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PubMed1999

Ooplasmic injections of rabbit round spermatid nuclei or intact round spermatids from fresh, cryopreserved and cryostored samples.

We compared the outcome of ooplasmic round spermatid nuclear injections (ROSNI) versus intact round spermatid injections (ROSI). Rabbit round spermatid nuclei and intact round spermatids were recovered and injected into rabbit oocytes (groups A and B, respectively). Fertilization, cleavage and embryonic development rates were compared. In additional studies, five protocols for cryopreservation of round spermatids and two protocols for cryostorage of round spermatids were applied. The outcome of ROSNI techniques using frozen-thawed or cryostored-warmed round spermatids was evaluated. The cleavage rate and the overall morula plus blastocyst development rate were significantly larger in group A than group B. ROSNI procedures are superior to ROSI techniques in the rabbit. The largest fertilization, cleavage and embryonic development rates after ROSNI techniques using cryopreserved or cryostored round spermatids were demonstrated in groups of round spermatids in which a mixture of seminal plasma plus test yolk buffer was employed as an extender, and dimethyl sulphoxide plus a high concentration of glycerol served as cryoprotectants. It appears that the seminal plasma contains factors protecting round spermatids during cryopreservation or cryostorage, and/or the employment of two cryoprotectants has a beneficial role in the maintenance of round spermatid reproductive capacity.

Animals↗
PubMed1998

Functional studies of maturing myeloid cells during ex vivo expansion for treatment of aplasia: feasibility of ex vivo expansion from cryopreserved bone marrow cell samples.

Ex vivo expanded CD34+ progenitor cells from fresh or cryopreserved primate bone marrow, induced to granulocytic differentiation with growth factors, were investigated to determine whether myeloid cells produced in liquid cultures have the normal biologic functions needed for the treatment of patients with neutropenia following high-dose chemotherapy or therapeutic or accidental radiation exposure. Human and simian (baboons or macaques) CD34+ cells were cultured with granulocyte-colony stimulating factor (G-CSF), stem cell factor (SCF), interleukin-1 (IL-1), IL-3, and IL-6, and assessed at 14 days of culture for their capacity to respond to different functional tests. Immunostaining revealed that human ex vivo expanded cells contained myeloperoxydase (MPO, 82% +/- 8%) and lactoferrin (LF, 30% +/- 6%) in their granules. Maturation of cultured cells was associated with stimulated chemotactic responsiveness and respiratory burst activity (superoxide anion and hydrogen peroxide production) in expansions from human, baboon, and macaque CD34+ progenitor cells. Mature cells obtained from ex vivo expansion of selected cryopreserved human bone marrow CD34+ cells presented reduced but significant functional activities (chemotactic responsiveness and hydrogen peroxide production) when compared with human peripheral blood neutrophils. The validation of nonhuman primate ex vivo expansion systems may permit their use as models of irradiation. The feasibility of ex vivo expansion from cryopreserved bone marrow cell samples may offer considerable opportunity for banking bone marrow for autologous transfusion.

Anemia, Aplastic↗
PubMed1995

Prediction of post-thaw sperm motility and sperm cryosurvival rate using the pre-freeze sperm parameters.

To determine the relationships between pre-freeze semen variables and cryosurvival rate and post-thaw motility and examine whether they have any predictive value for the cryosurvival rate and post-thaw sperm motility, conventional semen analysis, supravital staining for sperm viability and hypo-osmotic swelling test were performed on 50 semen samples before cryopreservation. Thawed semen samples were examined for post-thaw sperm motility and cryosurvival rate. Significant correlations were observed between post-thaw sperm motility and several pre-freeze semen variables, such as, hypo-osmotic swelling test, pre-freeze sperm motility and sperm viability. In a stepwise regression analysis, an accurate prediction of post-thaw sperm motility (R = 0.826) was obtained using a multiple regression equation incorporating 3 variables including hypo-osmotic swelling test, pre-freeze sperm motility and sperm concentration. In conclusion, a set of criteria have been identified that accurately predicts post-thaw sperm motility and which place particular emphasis on hypo-osmotic swelling test. Conventional semen analysis and hypo-osmotic swelling test are simple and effective assays for the prediction of post-thaw motility.

Cryopreservation↗
PubMed1996

Optimal cryopreservation of human umbilical cord blood.

