Developmental potential of elongating and elongated spermatids obtained after in-vitro maturation of isolated round spermatids

BACKGROUND: Round spermatid injections are associated with disappointing clinical outcomes, and although these cells have been shown to mature into late spermatids in vitro, the developmental potential of such gametes remains to be demonstrated. METHODS: Round spermatids were isolated from 12 testicle samples of patients with obstructive azoospermia, hypoplasia, complete maturation arrest, and incomplete Sertoli cell-only syndrome. They were cultured for 7 days at 32 degrees C, 5% CO(2)in air, in microdrops of Vero cell-conditioned medium containing 10% synthetic serum substitute. RESULTS: From the 238 round spermatids cultured, 25.2% attained the elongating and 5.5% the elongated spermatid stage (3-4 days per step). Relatively higher maturation rates were found in cases with obstructive azoospermia, but differences were significant only for elongated spermatids (9.3%). No differences were found in maturation rates between cases with non-obstructive azoospermia (4.3% of elongated spermatids). Experimental microinjections with elongating and elongated spermatids revealed a low fertilization rate (40.9%) but a normal blastocyst formation rate (60%). CONCLUSIONS: Late spermatids resulting from in-vitro culture of round spermatids in conditioned medium, either in controls in cases with a spermiogenetic block, appeared able to successfully fertilize the human oocyte and elicit normal embryo development.     

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.     

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.     

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.     

Assisted reproduction with in-vitro-cultured testicular spermatozoa in cases of severe germ cell apoptosis: a pilot study.

BACKGROUND: Apoptosis-related cell damage is known to compromise success rates of assisted reproduction with ejaculated spermatozoa. This study was undertaken to determine whether the frequency of apoptosis-related cell damage and reproductive performance of testicular spermatozoa from men with non-obstructive azoospermia can be improved by in-vitro culture. METHODS: Testicular tissue samples were cultured for 2 days in the presence of 50 IU/l FSH and 1 micromol/l testosterone. The frequency of spermatozoa showing DNA strand breakage and plasma membrane phosphatidylserine externalization was compared in before-culture and after-culture samples. The after-culture samples were used in assisted reproduction attempts. RESULTS: In a group of 11 azoospermic patients with at least two previous intracytoplasmic sperm injection (ICSI) failures, the incidence of DNA strand breakage was high in living testicular spermatozoa from before-culture samples, but significantly lower in after-culture samples (96 versus 30%, P < 0.001). The same applied to the incidence of phosphatidylserine externalization in the motile sperm subpopulation from the before-culture and after-culture samples (83 versus 6%, P < 0.001). Seven ongoing clinical pregnancies (six with fresh embryos and one with cryopreserved embryos) were established. CONCLUSIONS: Severe testicular sperm apoptosis may become a new indication for testicular tissue in-vitro culture before ICSI.     

A homozygous loss-of-function mutation in FBXO43 causes human non-obstructive azoospermia

Non-obstructive azoospermia (NOA) represents one of the most serious forms of male infertility caused by spermatogenic failure. Despite multiple genes found to be associated with human NOA, the genetic basis of this idiopathic disease remains largely unknown. FBXO43 is a direct inhibitor of the anaphase-promoting complex/cyclosome (APC/C) E3 ligase and crucially important in mouse spermatogenesis. In this study, for the first time, we identified a homozygous nonsense mutation in FBXO43 c.1747C > T:p.Gln583X in two NOA brothers from a Chinese consanguineous family via whole-exome sequencing. FBXO43 was absent from testicular tissue of the proband, and FBXO43-immunostaining signals were invisible in the affected seminiferous tubules. Furthermore, in humans, FBXO43 defects cause meiotic arrest within early diplotene of prophase I. The results here demonstrate the pathogenicity of this loss-of-function mutation and confirmed that spermatocytes were unable to complete meiotic divisions without FBXO43 in humans. In mouse testicular protein extracts, three subunits of the APC/C, including ANAPC2, ANAPC8 and ANAPC10, were validated to interact directly with FBXO43, whereas no interactions were detected for FBXO43 and SKP1. This study furthers our understanding of the genetic basis of human NOA and provides insights into FBXO43 and male infertility.     

