A 10-year experience in testicular tissue cryopreservation for boys under 18 years of age: What can be learned from 350 cases?

BACKGROUND: A growing number of centers worldwide are preserving testicular tissue (TT) of young boys at risk of fertility loss to preserve their fertility. Data in this regard are scarce and experience sharing is essential to the optimization of the process. OBJECTIVES: This report of our 10-year activity of pediatric fertility preservation (FP) has the objective to (1) improve knowledge regarding the feasibility, acceptability, safety, and potential usefulness of the procedure; (2) analyze the impact of chemotherapy on spermatogonia in the cryopreserved TT. MATERIALS AND METHODS: For this retrospective study of data prospectively recorded, we included all boys under 18 years of age referred to the FP consultation of our academic network between October 2009 and December 2019. Characteristics of patients and cryopreservation of testicular tissue (CTT) were extracted from the clinical database. Univariate and multivariate analyses were used to assess factors associated with the risk of absence of spermatogonia in the TT. RESULTS: Three hundred and sixty-nine patients (7.2 years; 0.5-17.0) were referred to the FP consultation for malignant (70%) or non-malignant (30%) disease, of whom 88% were candidates for CTT, after a previous chemotherapy exposure (78%). The rate of recorded immediate adverse events was 3.5%, with painful episodes dominating. Spermatogonia were detected in the majority of TTs: 91.1% of those exposed to chemotherapy and 92.3% of those not exposed (p = 0.962). In multivariate analysis, the risk of absence of spermatogonia was almost three-fold higher in boys > 10 years of age ([OR] 2.74, 95% CI 1.09-7.26, p = 0.035) and four-fold higher in boys exposed to alkylating agents prior to CTT ([OR] 4.09, 95% CI 1.32-17.94, p = 0.028). DISCUSSION/CONCLUSION: This large series of pediatric FP shows that this procedure is well accepted, feasible, and safe in the short term, strengthening its place in the clinical care pathway of young patients requiring a highly gonadotoxic treatment. Our results demonstrate that CTT post-chemotherapy does not impair the chance to preserve spermatogonia in the TT except when the treatment includes alkylating agents. More data on post-CTT follow-up are still required to ensure the long-term safety and usefulness of the procedure.

Factors associated with the collection of isolated immature oocytes during ovarian tissue cryopreservation.

PURPOSE: To identify patient characteristics associated with successful isolated immature oocyte retrieval (IsO) during ovarian tissue cryopreservation (OTC) and to determine whether they are predictive of the collection of larger numbers of oocytes. METHODS: We retrospectively analyzed all patients undergoing OTC with IsO for fertility preservation over three years of activity at a university hospital. Univariate and multivariate analyses were used to identify the patients with the highest and lowest chances of oocyte recovery, and those with the largest numbers of oocytes. We also analyzed the correlation of IsO with the number of ovarian fragments collected and histological parameters. RESULTS: We analyzed 257 consecutive patients undergoing these procedures, at a median age of 17.1 years [0.3-38.3 years]. Isolated oocytes were obtained from 47.1% of patients, and IsO was more likely in patients with ovulatory cycles (63.0% vs. 38.6%; P≤ .001), without chemotherapy before OTC (61.4% vs. 33.1; P< .001) and with non-malignant diseases other than Turner syndrome (77.5%). Oocyte collection failure rates were highest in patients with Turner syndrome (OR 25.0, 95% CI 3.99-157.0; P< .001) or undergoing chemotherapy with alkylating agents before OTC (OR 37.6, 95% CI 8.36-168.8; P< .001). Prepubescent status (P= .043) and large numbers of ovarian fragments (P< .001) were associated with the retrieval of larger numbers of oocytes. Oocyte recovery was correlated with the presence of follicles in the medulla, but not with follicular density. CONCLUSION: The chances of IsO differ between patients. Identifying patients with the highest chances of success facilitates appropriate resource allocation.

Spermatozoa isolation with Felix™ outperforms conventional density gradient centrifugation preparation in selecting cells with low DNA damage.

