the fertilization and cleavagerates were found to be reduced in the group with previous failedIVF cycles. It is therefore concluded that previous fertilizationfailure despite normal sperm parameters in an 1VF cyde may notbe alleviated by the intracytoplasmlc sperm injec tion (ICSI)procedure. These patients might suffer from oocyte defects aswell.     

Oocyte activation and Ca(2+) oscillation-inducing abilities of mouse round/elongated spermatids and the developmental capacities of embryos from spermatid injection

To investigate differences in fertilization mechanisms and the potential clinical use of round/elongated spermatid, we conducted detailed studies of oocyte activation and Ca(2+) oscillation-inducing abilities in these immature sperm cells and compared these abilities against those of mature spermatozoa. When round spermatids from B(6)D(2)F(1) mice were injected, none of the oocytes was activated and no intracellular Ca(2+) ([Ca(2+)](i)) increases were observed. Elongated spermatids could induce activation normally in 87% of injected oocytes, but Ca(2+) oscillation could not be induced at all and most of the oocytes (94%) exhibited only several transient [Ca(2+)](i) rises (transient patterns). Because normal offspring could be obtained when embryos through elongated spermatid injection were transferred to foster mothers, it seems that a normal oscillation pattern of [Ca(2+)](i) is not essential for normal fertilization and embryo development. [Ca(2+)](i) patterns of injected oocytes changed from transient patterns to oscillation patterns while the injected immature sperm cells were maturing to spermatozoa. Dissociations were seen between the timing of appearance of oocyte activation and that of Ca(2+) oscillation-inducing abilities in maturing sperm cells. These dissociations may be due to differences in the thresholds to oocyte activation and Ca(2+) oscillation-inducing factor for inducing oocyte activation and Ca(2+) oscillation.     

Fertilization and early embryology: Time-course of oocyte activation, pronucleus formation and cleavage in human oocytes fertilized by intracytoplasmic sperm injection

Knowledge of the timing of the stages of fertilization in humansis still limited because the time of gamete fusion is not knownwhen pre-ovulatory or in-vitro matured cumulus-enclosed oocytesare inseminated. We therefore studied the morphological nuclearchanges in 14 patients' oocytes by means of light microscopicobservation at 2, 4, 6, 8, 16, 18 and at 20 h after intracytoplasmicsingle sperm injection (ICSI). A total of 144 metaphase II oocyteswere injected with the spermatozoa of the patients' partners.Out of the 134 oocytes that survived the injection, 93 displayedtwo pronuclei in the course of the observation period (69%).Out of the 93 normally fertilized oocytes, 21 extruded the secondpolar body at 2 h after micro-injection (23%) and 63 oocytesat 4 h (68%). Pronuclei appeared as early as 6 h after ICSIin 16 normally fertilized oocytes (17%). At 8 h, 75 (80%) oocyteshad two visible pronuclei, at 16 h 92 (99%), at 18 h 76 (82%)and at 20 h 63 (68%). In 24 oocytes (26%) the appearance ofpronuclei was asynchronous, while the disappearance of the pronucleiwas always synchronous, except in one oocyte. Nine of the 134successfully injected oocytes showed three equal-sized pronuclei(6.7%). Four of the nine multi-pronucleated oocytes did notextrude the second polar body at all, while the time sequenceof appearance of pronuclei was similar to that of the normallyfertilized oocytes. From this study it can be concluded thatmany of the oocytes with two pronuclei complete meiosis II by2 h and that pronucleus formation is complete imeiosis II by2 h and that pronucleus formation is complete in the majorityof these oocytes by 8 h after ICSI.     

Calcium responses of human oocytes after intracytoplasmic injection of leukocytes, spermatocytes and round spermatids

Oocyte activation in mammals involves the action of a solublesperm factor (SSF) that enables oocytes to develop a characteristicseries of Ca²⁺ spikes (Ca²⁺ oscillations driving oocyte activationafter intracytoplasmic sperm injection (ICSI). With an appropriateinjection technique, Ca²⁺ oscillations do not develop spontaneouslyafter ICSI but can be triggered by subsequent treatment of sperm-injectedoocytes with Ca²⁺ ionophore. Here we show that Ca²⁺ oscillations,quite similar to those developing after ICSI, can be triggeredby the ionophore treatment in human oocytes previously injectedwith human round spermatids. In contrast, oocytes injected withearlier spermatogenic cells (primary and secondary spermatocytes)and with non-germ cells (polymorphonuclear leukocytes) did notdevelop Ca²⁺ oscillations after the ionophore challenge althoughthe subsequent injection of SSF did induce typical Ca²⁺ oscillationsin these oocytes. Disintegration of the plasma membrane of theinjected cells was detected in all cases by transmission electronmicroscopy. Thus, the absence of the typical oscillatory Ca²⁺response in spermatocyte-injected oocytes was due to the actualdeficiency of SSF in the spermatocytes rather than to a defectiveresponsiveness of the injected oocytes or to the failure ofSSF release into the oocyte cytoplasm. The ability of humanround spermatids to induce a response to calcium in oocytesthat is similar to that induced by mature spermatozoa may beimportant for normal embryonic development after spermatid conception. calcium/leukocyte/oocyte activation/spermatid/spermatocyte     