Differentiation of spermatogenic cells during in-vitro culture of testicular biopsy samples from patients with obstructive azoospermia: effect of recombinant follicle stimulating hormone

In-vitro differentiation of spermatogenic cells is a potentialapproach to the treatment of male sterility due to spermatogenicarrest. This is a pilot study evaluating meiotic, morphogeneticand cytoplasmic maturation of spermatogenic cells from 18 patientswith obstructive azoospermia, during in-vitro culture of partlydisintegrated testicular biopsy samples in the presence or absenceof recombinant follicle stimulating hormone (rFSH). Meioticprogression was detectable only in the presence of rFSH in culturemedium. FSH-dependent condensation, peripheral migration andprotrusion of spermatid nuclei, together with FSH-independentflagellar growth, were the main events indicating post-meioticsperm cell differentiation. rFSH also promoted the progressionof spermatid cytoplasmic maturation, reflected by accelerationof acrosomal development. These differentiation events appearedto be mediated by humoral activity of Sertoli cells, withoutthe need for a direct Sertoli-sperm cell contact. These findingsprovide a background for similar studies in patients with non-obstructiveazoospermia. If reproducible in the latter group, transmeioticin-vitro differentiation of primary spermatocytes may be usefulin cases of complete maturation arrest, whereas the developmentof culture-specific forms may help select viable spermatidsin cases of complete spermiogenesis failure.     

The effects of meiosis activating sterol on in-vitro maturation and fertilization of human oocytes from stimulated and unstimulated ovaries

The object of this study was to assess functional maturation in vitro by obtaining data on the fertilization and embryonic competence of human oocytes with or without exposure to meiosis activating sterol (MAS) during maturation in vitro. Immature oocytes were either collected from unstimulated patients with polycystic ovaries (PCO) during gynaecological surgery, or were donated by patients undergoing a cycle of intracytoplasmic sperm injection (ICSI) treatment including ovarian stimulation with gonadotrophins. PCO oocytes had variable cumulus cover, which was retained during culture while those from ICSI patients were cultured without cumulus. The study included 119 oocytes from PCO patients and 72 from ICSI patients. The oocytes were allowed to mature in vitro for up to 46 h in the presence or absence of MAS. Mature oocytes were inseminated by ICSI with fertile donor spermatozoa and embryo development was monitored in vitro. MAS (30 microg/ml) significantly increased the survival of oocytes from PCO patients (P < 0.01) but did not significantly affect the proportion completing maturation in vitro. For the ICSI patients, >90% of oocytes survived in all culture groups, regardless of MAS addition, however MAS (10 or 30 microg/ml) significantly increased the proportion of oocytes maturing in vitro (P < 0.05). The apparent tendency towards improved subsequent development in vitro will require larger numbers of oocytes for evaluation. Oocytes from ICSI patients matured more rapidly in vitro than those from PCO patients. Our results show positive effects of MAS on human oocytes, confirming previous data in mice. This work may have implications for the future clinical application of IVM.     

Predictive value of testicular histology in secretory azoospermic subgroups and clinical outcome after microinjection of fresh and frozen-thawed sperm and spermatids