BACKGROUND: The optimization of spermatozoa preparation techniques in order to obtain cell fractions enriched with structurally and functionally "superior" spermatozoa is a key objective of the assisted reproduction industry. OBJECTIVES: The purpose of this study was to evaluate a recent development of an electrophoretic spermatozoa separation device (Felix™, Memphasys Ltd, Sydney, Australia) and to compare its performance with conventional spermatozoa preparation by density gradient centrifugation (DGC). Particular attention was paid to the evaluation of sperm DNA/nuclear integrity. MATERIALS & METHODS: A cohort of 29 human semen samples was studied. Semen samples were analyzed fresh and after DGC or Felix™ preparation. Semen parameters monitored included sample volume, sperm count, total motility, progressive motility, sperm DNA fragmentation using the Sperm Chromatin Structure Assay and sperm DNA oxidation. RESULTS: Spermatozoa preparation with Felix™ resulted in significantly improved spermatozoa fractions with higher progressive motility, lower sperm DNA fragmentation, and lower sperm DNA oxidation compared with raw semen and DGC-prepared spermatozoa. DISCUSSION & CONCLUSION: The data collected in this study support the preparation of spermatozoa by the Felix™ system as it allows selection of spermatozoa with the highest progressive motility as well as the lowest nuclear/DNA damage. These improved sperm parameters, along with the fact that the Felix™ separation process is very fast and highly standardized, should be of great interest to the assisted reproduction technologies industry.

Computer-aided sperm analysis, the new key player in routine sperm assessment.

For sperm analysis, important inter-laboratory variations have been observed in manual analyses. In this study, a computer-aided sperm analysis (CASA) system was assessed versus manual technique, and specific software modifications were operated to fit the David's classification already used in the laboratory. Four parameters were studied (concentration, motility, vitality and morphology), and at least 30 semen samples from 30 different patients have been tested. Manual and automated analyses were compared using a least-squares regression line analysis, Student's t test, Bland-Altman plots and Passing-Bablok regressions. Repeatability was also assessed, and coefficients of variation (CV) were calculated. Both manual and automated methods gave similar results for sperm concentration (n = 150), motility (n = 30), vitality (n = 90) and morphology (n = 90). Repeatability always showed a decrease in the CV with automated analysis; for example in normal range of sperm values, CV for manual and CASA analyses were, respectively, 9.0% versus 4.4% for sperm concentration, 5.2% versus 4.1% for motility, 7.3% versus 4.2% for vitality and 11.4% versus 4.1% for morphology. All parameters were comparable between automated and manual analysis, and repeatability measures confirm the more reliable values of the SCA compared to those of manual analysis.

Detection of HIV-1 RNA in seminal plasma samples from treated patients with undetectable HIV-1 RNA in blood plasma on a 2002-2011 survey.

OBJECTIVES: To estimate the frequency of detectable seminal HIV-1 viral load in men with repeatedly undetectable blood viral load, in the recent past years and over a 10-year period (2002-2011) in a large cohort of HIV-1-infected men from couples requesting assisted reproduction technologies. We also searched for an association between HIV-1 RNA seminal viral load, HIV-1 RNA plasma viral load measured by ultrasensitive assay, and blood HIV-1 DNA in a subgroup of 98 patients. METHODS: Three hundred and four HIV-1 infected men have provided 628 paired blood and semen samples. In a subset of 98 patients for which a blood sample was available, residual viremia, HIV-1 RNA in semen and HIV-1 DNA were studied. RESULTS: Twenty of 304 patients (6.6%) had detectable HIV-1 RNA in semen, ranging from 135 to 2365 copies/ml, corresponding to 23 samples, although they had concomitantly undetectable HIV-1 RNA in blood while they were under antiretroviral therapy. This prevalence was stable over time even in recent years. There was an association between HIV-1 RNA plasma viral load measured by ultrasensitive assay and HIV-1 DNA in blood, but both were not associated with seminal HIV-1 RNA viral load. CONCLUSION: It seems cautious individually to maintain the recommendations of safe sex and the recourse to ART, or at least to inform the couple of the residual potential risk, in serodiscordant couples desiring a child.

Effect of antiretroviral drugs on the quality of semen.