The nature and dissemination of UHMWPE wear debris retrieved from periprosthetic tissue of THR

The role of wear debris in provoking joint replacement failure through bone resorption is now supported by much research. This study presents the analysis of 104 tissue samples using laser diffraction wear particle analysis in conjunction with standard histologic methods. The number and volume distributions were correlated to a range of joint and patient parameters. The median particle diameter by number was 0.69 microm. No particles smaller than 0.113 microm were resolved. No variation in terms of particle distribution was found among joint types. The ability of particles to migrate away from their point of origin was found to be inversely proportional to their size. The numbers of particles per gram of tissue found in various regions around the prosthesis varied little. Further, the numbers of particles in tissue samples shown to have a chronic foreign-body reaction was > 1 x 10(9) particles/gram.     

Timing of oocyte activation, pronucleus formation and cleavage in humans after intracytoplasmic sperm injection (ICSI) with testicular spermatozoa and after ICSI or in-vitro fertilization on sibling oocytes with ejaculated spermatozoa

In the first study, we evaluated 101 oocytes [2, 4, 6, 8, 16, 18 and 20 h after intracytoplasmic sperm injection (ICSI)] that had been microinjected with testicular spermatozoa. Of the 70 normally fertilized oocytes (69%) 30 (43%) had two pronuclei by 6 h after ICSI. Fifty-one (73%) by 8 h, 69 (99%) by 16 h and four of them by 20 h cleaved to the 2-cell stage. In the second study, 95 cumulus-corona- oocyte complexes (CCOC) were divided into two groups. Forty-seven CCOC were inseminated by conventional in-vitro fertilization (IVF) and 40 metaphase-II oocytes by ICSI. Oocytes were evaluated at 2, 4, 6 (only after ICSI), 8, 10, 12, 18, 20, 22, 24, 26, 28 and 30 h after both ICSI and IVF. After IVF, 35 oocytes were fertilized normally (75%), four of which (11%) had two pronuclei by 8 h, 11 (31%) by 10 h, 27 (77%) by 12 h and 35 (100%) by 14 h. The first cleavages had occurred by 24 h after insemination (four oocytes, 11%). After ICSI, 34 oocytes were fertilized normally (79%), 13 of which (38%) had two pronuclei by 6 h, 27 (79%) by 8 h and 32 (94%) by 10 h. Three oocytes cleaved by 20 h after microinjection (9%) and 19 by 24 h (56%). Pronuclei developed asynchronously in six oocytes after ICSI (18%) as opposed to 16 oocytes after IVF (46%). The results of this study suggest that the timing of pronuclear formation is no different when a testicular spermatozoon is microinjected into the oocytes from when an ejaculated spermatozoon is injected. Secondly, pronuclear development and first cleavage generally take place 4 h sooner after ICSI than after IVF. On the other hand, a higher proportion of oocytes develop two pronuclei asynchronously after IVF than after ICSI.      

Endometriosis-related pathology: a discussion of selected uncommon benign,premalignant and malignant lesions

Endometriosis is an extremely common condition and, in most cases, establishing a histological diagnosis is straightforward, although a variety of benign alterations may result in problems with interpretation. In this review, I discuss selected uncommon variants of endometriosis or benign alterations that may result in diagnostic problems. The topics covered include the contentious issue of so-called atypical endometriosis, stromal endometriosis, polypoid endometriosis, and the association of endometriosis with florid mesothelial hyperplasia. The propensity of endometriosis to undergo neoplastic transformation (especially to endometrioid and clear cell carcinoma) is well known. Selected issues relating to the various neoplasms that can arise in endometriosis are discussed, with a particular concentration on unusual variants of endometrioid carcinoma that result in a disproportionately high number of issues in referral practice. The propensity of ovarian endometrioid carcinomas to show an unexpected (‘aberrant’) immunophenotype with positive staining with ‘intestinal’ markers and negative staining with Mullerian markers is also discussed. Uncommon tumour types that may arise in endometriosis, namely seromucinous neoplasms, mesonephric-like carcinomas, and somatically derived yolk sac tumours, are also covered.     