Cryopreservation techniques for umbilical cord blood (UCB) have been based on methods established for marrow (BM) and peripheral blood progenitor cells (PBPC) with varying degrees of success. The aim of this study was to optimise cryopreservation of UCB haemopoietic cells based on sound cryopreservation principles. UCB samples were cryopreserved with different combinations of DMSO and hydroxyethyl starch (HES) by a variety of freezing protocols. After cooling at 1 degree C/min in solutions containing 4% HES and various concentrations of DMSO there was a dramatic fall in CD34+ recovery from 85.4% (s.d. 28.4) to 12.2% (s.d. 10.0) as DMSO concentration was reduced from 5 to 2.5%. Varying HES concentration in solutions containing 5% DMSO did not have a significant effect on CD34+ cell recovery. Increasing cooling rate from 1 to 10 degrees C/min significantly reduced CD34+ recovery (P < 0.0001) while increasing DMSO concentration up to 10% had little effect (P = 0.8, two-way ANOVA). Good recovery of UCB CD34+ cells can be achieved with 5-10% DMSO at a controlled cooling rate of 1 degrees C/min. There was a significant difference (P < 0.0001) in the apparent recovery of CD34+ cells between paired aliquots thawed in the presence (recovery = 76.8%, s.d. 26.0) and absence (32.5%, s.d. 18.7) of DNase. In conclusion, conditions for cryopreserving UCB for clinical banking that yield optimal recovery of CD34+ cells have been established.

Antigens, CD34↗
PubMed1994

Relationship between stimulated hyperactivated motility of human spermatozoa and pregnancy rate in donor insemination: a preliminary report.

The proportion of spermatozoa exhibiting the vigorous motility behaviour termed 'hyperactivation' (HA) has been shown to be increased following removal of seminal plasma and stimulation with chemical agents such as pentoxifylline. The aim of this study was to examine the relationship between the proportion of HA in cryopreserved semen samples from sperm donors and the corresponding pregnancy rates achieved by donor insemination. Cryopreserved samples from 20 men were incubated in the presence or absence of 3 mM pentoxifylline for 1 h and the %HA determined in each sample. The relationship between pregnancy rate, the proportion of HA spermatozoa in control and pentoxifylline-treated groups and the change in %HA following pentoxifylline treatment (delta HA) as well as the mean semen characteristics for each donor [sperm count, motility (%), motility index, normal morphology (%), post-thaw motility (%) and post-thaw motility index] were examined by logistic regression of the occurrence of clinical pregnancy with each insemination. Both delta HA and mean post-thaw motility index were significantly related to pregnancy rates and together accounted for 64% of the observed variation in pregnancy rates.

Cryopreservation↗
PubMed2004

Effects of cryopreservation and thawing on the structure of vascular segment.

The objective of this study was to analyze the importance of the quality of the vascular segment to be cryopreserved and the influence of storage in a gas phase, a liquid phase, or after accidental immersion in liquid nitrogen. In addition, we investigated the effects of rapid versus slow thawing on the occurrence of fractures and changes in the structure of the vessel wall. The tissue sources were whole thoracic and abdominal aortas from 15 pigs. Each aorta was cut into equal segments and randomly assigned to each study group. One segment of fresh unfrozen aorta of the same size was used as a control. The samples were cryopreserved using a programmed apparatus. After 2 weeks the arterial segments were thawed rapidly or slowly. A great variation in the results was obtained depending on the quality of the control. Although endothelial cells were better preserved in the liquid phase, the internal elastic lamina and elastic lamelli showed better preservation and fewer microfractures in the gas phase. The internal elastic lamina showed a greater number of microfractures when an accidental immersion in liquid nitrogen had taken place. Furthermore, better preservation of the structure of the vascular segment was observed with a slow thawing method. In general, the conditions of storage and the method of thawing seem to damage the structure of vascular segments. It is necessary to use a severe protocol of donor and vascular segment selection to optimize the post-thaw quality of the cryopreserved samples.

Animals↗
PubMed2003

Increased transmigration of G-CSF-mobilized peripheral blood CD34+ cells after overnight storage at 37 degrees C.