The aim of this cross-sectional study was to determine which antiretroviral drugs (ARVs) are associated with changes in the characteristics of semen and the impact of these ARVs according to their score penetration into the male genital compartment. Data from 144 men infected with HIV-1 enrolled in an Assisted Reproductive Technology program were analyzed retrospectively. A seminal penetration score of ARV was based on the available literature. The nonparametric Kruskal-Wallis test and chi-square test were used. There was no difference on sperm parameters between NRTI, NNRTI, or PI regimen. In patients receiving NRTIs or PIs no differences were observed between antiretrovirals of these classes. However, in patients receiving NNRTIs, nevirapine (n = 22) was associated with a higher percentage of progressively motile spermatozoa (P < 0.0001) versus efavirenz (n = 38) as well as vitality (P = 0.0004). No relationship was observed between semen quality and the penetration score. NRTIs and PIs were not associated with any semen changes. Nevirapine was associated with a better quality of semen versus efavirenz. It would be of interest to validate, improve and test our penetration score in a prospective study.

[Fertility preservation in prepubertal children]. / Préservation de la fertilité chez l'enfant prépubère.

Gonadotoxic therapies during childhood may impair future fertility in adult life and fertility preservation techniques should be discussed before starting gonadotoxic therapies. In both sexes, fertility preservation often means immature gametes cryopreservation. For girls, ovarian tissue cryopreservation is the only existing option to preserve fertility in prepubertal girls at risk of premature ovarian failure. This promising approach involves the storage of a large number of follicles, which could subsequently be transplanted or cultured to obtain mature oocytes. The results of ovarian tissue cryopreservation in adults are encouraging. At least nine children have been born after orthotopic reimplantation of frozen-thawed ovarian cortex. None of these pregnancies were obtained by reimplantation of ovarian tissue harvested before puberty; however, the probability of restoring fertility should be higher for younger girls, as their ovarian cortex clearly contains a large number of follicules. In vitro growth of primordial follicles to mature oocytes could be an option but this goal has not yet reached in humans. This option may be reach in the future, when young patients are in their twenties or thirties. For boys, spermatogonial stem cells can be cryopreserved and uni or bilateral testicular pieces can be stored for future use. Animal data reveals that healthy offspring were reported after grafting of frozen testicular cell suspensions or tissue pieces in different species. Although recent data show promising results, restoring fertility by using frozen testicular cells after transplantation or in vitro culture is not shown yet. Then, immature testicular tissue cryopreservation for prepubertal boys is still an experimental procedure. However, as their use for restoring fertility should not be requested before 10-30 years, a long time is given for advances in medical research.

Embryo cryopreservation: proposal for a new indicator of efficiency.

OBJECTIVE: To determine whether "pregnancy rate (PR) per thawed embryo," accounting for embryo losses, is more relevant than "PR per ET," generally used in the literature for expressing embryo cryopreservation results. DESIGN: Systematic review. SETTING: University teaching hospital. PATIENT(S): None. INTERVENTION(S): Analysis of reports from January 1983 to February 2009 involving thawing of human cryopreserved embryos. Of the 1,275 articles that were initially identified, 102 met the inclusion criteria. MAIN OUTCOME MEASURE(S): Comparisons between PR per ET and PR per thawed embryo, contribution of embryo loss to PR. RESULT(S): Overall PR, expressed per ET and per thawed embryo, was compared according to the different freezing speeds and cryoprotectants used. Statistical analysis revealed significant differences for four comparisons with one approach to expressing results, not identified with the other. All other statistical analyses gave similar results whichever way the results were expressed. The rate of thawed transferred embryos, a measure accounting for embryo losses, is a good prognostic factor for PR. CONCLUSION(S): Statistical analysis accounting for embryo losses can give results different from those published in the literature. Consequently, the clinical PR per thawed embryo may be a more informative measure for accurate analysis of practices.

Oxidative stress status in normal ovarian cortex surrounding ovarian endometriosis.

Expression levels of 8-hydroxydeoxyguanosine, a sensitive indicator of DNA damage resulting from oxidative stress, were significantly higher in samples of normal ovarian cortex surrounding endometriotic cysts when compared with ovarian cortex surrounding dermoid and serous ovarian cysts. These findings suggest that the normal ovarian cortex surrounding endometriotic tissues is more severely affected by oxidative stress than ovarian cortex adjacent to other benign ovarian cysts.