Correlation between motility of testicular spermatozoa, testicular histology and the outcome of intracytoplasmic sperm injection

The objective of the present study was to analyse the influence of motility on the results of intracytoplasmic sperm injection (ICSI) when testicular spermatozoa are used for microinjection and to correlate this with testicular histology. A total of 197 ICSI treatment cycles (167 couples) was analysed retrospectively in which testicular spermatozoa were used, because of complete azoospermia, for microinjection. Fertilization, embryo cleavage, transfer and pregnancy rates were evaluated and compared in relation to motility of testicular spermatozoa. In 170 cycles, histological diagnoses were compared with findings on motility. Injection of motile testicular spermatozoa (in 159 cycles) provided a higher normal fertilization rate than did injection of non-motile spermatozoa (in 14 cycles; 65 versus 45% respectively). Normal spermatogenesis was diagnosed in a significantly higher proportion and incomplete maturation arrest in a significantly lower proportion in the group of patients in which only motile spermatozoa were used for microinjection (65 and 10%), as compared to the group where exclusively non-motile spermatozoa were used (36 and 36%). Fertilization rate after ICSI was relatively high when non-motile testicular spermatozoa were used for microinjection, but use of motile testicular spermatozoa was associated with a still higher fertilization rate (except when histology of the testicular biopsy showed normal spermatogenesis), and therefore selection of motile testicular spermatozoa is always preferable for ICSI. Normal spermatogenesis predicts a greater probability, and maturation arrest a lower probability of recovering motile testicular spermatozoa.      

Fertilization and early embryology: Intracytoplasmic single sperm injection of 1-day-old unfertilized human oocytes

Although the average fertilization rate in most in-vitro fertilization(IVF) centres is 60–70%, there are cases of complete orvirtually complete fertilization failures. The aim of our workwas to study the fertilization and the subsequent cleavage characteristicsof 1-day-old human oocytes treated by intracytoplasmic singlesperm injection (ICSI) after failing to fertilize during thestandard IVF procedure. A total of 115 metaphase II 1-day-oldunfertilized oocytes were collected from 23 patients. No additionaltreatment was applied to the oocytes or to the semen sample.A single spermatozoon from the patient's husband was injectedinto the cytoplasm of each of these oocytes 21–33 h afterovum retrieval. Injected oocytes were observed at 16–18h and again 42–44 h after the ICSI procedure. Of the injectedoocytes, 92% (n = 106) were intact after ICSI, 38% (n = 44)had two distinct pronuclei and there was no difference in thefertilization rate of oocytes when andrological and non-andrologicalpatients were compared. Similarly, there was no difference inthe fertilization rate after ICSI where patients with acceptableor good (> 15%) fertilization after standard IVF were comparedto patients who had poor (<15%) fertilization after IVF.There was no significant difference in the sperm concentrationor in the progressive forward motility (a + b motility) in thesegroups except where a + b motility of andrological and non-adrologicalpatients was compared. The majority (84%) of the normally fertilizedoocytes cleaved and most (77%) of these embryos showed <20%fragmentation 2 days after the ICSI procedure. From this studyit can be concluded that 1-day-old metaphase II oocytes whichhave failed to be fertilized after standard IVF procedure canbe fertilized and cleave when ICSI is performed on them theday after oocyte retrieval.     

Intracytoplasmic sperm injection allows fertilization and development of a chromosomally balanced embryo from a binovular zona pellucida

A binovular zona pellucida was found in two in-vitro fertilization (IVF) treatment cycles. In both cases, two oocytes of slightly unequal size were enclosed within a single zona pellucida, the larger oocyte appearing as a metaphase II oocyte while the smaller one as an immature oocyte with a germinal vesicle. Intracytoplasmic sperm injection performed in the mature oocyte of each pair led to normal fertilization and embryonic development in both cases. Results of genetic analysis performed by fluorescence in-situ hybridization in one of the two treatment cycles were consistent with a diploid chromosomal status of both the non-injected immature oocyte as well as the embryo which developed following the microinjection. These results indicate that, in this case, the binovular zona pellucida was most probably created when granulosa cells failed to separate two distinct oocytes during follicular formation. It may also imply that selective fertilization of a single mature oocyte in a binovular zona pellucida by intracytoplasmic sperm injection can lead to the development of a chromosomally balanced embryo and can prevent the undesired consequences that may result if the two oocytes are fertilized in the course of standard IVF.      

Case report: Pregnancy resulting from intracytoplasmic injection of spermatozoa from a frozen-thawed testicular biopsy specimen

A testicular biopsy specimen was taken in connection with scrotalexploration of a healthy 35 year old man who had azoospermia.Bilateral severe scarring of unknown aetiology was found inthe exploration, and no epididymal spermatozoa could be obtained.Spermatozoa from the fresh biopsy specimen were used for intracytoplasmicsperm injection (ICSI) on the same day. Two-embryo transferresulted in biochemical pregnancy. The rest of the biopsy specimenwas frozen as small pieces of tissue using glycerol as a cryoprotectant.ICSI was then performed with spermatozoa prepared from the frozen-thawedtissue. One embryo was obtained and transferred. The transferresulted in pregnancy, and a living fetus was seen in ultrasoundscans at the seventh and 16th weeks of pregnancy. It is possibleto avoid repeated testicular biopsies by using cryopreservationof testicular tissue.