BACKGROUND: G-CSF-mobilized PBPCs are utilized in allogeneic and autologous PBPC transplants. Homing, adhesion, and transmigration of hematopoietic CD34+ cells are required for successful engraftment. Hematopoietic CD34+ cells undergo directional migration toward the CXCR4 receptor ligand stromal-derived factor-1 (SDF-1). Limited data are available on the effects of liquid storage and cryopreservation on PBPC CD34+ cells. STUDY DESIGN AND METHODS: Magnetic-assisted cell sorting (MACS)-selected CD34+ cells were assayed for retention of in vitro transmigration and phenotypic changes of unit-matched liquid-stored and cryopreserved PBPC samples from healthy donors. Studies evaluated whether transmigration of CD34+ cells in Iscove's modified Dulbecco's medium plus 1 percent HSA alone or in medium supplemented with SCF or allogeneic plasma was affected by overnight incubation at 37 degrees C, relative to nonincubated conditions. RESULTS: Transmigration was maintained during liquid storage at 1 to 6 degrees C during a 2-day period and in unit-matched cryopreserved-thawed samples that had been initially liquid stored. Overnight incubation at 37 degrees C of MACS-selected unit-matched liquid-stored or cryopreserved-thawed CD34+ cells resulted in substantially increased transmigration, in particular with noncoated filters chemoattracted with the chemokine SDF-1. CONCLUSION: CD34+ cell transmigration was comparable between liquid-stored and cryopreserved samples, and both demonstrated similar increases after overnight incubation at 37 degrees C.

Antigens, CD34↗
PubMedDate not supplied

An immunomagnetic epithelial tumor cell enrichment model for minimal residual disease detection of cytokeratin 8+ malignancies.

Immunocytochemical detection of isolated tumor cells in peripheral blood and bone marrow is currently the most established method for monitoring early dissemination in epithelial cancer. In this study we used an immunomagnetic selection technique to develop an enrichment model for disseminated tumor cells in blood. Buffy coat cells spiked with varying numbers of BT-474 carcinoma cells were permeabilized and fixed, following which carcinoma cells were magnetically labelled with an anti-cytokeratin 8 mAb. Labelled cells were enriched by the use of magnetic columns. The eluted cytokeratin 8+ tumor cells were detected by flow cytometry and immunocytochemistry. Spiked samples were split and processed freshly in the immunomagnetic enrichment assay, as well as cryopreserved and processed in the assay after thawing. Enumeration of BT-474 cells demonstrated a detection limit of one BT-474 cell in 1.0 x 10(7) leukocytes in both fresh and cryopreserved-thawed samples. The pair wise comparison showed a significantly higher recovery of spiked BT-474 cells from freshly processed samples than from cryopreserved and thawed samples (57% vs 21%). Viability tests suggested that this outcome might be due to a greater susceptibility of BT-474 cells than buffy coat cells to the used cryopreservation and thawing technique. Altogether our findings show that the performance of the immunomagnetic enrichment assay on fresh samples is satisfactory with a recovery rate of almost 60% and a sensitivity of 10(-7). However, performance of the assay on cryopreserved and thawed cells needs to be improved.

Biomarkers, Tumor↗
PubMed1989

Longitudinal study of the in vivo hprt mutant frequency in human T-lymphocytes as determined by a cell cloning assay.

The in vivo frequency of mutants resulting from mutation at the hprt locus in human T-lymphocytes can be determined by a cloning assay. This assay quantifies the frequency of 6-thioguanine-resistant (TGr) T-cells through growth of colonies in 96-well microtiter dishes. The reproducibility of the TGr mutant frequency values has now been assessed in a longitudinal study of six individuals (three male, three female, aged 22-33 years) employing 4-5 blood samples over a 26-37 week time period. Cloning assays were performed with both fresh and cryopreserved cell samples. No significant differences were found among the mutant frequency values for multiple samples from each individual with both fresh and cryopreserved cell samples. These results demonstrate the reproducibility of this cloning assay for in vivo mutant frequency determinations in human T-lymphocytes.

Adult↗
PubMed2001

Enrichment of non-apoptotic human spermatozoa after cryopreservation by immunomagnetic cell sorting.