BACKGROUND: A retrospective study was carried out on 159 treatment cycles in 148 secretory azoospermic patients to determine whether histopathological secretory azoospermic subgroups were predictive for gamete retrieval, and to evaluate outcome of microinjection using fresh or frozen-thawed testicular sperm and spermatids. METHODS: Sperm and spermatids were recovered by open testicular biopsy and microinjected into oocytes. Fertilization and pregnancy rates were assessed. RESULTS: In hypoplasia, 97.7% of the 44 patients had late spermatids/sperm recovered. In maturation-arrest (MA; 47 patients), 31.9% had complete MA, and 68.1% incomplete MA due to a focus of early (36.2%) or late (31.9%) spermiogenesis. Gamete retrieval was achieved in 53.3, 41.2 and 93.3% of the cases respectively. In Sertoli cell-only syndrome (SCOS; 57 patients), 61.4% were complete SCOS, whereas incomplete SCOS cases showed one focus of MA (5.3%), or of early (29.8%) and late (3.5%) spermiogenesis. Only 29.8% of the patients had a successful gamete retrieval, 2.9% in complete and 77.3% in incomplete SCOS cases. In total, there were 87 ICSI, 39 elongated spermatid injection (ELSI) and 33 round spermatid injection (ROSI) treatment cycles, with mean values of fertilization rate of 71.4, 53.6 and 17%, and clinical pregnancy rates of 31.7, 26.3 and 0% respectively. CONCLUSIONS: Histopathological subgroups were positively correlated with successful gamete retrieval. No major outcome differences were observed between testicular sperm and elongated spermatids, either fresh or frozen-thawed. However, injection of intact round-spermatids showed very low rates of fertilization and no pregnancies.     

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.     

Live human germ cells in the context of their spermatogenic stages

BACKGROUND: Various types of live, dispersed, human testicular cells in vitro were previously compared with the morphologic characteristics of human spermatogenic germ cells in situ within seminiferous tubules. The current study extends those observations by placing live human germ cells in the context of their developmental steps and stages of the spermatogenic cycle. METHODS: Live human testicular tissue was obtained from an organ-donating, brain-dead person. A cell suspension was obtained by enzymatic digestion, and dispersed cells were observed live with Nomarski optics. Testes from 10 men were obtained at autopsy within ten hours of death, fixed in glutaraldehyde, further fixed in osmium, embedded in Epon, sectioned at 20 microm, and observed unstained by Nomarski optics. RESULTS: In both live and fixed preparations, Sertoli cells have oval to pear-shaped nuclei with indented nuclear envelopes and large nucleoli, which makes their appearance distinctly different from germ cells. For germ cells, size, shape, and chromatic pattern of nuclei, the presence of meiotic metaphase figures, acrosomic vesicles/structures, tails, and/or mitochondria in the middle piece are characteristically seen in live dispersed cells and those in the fixed seminiferous tubules. These lead to identification of live germ cells in man and placement of each in the context of their developmental steps of spermatogenesis at corresponding stages of the spermatogenic cycle. CONCLUSIONS: This comparative approach allows verification of the identity of individual germ cells seen in vitro and provides a checklist of distinguishing characteristics of live human germ cells to be used in clinical procedures or by scientists interested in studying live cells at known steps in spermatogenic development characteristic of germ cells in specific stages of the spermatogenic cycle.     

A macaque model for studying mechanisms controlling oocyte development and maturation in human and non-human primates.

A model to study mechanisms controlling nuclear and cytoplasmic maturation of primate oocytes is being developed in our laboratory. The high incidence of pregnancy failure in women following in-vitro fertilization (IVF) may be partly attributed to inadequate cytoplasmic maturation of oocytes. Advancement of knowledge of mechanisms controlling primate oocyte maturation would have important implications for treatment of human infertility, and would potentially increase numbers of viable non-human primate embryos for biomedical research. Use of a non-human primate model to study oocyte and embryo biology avoids legal, ethical and experimental limitations encountered in a clinical situation. Using this model, the meiotic and developmental capacity of oocytes from three sources have been compared: (i) in-vivo matured oocytes from monkeys stimulated with follicle-stimulating hormone (FSH) and human chorionic gonadotrophin, (ii) in-vitro matured oocytes from monkeys primed with FSH, and (iii) in-vitro matured oocytes from non-stimulated monkeys. This work demonstrates that oocyte developmental competence is likely acquired both during follicle development, before meiotic resumption, and during meiotic progression, concurrent with nuclear maturation. Potential causes of developmental failure of in-vitro matured oocytes, implications for human infertility, and future strategies to study the regulation of primate oocyte maturation are discussed.     