Follicular growth and estradiol follow-up after subcutaneous xenografting of fresh and cryopreserved human ovarian tissue.

OBJECTIVE: To assess ovarian cortex surrounding benign ovarian cysts after cryopreservation and grafting to severe combined immunodeficient (SCID) mice. DESIGN: Animal study. SETTING: Academic research laboratories. PATIENT(S): Ovarian tissue obtained from 15 patients. INTERVENTION(S): Grafting of fresh and frozen/thawed ovarian tissue into the subcutaneous space of 22 SCID mice for 80 days. MAIN OUTCOME MEASURE(S): Histologic analysis before and after grafting. Serum E(2) measured before (after 37 days of grafting) and after FSH/LH supplementation (end of the study). RESULT(S): After grafting, follicular density had decreased for frozen/thawed tissue in all cases. The follicular distribution was modified in fresh tissue: Primordial follicles proportion was reduced (79% vs. 17%), whereas the primary and secondary ones were increased (21% vs. 57% and 0% vs. 23%, respectively). The same tendency was observed in frozen/thawed tissue. Significant E(2) secretion was obtained before and after FSH/LH supplementation in castrated mice, grafted with either fresh or frozen/thawed tissue. CONCLUSION(S): Fresh and cryopreserved ovarian cortex surrounding benign ovarian cysts grafted into the subcutaneous space of SCID mice is able to sustain ovarian tissue function, although follicular growth appears lower with frozen/thawed tissue.

Human ovarian tissue from cortex surrounding benign cysts: a model to study ovarian tissue cryopreservation.

BACKGROUND: The scarcity of human ovarian tissue is a major problem in developing research on ovarian cryopreservation. We were interested in ovarian cortex surrounding benign ovarian cysts harvested during their requisite operations. METHODS: Ovarian tissue was collected from 25 women (mean age = 27.7 +/- 1.0 SEM) and frozen in serum-free cryoprotective medium. Histological and viability analysis were performed on fresh and frozen-thawed slices of tissue. RESULTS: Dermoid (n = 7), endometriosis (n = 13) and serous (n = 5) cysts were observed. Follicular densities (expressed per mm3) in ovarian cortex surrounding dermoid cysts were higher than in endometriosis and serous cysts for both histological (median of follicular densities: 13.04, 0.31 and 0.89 respectively) and viability analysis (2.93, 0.05 and 0.71 respectively). Freezing-thawing did not result in gross abnormality of follicle population either in number or morphology (80% of follicles preserved a normal pattern). However, a slight decrease of the density of living follicles (expressed per mm2) was reported. CONCLUSIONS: Ovarian cortex surrounding ovarian cysts, especially dermoid cysts, could be considered a source of ovarian tissue for future research. In our study, the cryopreservation procedure resulted in high follicular survival assessed by both histological and viability analysis. Nevertheless, further studies of in vivo and in vitro follicular maturation are needed to strengthen this model.

Sperm analysis by FISH in a case of t(17; 22) (q11; q12) balanced translocation: case report.

Individual sperm from men with balanced translocations have different chromosomal contents. Thus, an estimation of the overall sperm chromosomal imbalance of such patients could help to give the couple an adapted genetic counselling. We report here the study of a balanced translocation carrier, t(17;22) (q11;q12) whose reproductive history reported four miscarriages. Moreover, he had an abnormal semen analysis with oligoteratozoospermia. The meiotic segregation pattern was examined in 700 sperm, using fluorescence in-situ hybridization (FISH). Nineteen percent of the sperm had balanced translocations or were normal. All other sperm were unbalanced (81%) and their distribution was observed as follows: the frequencies of adjacent 1, adjacent 2 and 3:1 segregations were 12.9, 5.8 and 46.8% respectively. Among the segregations scored, 13.7% were related to second meiotic division abnormalities. Less than 2% of the total sperm scored were not explained. The 3:1 segregation was present at a very high rate, which is very unusual. In cases of balanced translocations, we believe that no general features can be drawn. Thus, the FISH technique may be very helpful for genetic counselling, which remains an important step and must be done with care.