Cryopreservation increases the rate of apoptotic spermatozoa with decreased capability to fertilise oocytes. In order to optimise the fertilisation rates, especially in assisted reproduction the use of apoptotic sperms should be avoided. Early events of apoptosis in cryopreserved spermatozoa are not detectable by conventional methods. However, the surface of apoptotic spermatozoa is characterised by externalisation of phosphatidylserine (PS), which has a high affinity to Annexin V. Therefore, colloid paramagnetic Annexin-V-conjugated microbeads (AN-MB) were tested for their ability to eliminate apoptotic spermatozoa from a total of 40 fresh and in TEST yolk buffer cryopreserved semen samples which were provided by 15 healthy volunteers. By passing through a magnetic field (MiniMACS, Miltenyi Biotec) the sperm suspensions were divided into 2 sperm fractions depending on bound magnetic Annexin V-microbeads (AN-MB) to spermatozoa. As additional markers of apoptosis CD95 (Fas, APO-1) on the sperm surface and activated caspases in the cytosol were detected in both fractions. Supplementary investigations comprised eosin-supravital staining and computer assisted sperm motion analysis. The separation was supervised by flow cytometric analysis of spermatozoa labelled with FITC-conjugated anti Annexin V-antibodies. Analyses of the magnetic inactive sperm fraction (AN-MB-negative) showed CD95 on 0.6 +/- 0.3% (X +/- SEM) of spermatozoa and only 3.2 +/- 0.5% were stainable with eosin, whereas, 40.6 +/- 6.7% of the remaining cells in the column appeared to be CD95 positive and 99.8 +/- 0.1% stainable with eosin after cryopreservation. Indeed the overall amount of CD95 positive spermatozoa did not significantly increase after cryopreservation (2.5 +/- 0.5% vs. 4.3 +/- 1.2%; p > 0.05). Activated caspases were found in 21.8 +/- 2.6% of the spermatozoa in fresh and in 47.7 +/- 5.8% of cryopreserved semen samples (p < 0.01). The separation procedure of the cryopreserved spermatozoa reduced significantly the quantity of those containing activated caspases to 9.3 +/- 2.2% within the AN-MB-negative fraction. In contrast 89.1 +/- 2.3% of AN-MB-positive sperms showed activation of these proteolytic enzymes. Flow cytometric analyses using FITC-conjugated anti Annexin V-antibodies for monitoring of AN-MB-binding to spermatozoa showed 5.2 +/- 1.0% labelled spermatozoa in the AN-MB negative fraction and 72.6 +/- 2.7% labelled spermatozoa in the AN-MB positive one. There was no significant influence of the separation column and the magnetic field on the sperm functions. The passage through the column led to a sperm loss of 0.8 +/- 1.2%. Conclusion: The binding of paramagnetic Annexin V-conjugated microbeads is an excellent method to eliminate spermatozoa at early apoptotic stages from cryopreserved semen samples. A deleterious influence of the separation column and the magnetic field on the spermatozoa was not observed.

Journal Article↗
PubMed1985

Frequency and coordination of ciliary beat after cryopreservation of respiratory epithelium.

The effect of cryopreservation on human nasal mucosal biopsies was evaluated by determining the frequency and coordination of the ciliary beat. Samples were cryopreserved in a medium containing 80% Gey's balanced salt solution, 10% dimethyl sulfoxide, and 10% fetal calf serum. After thawing, the samples were put in a solution of 90% Gey's balanced salt solution and 10% fetal calf serum. Video recordings of the samples before and after cryopreservation were compared using a semiquantitative method. All the frequencies and coordination patterns seen before cryopreservation could be found in the sample after cryopreservation. It is concluded that ciliated epithelial biopsies can be stored in liquid nitrogen with the maintenance of ciliary beat frequency. In the recorded ciliated cells the ciliary beat coordination was slightly reduced; a lack of coordination was present in 20% of cells after cryopreservation as compared to 10% before cryopreservation.

Adult↗
PubMed2002

Repeat intracytoplasmic sperm injection. Clinical perspective.

OBJECTIVE: To report the results of repeat intracytoplasmic sperm injection (ICSI) after complete failure of fertilization with initial ICSI. METHODS: The medical records of the couples undergoing repeat ICSI at the Human Reproductive Biology Unit, Soliman Fakeeh Hospital, Jeddah, Kingdom of Saudi Arabia, after complete failure of fertilization by initial ICSI between December 1994 and December 2000 were retrospectively examined. RESULTS: Seven hundred and eighty two oocytes from 146 women failed to fertilize by initial ICSI. The main indications for the procedure were severe oligoasthenoteratozoospermia or azoospermia. Fresh sperms were used in 136 cases, of which 98 (72%) were ejaculated, 33 (24.3%) were obtained by testicular sperm extraction, and 5 (3.7%) by testicular sperm aspiration. Of the remaining 10: 3 were from cryopreserved semen samples and 7 were from cryopreserved testicular biopsies. The age of the women (mean standard deviation) was 31 16.2 years. The duration of infertility was 10.5 9.4 years. A total of 151 (19.3%) oocytes were fertilized after repeat ICSI. The number of cleaved embryos was 125 (15.9%); of which 2 (1.6%) were grade 5, 47 (37.6%) were grade 4, 65 (52%) were grade 3, and 11 (8.8%) were grade 2. A total of 122 embryos were transferred to 71 women. This resulted in one pregnancy and the birth of a healthy full term baby. CONCLUSION: In cases of complete failure of fertilization with initial ICSI, fertilization and pregnancy can follow repeat ICSI. Further clinical and cytogenetic studies in this area are necessary.