Embryo development and pregnancies from in-vitro matured and fertilized human oocytes.

There is an increasing interest in retrieving immature oocytes in the absence of or with limited gonadotrophin exposure, with the aim of maturing them in vitro for embryo transfer purposes. The aim of this report is to present our experience of fertilization, embryonic development and pregnancies from in-vitro maturation cycles. A total of 18 patients underwent 21 cycles in which an average of 8.1 immature oocytes was retrieved after limited exposure to human menopausal gonadotrophin (HMG) and no exposure to human chorionic gonadotrophin (HCG). In one cycle, no oocytes were recovered. The oocytes were cultured for 44 h and 121 oocytes which reached MII were injected with a single spermatozoon. A total of 71 oocytes showed two pronuclei and 53 zygotes cleaved. Forty-four embryos were transferred in 17 cycles. Five weeks after embryo transfer, ultrasound examination indicated the presence of one gestational sac and one fetal heart beat in two patients. The results suggest that in-vitro matured oocytes can undergo fertilization and the resulting embryos may result in pregnancies. However, the success rate was not sufficient to recommend widespread use of the technique without further research.     

Testicular sperm extraction: microdissection improves sperm yield with minimal tissue excision.

Testicular sperm extraction (TESE) is often an effective method for sperm retrieval from men with non-obstructive azoospermia. However, TESE has been a blind procedure that does not identify the focal sperm-producing areas of the testicle until after tissue has been excised from the patient. Experience with a new technique of microdissection of testicular tubules is presented here that identifies sperm-containing regions before their removal. Identification of spermatogenically active regions of the testicle is possible by direct examination of the individual seminiferous tubules. The underlying concept for this technique is simple: seminiferous tubules containing many developing germ cells, rather than Sertoli cells alone, are likely to be larger and more opaque than tubules without sperm production. In a sequential series of TESE cases for men with non-obstructive azoospermia, the ability to find spermatozoa increased from 45% (10/22) to 63% (17/27) after introduction of the microdissection technique. Microdissected samples yielded an average of 160,000 spermatozoa per sample in only 9.4 mg of tissue, whereas only 64,000 spermatozoa were found in standard biopsy samples that averaged 720 mg in weight (P < 0.05 for all comparisons). For men where microdissection was attempted, successful identification of enlarged tubules was possible in 56% (15/27) of cases. However, spermatozoa were retrieved with microdissection TESE for six men in whom sperm retrieval was unsuccessful with standard TESE approaches (35% of all men with spermatozoa retrieved). These findings suggest that microdissection TESE can improve sperm retrieval for men with non-obstructive azoospermia over that achieved with previously described biopsy techniques.     

Fertilization and early embryology: Maturation of human cumulus-free germinal vesiclestage oocytes to metaphase II by coculture with monolayer Vero cells

The objective of this study was to evaluate and compare theefficacy of coculture with Vero cells (monkey kidney cells)and in-vitro culture in B2 Ménézo medium for thematuration of germinal vesicle (GV)-stage cumulus-free humanoocytes to metaphase II. Cumulus-free human GV-stage oocytesobtained after ovulation induction and retrieval via vaginalultrasound echo-guided puncture were put either in coculturewith Vero cells or in 25 µl droplets of B2 Ménézomedium. In a controlled randomized study, 145 GV-stage oocyteswere matured in vitro: 72 were placed in the coculture systemwith Vero cells and 73 were cultured in B2 alone. After 30 hof in-vitro culture, 82.4% had dissolved the nuclear membraneand extruded the first polar body in the coculture system comparedwith 37% in B2 medium (P < 0.001). A significant differencewas also noticed in the number of GV-stage oocytes which underwentGV breakdown; the proportion of oocytes which did not extrudethe first polar body (metaphase I) was 1.6% in the coculturesystem compared to 44.0% in the B2 medium. It can be concludedthat coculture with Vero cells improves the in-vitro maturationof cumulus-free human GV-stage oocytes.     

