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.     

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.     

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.     

Fetal cells in the maternal circulation: isolation by multiparameter flow cytometry and confirmation by polymerase chain reaction.

During pregnancy, nucleated fetal erythrocytes enter the maternal circulation and can be isolated efficiently from the maternal cells by multiparameter flow cytometry. Male DNA, implying presence of a male fetus, can be identified in flow-sorted maternal blood by polymerase chain reaction with oligonucleotide primers flanking single-copy Y-specific DNA sequences. Among flow-sorted samples, we correctly identified fetal sex in 17/18 (94%) pregnancies of 10-21 weeks gestation. Maternal blood thus provides a potential opportunity for prenatal diagnosis that could preclude the need for invasive procedures in current use.     

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.     

Chromatin status in human ejaculated spermatozoa from infertile patients and relationship to seminal parameters

The aim of this study was to evaluate the chromatin status in different groups of patients. Five groups of men were selected: pre-vasectomy; male factor infertility; varicocele; immunological male infertility; and idiopathic infertility. Chromatin status was evaluated using flow cytometry after staining the DNA with the fluorochrome propidium iodide. Differences were observed in the state of sperm chromatin between the male factor and varicocele groups with respect to the others. These two groups presented poorer quality chromatin, as evidenced fundamentally by a lower degree of condensation. These deficiencies in chromatin status were usually accompanied by alterations in the other standard parameters of semen analysis. Individuals who are infertile due to male factor and those presenting varicocele have spermatozoa with less condensed chromatin which might, in part, explain their sterility.     

DNA and chromatin structure: Influence of incubation on the chromatin condensation and nuclear stability of human spermatozoa by flow cytometry

Flow cytometry analysis was used for the acurate and objectiveevaluation of sperm chromatin condensation and chromatin stabilityof sperm nuclei. It was also possible to determine the influenceof incubation on sperm chromatin. Different types of spermatozoawere studied: unprocessed spermatozoa at 1 and 45 min afterejaculation, after swim-up (migrated), spermatozoa incubatedfor 6 h in non-capacitating conditions (aged), or in B2 medium(capacitated) or B2 medium followed 1 h later with A23187 (reacted).All types of spermatozoa were analysed before and after treatmentwith various decondensation agents: sodium dodecyl sulphate(SDS), SDS plus EDTA and SDS plus disulphide-reducing agent[dithiotreitol (DTT)). Sperm nuclei were enzymatically isolatedand stained with propidium iodide. Three flow cytometric parameterswere then measured: forward light scatter (cellular size), sidelight scatter (cellular complexity) and fluorescence (uptakeof propidium iodide). Fluorescence was the most suitable parameterto study the degree of condensation and resistance to decondensationof DNA in the spermatozoa. Unprocessed spermatozoa 1 min afterejaculation underwent decondensation by all assessed treatments(anionic detergent, chelating or disulphidereducing agents).Unprocessed spermatozoa 45 min after ejaculation and migratedspermatozoa did not undergo decondensation with SDS treatment,but decondensation occurred after treatment with SDS + EDTAor SDS + DTT. Spermatozoa incubated for 6 h under both non-capacitating(aged spermatozoa) and capacitating conditions (capacitatedspermatozoa) and reacted spermatozoa were decondensed only aftertreatment with SDS + DTT. In conclusion, the post-ejaculationand incubation time have to be taken into account when clinicalinterpretation of the effect of different treatments on spermchromatin condensation is made.     

Single nucleotide polymorphisms in the protamine-1 and -2 genes of fertile and infertile human male populations

Although various genetic factors have been implicated in human male infertility, the causative genes for the different types of idiopathic male infertility have not been elucidated. Protamines, which are the major DNA-binding proteins in the sperm nucleus, package the DNA into the sperm head. Analysis of the human protamine-1 (PRM1) and -2 (PRM2) gene sequences in 226 sterile male patients and in 270 proven-fertile male volunteers revealed four single nucleotide polymorphisms (SNPs) in the PRM1 coding region, which did not cause any amino acid substitutions, and one SNP in the PRM2 gene, which produced translation termination. We also observed one SNP in the 3' non-coding region of the PRM1 gene, and two SNPs within the intron of the PRM2 gene. The prevalence of these SNPs was similar in both infertile patients and in proven-fertile volunteers, except that the c248t alteration in the PRM2 gene induced a nonsense codon under conditions of heterozygosity in one infertile patient. Although the PRM1 and PRM2 genes are highly conserved, the single SNP in the PRM2 gene that induces translation termination may result in male infertility due to haploinsufficiency of PRM2.     

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.     