Adult↗
PubMed2006

Transcriptional profiling of degraded RNA in cryopreserved and fixed tissue samples obtained at autopsy.

BACKGROUND: Traditional multiplexed gene expression methods require well preserved, intact RNA. Such specimens are difficult to acquire in clinical practice where formalin fixation is the standard procedure for processing tissue. Even when special handling methods are used to obtain frozen tissue, there may be RNA degradation; for example autopsy samples where degradation occurs both pre-mortem and during the interval between death and cryopreservation. Although specimens with partially degraded RNA can be analyzed by qRT-PCR, these analyses can only be done individually or at low levels of multiplexing and are laborious and expensive to run for large numbers of RNA targets. METHODS: We evaluated the ability of the cDNA-mediated Annealing, Selection, extension, and Ligation (DASL) assay to provide highly multiplexed analyses of cryopreserved and formalin fixed, paraffin embedded (FFPE) tissues obtained at autopsy. Each assay provides data on 1536 targets, and can be performed on specimens with RNA fragments as small as 60 bp. RESULTS: The DASL performed accurately and consistently with cryopreserved RNA obtained at autopsy as well as with RNA extracted from formalin-fixed paraffin embedded tissue that had a cryopreserved mirror image specimen with high quality RNA. In FFPE tissue where the cryopreserved mirror image specimen was of low quality the assay performed reproducibly on some but not all specimens. CONCLUSION: The DASL assay provides reproducible results from cryopreserved specimens and many FFPE specimens obtained at autopsy. Gene expression analyses of these specimens may be especially valuable for the study of non-cancer endpoints, where surgical specimens are rarely available.

Journal Article↗
PubMed2004

Quality control of bacterial contamination in autologous peripheral blood stem cells for transplantation.

BACKGROUND AND OBJECTIVES: Microbiological follow-up is part of quality control of peripheral blood stem cell (PBSC) manipulation. DESIGN AND METHODS: We prospectively studied microbiological cultures performed in 865 consecutive untreated autologous PBSC harvests from 348 patients. Our aim was to know the rate of microbiological contamination, the optimum moment to evaluate the sample and the clinical significance of the positive findings. RESULTS: Fifty-nine of the 852 samples (6.9%) yielded a positive culture after PBSC collection (sample 1) and 62 samples also yielded positive results before cryopreservation (7.2%) (sample 2). At the time of the analysis, a total of 520 aphereses had been infused and the number of positive cultures after thawing (sample 3) and after washing (sample 4; 82 aphereses) was 5.4% and 2.3%, respectively. Most of the positive cultures were due to coagulase-negative staphylococci (48 isolates). After thawing 15 coagulase-negative staphylococci and 2 enterococci isolates were recovered. Comparison between samples using a marginal homogeneity test showed no differences in the rate of contamination observed at the different sampling points. INTERPRETATION AND CONCLUSIONS: Positive microbiological findings in collected PBSC are not due to contamination within the laboratory. Cryopreservation using DMSO does not eradicate bacteria and manipulation does not seem to affect results. To simplify the procedure it would be possible to eliminate the microbiological controls performed immediately before cryopreservation.

Antibiotic Prophylaxis↗
PubMed1998

Optimal utilization of cryopreserved human semen for assisted reproduction: recovery and maintenance of sperm motility and viability.

PURPOSE: Our purpose was to evaluate sperm motility and viability and the maintenance of these parameters in already cryopreserved semen samples following repeated freezing/thawing cycles. METHODS: Human spermatozoa were subjected to five cycles of cryopreservation/thawing. Recovery of sperm motility and viability and the proportion of viable nonmotile sperm were determined up to 6 hr after thaw. RESULTS: Sperm motilities (prefreeze motility, 70.1%; n = 9 samples) after each of five freeze/thaw cycles were 24.4, 8.0, 3.5, 1.5 and 1.8%. The recovery of sperm viability was higher than that of motility after each cycle: 39.1, 25.3, 22.6, 17.8, and 16.5%. Recoveries of motility and viability were improved if the thawed samples were left in the original cryopreservation medium prior to refreezing vs. if a washing/ resuspension step was included. The recovery of sperm motility in the first thawing cycle was indicative of the expected motile sperm recovery in the second thawing cycle. CONCLUSIONS: Cryopreserved semen that is intended to be reused in future assisted reproduction treatments should be thawed only once and aliquoted in the original freezing medium before refreezing. The recovery of sperm motility and viability in the second thawing cycle, thus the applicability of the sample in conventional in vitro fertilization or intracytoplasmic sperm injection may be anticipated in > 90% of the samples. In view of intracytoplasmic sperm injection it is important that sperm viability is maintained better than motility; after the first, second, and third thawing cycles the ratios of motile:nonmotile viable sperm were 1:1, 1:4, and 1:7, respectively.