Spermatid injection into human oocytes. I. Laboratory techniques and special features of zygote development

Spermatid injection into the oocyte cytoplasm has been shownrecently to yield viable human embryos developing to term aftertransfer to the mother. This study provides details of the laboratorytechniques related to round spennatid injection (ROSI) and elongatedspennatid injection (ELSI) and focuses on some special featuresof zygote development associated with the use of these typesof sperm precursor cells for fertilization. A spermatid-enrichedfraction was obtained by centrifugation of cells from azoospermicejaculates through a discontinuous Per-coll gradient column.Individual round or elongated sperm-atids were identified inthis fraction and injected deep into oocytes, Oocyte activationwas boosted by a vigorous aspiration of the ooplasm at the timeof injection. The fertilization rates after ROSI and ELSI were45 and 44% respectively. A single large syngamy nucleus wasdetected in 36% of the zygotes that previously showed two normal-sizedpronuclei. This condition did not appear to delay the firstcleavage division. These observations underscore the importanceof distinguishing the syngamy nucleus of diploid zygotes fromthe female pronudeus of haploid, partheno-genetically activatedeggs.     

Factors influencing the outcome of ICSI in patients with obstructive and non-obstructive azoospermia: a comparative study

BACKGROUND: Factors influencing success of sperm retrieval in azoospermic patients and outcome of ICSI were evaluated. METHODS AND RESULTS: Uni- and multifactorial analysis were performed using logistic and stepwise analysis, following surgical sperm retrieval by percutaneous epididymal sperm aspiration (55 cycles) or testicular sperm extraction (142 cycles) in 52 and 123 patients with obstructive azoospermia (OA) and non-obstructive azoospermia (NOA) respectively. ICSI cycles using fresh or cryopreserved-thawed sperm were included. Sperm were retrieved to allow ICSI in 100 and 41% of OA and NOA patients, with no significant correlation with patients' age or FSH level. Occurrence of pregnancy was significantly correlated with female age (90th quantile: 38 years), number of oocytes retrieved (10th quantile: five oocytes) and number of oocytes injected (10th quantile: four oocytes). Sperm origin (epididymal versus testicular), status (fresh or thawed), male partner's age, and serum FSH had no significant effect upon implantation rate, pregnancy rate per embryo transfer or spontaneous miscarriage rate. CONCLUSIONS: In OA patients ICSI should be planned in conjunction with surgical sperm retrieval. In contrast, the lack of efficient non-invasive parameters to predict sperm retrieval in NOA suggests that elective surgical sperm retrieval may be offered to these patients prior to ovarian stimulation of their partners, especially when donor back-up is not an alternative. Female factors such as age and ovarian reserve have significant impact upon clinical success rates.     

Outcome of testicular sperm recovery and ICSI in patients with non-obstructive azoospermia with a history of orchidopexy