Andrology: Changes in chromatin condensation of human spermatozoa during epididymal transit as determined by flow cytometry

Inasmuch as caput epididymal and even testicular spermatozoaare now being used to generate pregnancies by direct injectioninto the oocyte, differences in the chromatin of spermatozoafrom proximal and distal locations in the epidldymis were studied.Acridine Orange staining was used to investigate chromatin structurein human spermatozoa which had left the testis and were undergoingmaturation in the epididymis. Measurement of green and red fluorescenceIntensities of human spermatozoa by flow cytometry demonstrateda decrease in binding of Acridine Orange to DNA as the spermatozoatraversed the epididymis. Using spermatozoa from the cauda epididymisas the standard, the percentages of spermatozoa from the efferentduct, proximal corpus epididymis, midcorpus epididymis, distalcorpus epididymis, proximal cauda epidldymis and distal caudaepididymis that had matured with regard to chromatin condensationwere 28 ± 5, 39 ± 3, 49 ± 5, 64 ±5, 69 ± 6 and 74 ± 4% respectively. It may beconcluded that eggs fertilized by ejaculated spermatozoa receivea more highly condensed form of chromatin than that receivedby eggs Inseminated with proximal epididymal or testicular spermatozoa.     

Andrology: Assessment of the acrosomal status and viability of human spermatozoa simultaneously using flow cytometry

Acrosomal status and viability were evaluated simultaneouslyon human spermatozoa using flow cytometry. Samples were dividedinto three aliquots and randomly assigned to one of three treatments:(i) cryopreservation; (ii) 10 µM calcium ionophore [A23187in dimethylsulphoxide (DMSO)] or (iii) DMSO alone (control).Acrosomal status was evaluated using monoclonal antibodies recognizingMH61 and CD46, respectively. Fluorescein-conjugated goat anti-mouseimmunoglobulin (IgG) was used as a second antibody. Sperm viabilitywas assessed using Hoechst 33258 (H258) exclusion. The followingfactors were analysed: (i) the specificity of the monoclonalantibodies for the human acrosome; (ii) the relative effectivenessof flow cytometry and direct fluorescent microscopy scoringand (iii) the acrosomal status and viability of the control,ionophore-treated, and cryopreserved spermatozoa. Across alltreatments, the MH61 and CD46 monoclonal antibodies resultedin acrosomal status values (acrosome-reacted/viable spermatozoa)which were not significantly different (P > 0.05): control,1.0 ± 0.3% and 1.5 ± 0.6% (mean ± SEM);A23187, 42.8 ± 3.5% and 38.1 ± 3.5%; cryopreserved,8.2 ± 2.0% and 9.9 ± 1.3%; respectively. However,acrosomal status among treatments differed significantly (P< 0.01). Flow cytometric and direct fluorescent microscopyassessments were significantly correlated (r² = 0.96, P <0.01). These results indicate that flow cytometry, using anacrosome-specific monoclonal antibody and a supravital dye,provides an objective and efficient method to evaluate humansperm acrosomal and viability status simultaneously.     

Flow cytometry and its use in the diagnosis and management of mature lymphoid malignancies

The last decade has seen major advances in flow cytometric immunophenotyping and this has expanded the utility of flow cytometry to investigate the antigens present on normal and neoplastic haematopoietic cells. This review summarizes how flow cytometry is used currently in the diagnosis and management of mature lymphoid malignancies. The establishment of disease-specific phenotypes allows the creation of assays which can detect neoplastic cells with high specificity and sensitivity. Certain lymphoid neoplasms are well defined immunophenotypically, while others are more heterogeneous. The availability of more sophisticated cytometers and a wider selection of antibodies in routine diagnostic laboratories will lead to the resolution of these more complex disease entities.     

Etoposide (VP-16) is a potent inducer of micronuclei in male rat meiosis: Spermatid micronucleus test and DNA flow cytometry after etoposide treatment

The genotoxic and cytotoxic effects of etoposide (VP-16), a topoisomerase II inhibitor, on male rat spermatogenic cells were studied by analysing induction of micronuclei during meiosis. Micronuclei (MN) were scored in early spermatids offer different time intervals corresponding to exposure of different stages of meiotic prophase. Etoposide had a strong effect on diplotene-diakinesis I cells harvested 1 day after exposure, and a significant effect also on late pachytene cells harvested 3 days after exposure. The effect at 18 days corresponding to exposure of preleptotene stage of meiosis (S-phase) was weaker but also statistically significant. Adriamycin was used as a positive control in this study. The results indicate a different mechanism of action of etoposide compared with adriamycin and other chemicals studied previously with the spermatid micronucleus test. DMA flow cytometry was carried out to assess cytotoxic damage at the same time intervals (1, 3, and 18 days after treatment) at stages I and VII of the seminiferous epithelial cycle allowing a study of cytotoxicity to different spermatogenic cell stages. Damage of differentiating sper-matogonia was observed by a decrease in the cell numbers of the 2C peak 1 and 3 days after treatment and by a reduction of the number of 4C cells (primary spermatocytes) 18 d after etoposide treatment. Adriamycin also killed differentiating spermatogonia. Since the cell population which showed a high induction of MN by etoposide was not reduced in number, the genotoxic effect is remarkable. We conclude that etoposide is a potent inducer of genotoxicity and patients treated with this agent during cancer chemotherapy are at a risk of genetic damage. © 1994 Wiley-Liss, Inc.