Cryopreservation↗
PubMed1991

Cryopreservation of the occasionally improved semen samples for intrauterine insemination: a new approach in the treatment of idiopathic male infertility.

OBJECTIVE: To assess the value of treating idiopathic male infertility by intrauterine insemination (IUI) of the occasionally improved cryopreserved semen. DESIGN: Two groups of idiopathic oligospermic patients were chosen at random and treated by IUI using processed fresh semen in group A and the best available cryopreserved semen samples pooled with fresh samples in group B. SETTING: Egyptian IVF-ET Centre, Maadi, CAiro, Egypt. PATIENTS, PARTICIPANTS: One hundred fifty infertile couples because of idiopathic oligoasthenospermia. INTERVENTION: Intrauterine insemination. MAIN OUTCOME MEASURE: The pregnancy rate was evaluated after an average of three treatment cycles. RESULTS: The pregnancy rate (PR) was significantly higher in group B when compared with group A. The improvement in the PR was highly significant in the subgroup of patients for whom reasonable semen samples could be collected and cryopreserved. CONCLUSIONS: Our study indicates that IUI with fresh semen pooled with cryopreserved occasionally improved semen samples for the treatment of oligoasthenospermia results in an improved PR.

Cryopreservation↗
PubMed1989

The effect of cryopreservation on ciliary beat frequency of human respiratory epithelium.

The effect of cryopreservation on ciliary activity of human nasal respiratory epithelium was evaluated. Samples were cryopreserved in a solution containing nutrient medium, 10% fetal calf serum, and two different concentrations (10 or 20%) of dimethyl sulfoxide and stored in liquid nitrogen at -196 degrees C for 2 weeks. Ciliary beat frequencies (CBF) of the samples before and after cryopreservation were compared. Mean CBF values did not differ significantly with both concentrations of dimethyl sulfoxide. The mean intrasample coefficient of variation of the CBF decreased significantly after cryopreservation. After thawing, CBF remained unchanged for at least 4 hr. It is concluded that normal ciliated epithelial cells can be frozen and stored in liquid nitrogen at 196 degrees C while maintaining their CBF.

Adult↗
PubMed1996

[Semen preservation and gonadal toxicity in the treatment of non Hodgkin lymphoma. Experience at the Gustave-Roussy Institute from 1980 to 1993].

More than 50% of patients with non Hodgkin's lymphomas (NHL) are long-term survivors. We have retrospectively analysed the indication of semen cryopreservation and late gonadal toxicity for 213 males patients consecutively treated at the Gustave Roussy institute from 1980 to 1993 for NHL. The mean age was 30 years (15-42) and all patients received chemotherapy with or without radiotherapy. Initial spermograms and cryopreservation of semen were obtained in only 24 patients (half of them between 1991 and 1993). Spermogram characteristics were as follows: 11 normal; 13 abnormal with oligospermia (n = 7), asthenospermia (n = 7), and teratospermia (n = 8). No relation was found between the pretherapeutic status and the semen sample quality. Cryopreservation was possible in only 22 cases, and among the 16 surviving patients, two have undergone insemination and the remaining 14 are maintaining their cryopreserved semen samples. Long-term gonadal toxicity was assessed on spermograms of nine patients: three of whom had evidence of return to pretherapeutic status. FSH levels were assayed for 48 patients: at a threshold of 8 g/m2 of cyclophosphamide, 86% of patients had elevated values (P < 10(-6). Cumulated doxorubicin doses were not correlated with FSH elevation. Five patients have had children after treatment. In conclusion, chemotherapy for NHL seems to induce an intermediate level of gonadal toxicity which is between that of MOPP and ABVD. Complete information about gonadal toxicity of chemotherapy is warranted for young male patients who are to receive chemotherapy and semen cryopreservation should be suggested to this population.

Adolescent↗