BACKGROUND: Little is known about sperm recovery and ICSI using testicular sperm from men with non-obstructive azoospermia who had a previous orchidopexy. We therefore studied the sperm recovery in this subgroup and evaluated clinical parameters predicting successful sperm retrieval and the outcome of ICSI. METHODS: A total of 79 non-obstructive azoospermic men with a history of orchidopexy underwent a sperm recovery procedure. The predictive value of clinical parameters such as age at sperm retrieval, age at orchidopexy, testicular volume, FSH, FSH/LH ratio, testosterone and androgen sensitivity index (LH x testosterone) for successful testicular sperm retrieval was evaluated using receiver operating characteristics (ROC) curve analysis. A comparison between 64 ICSI cycles performed in these couples and 92 cycles performed in couples in which the men had an unexplained non-obstructive azoospermia was carried out. RESULTS: Testicular spermatozoa were recovered in 41 patients (52%). The mean age at orchidopexy of the patients with a positive sperm recovery was 10.6 years [95% confidence interval (CI) 7.3-13.8] versus 15.5 years (95% CI 11.3-19.8) for those where no spermatozoa were found. The mean testicular volume of the largest testis of patients with spermatozoa found was 10 ml (95% CI 8.3-11.9) versus 8.5 ml (95% CI 5.8-11.1) in patients with no spermatozoa found. The mean FSH and testosterone value for patients with successful and unsuccessful sperm recovery, respectively, was 24.1 IU/l (95% CI 17.9-30.3) and 4.4 ng/ml (95% CI 3.7-5.1) versus 28.8 IU/l (95% CI 19.4-38.2) and 3.4 ng/ml (95% CI 2.2-4.5). All clinical and biological parameters examined failed to predict the outcome of the testicular sperm extraction. No differences were observed between the orchidopexy and unexplained group for the number of oocytes retrieved, fertilization rate, embryo quality, pregnancy rate and implantation rate. CONCLUSIONS: As in the population of men with non-obstructive azoospermia, the sperm recovery rate for patients with a history of orchidopexy is approximately 50% and there are currently no clinical parameters predicting successful sperm retrieval in this subpopulation of patients. The outcome of the ICSI cycles is comparable with that in the population of men with non-obstructive azoospermia.     

Dynamics of human follicular growth and in-vitro oocyte maturation

The physiological trigger for meiotic resumption in the human oocyte is the surge of luteinizing hormone, but it can also occur spontaneously if oocytes are released from antral follicles and cultured in vitro. The development of novel techniques for the culture of murine oocytes has raised the possibility of growing human oocytes to maturity in vitro. Such a system could open the door to a number of techniques with revolutionary consequences. It would clearly be of benefit in basic physiological studies of follicular development, as well as being used to test the effect of toxicological substances on oocyte maturation. More significantly, such a system could provide a source of human oocytes for in-vitro fertilization (IVF) where immature or germinal vesicle oocytes are cultured to maturity before being fertilized. If this can be achieved, it might facilitate oocyte cryopreservation, where surplus oocytes are stored, thus avoiding the need for repeated superovulation. A combination of immature oocyte cryopreservation for later maturation and IVF will provide the opportunity to establish oocyte banks and help overcome some of the practical and ethical dilemmas that are currently shadowing the field of reproductive medicine.     

Pregnancies achieved after frozen-thawed pronuclear oocytes obtained by intracytoplasmic sperm injection with spermatozoa extracted from frozen-thawed testicular tissues from non-obstructive azoospermic men.

The use of frozen-thawed testicular tissue as a source of spermatozoa for intracytoplasmic sperm injection (ICSI) in non-obstructive azoospermia yields favourable fertilization and pregnancy rates while avoiding both repetitive biopsies and unexpected cycle cancellations. Spermatozoa were obtained from frozen-thawed testicular biopsy specimens from 67 non-obstructive azoospermic men. Following fertilization, supernumerary two pronuclear (2PN) oocytes were frozen. After thawing, 17 cycles of embryo transfer were carried out with a mean number of 2.7 embryos and a mean cumulative embryo score (CES) of 18.3 per transfer. The clinical pregnancy and implantation rates per transfer in these cycles (23.5 and 8.3% respectively) were comparable to those of fresh embryo transfers (35.7 and 12.7% respectively) with a mean number of 2.7 embryos and a mean CES of 28.7 per transfer. Abortion rates, although higher with cryopreserved 2PN oocytes were not significantly different. With this approach, cryopreservation of supernumerary 2PN oocytes can be used to improve the cumulative pregnancy rates in a severely defective spermatogenetic population. To our knowledge, these are the first pregnancies reported which have been obtained by the transfer of cryopreserved pronuclear oocytes obtained from ICSI using cryopreserved testicular spermatozoa.