Vitrification of Human Oocytes Before or After Rescue-IVM Does not Impair Maturation Kinetics but Induces Meiotic Spindle Alterations.

Cryopreservation of in vitro matured oocytes is still considered as an experimental alternative to mature oocyte vitrification after ovarian stimulation. Here, we investigated whether rescue-IVM should be performed before or after vitrification. For this, 101 immature oocytes (germinal vesicle stage) from women undergoing ICSI were used. Oocytes were divided into three groups: freshly in vitro matured oocytes (IVM), freshly in vitro matured oocytes subsequently vitrified (IVM + VIT) and vitrified/warmed GV oocytes then in vitro matured (VIT + IVM). Oocyte maturation rates and kinetics were assessed using time-lapse technology. Spindle dimensions and polarity, chromosome alignment and cytoplasmic F-actin filament length and density were determined using confocal microscopy and quantitative image analyses. No differences in IVM rates (fresh IVM: 63.16% and IVM post-VIT: 59.38%, p = 0.72) and timings (17.73 h in fresh IVM, 17.33 h in IVM post-VIT, p = 0.72) were observed whether IVM is performed freshly or after vitrification. Meiotic spindles were shorter in VIT + IVM (10.47 µm vs 11.23 µm in IVM and 11.40 µm in IVM + VIT, p = 0.012 and p = 0.043) and wider in IVM + VIT (9.37 µm vs 8.12 µm in IVM and 8.16 µm VIT + IVM, p = 0.027 and p = 0.026). The length-to-width ratio was lower in vitrified groups (IVM + VIT: 1.19 and VIT + IVM: 1.26) compared to IVM (1.38), p = 0.013 and p = 0.014. No differences in multipolar spindle and chromosome misalignment occurrence and cytoplasmic F-actin filament length and density were observed between groups. Our results suggest vitrification before or after rescue-IVM does not seem to impair maturation rates and kinetics parameters but induces meiotic spindle alterations.

Is large for gestational age in singletons born after frozen embryo transfer associated with freezing technique or endometrial preparation protocol? A longitudinal national French study.

STUDY QUESTION: Is large for gestational age (LGA) observed in babies born after frozen embryo transfer (FET) associated with either the freezing technique or the endometrial preparation protocol? SUMMARY ANSWER: Artificial cycles are associated with a higher risk of LGA, with no difference in rate between the two freezing techniques (vitrification versus slow freezing) or embryo stage (cleaved embryo versus blastocyst). WHAT IS KNOWN ALREADY: Several studies have compared neonatal outcomes after fresh embryo transfer (ET) and FET and shown that FET is associated with improved neonatal outcomes, including reduced risks of preterm birth, low birthweight, and small for gestational age (SGA), when compared with fresh ET. However, these studies also revealed an increased risk of LGA after FET. The underlying pathophysiology of this increased risk remains unclear; parental infertility, laboratory procedures (including embryo culture conditions and freezing-thawing processes), and endometrial preparation treatments might be involved. STUDY DESIGN, SIZE, DURATION: A multicentre epidemiological data study was performed through a retrospective analysis of the standardized individual clinical records of the French national register of IVF from 2014 to 2018, including single deliveries resulting from fresh ET or FET that were prospectively collected in fertility centres. Complementary data were collected from the participating fertility centres and included the vitrification media and devices, and the endometrial preparation protocols. PARTICIPANTS/MATERIALS, SETTING, METHODS: Data were collected from 35 French ART centres, leading to the inclusion of a total of 72 789 fresh ET, 10 602 slow-freezing FET, and 39 062 vitrification FET. Main clinical outcomes were presented according to origin of the transferred embryos (fresh, slow frozen, or vitrified embryos) and endometrial preparations for FET (ovulatory or artificial cycles), comparing five different groups (fresh, slow freezing-ovulatory cycle, slow freezing-artificial cycle, vitrification-ovulatory cycle, and vitrification-artificial cycle). Foetal growth disorders were defined in live-born singletons according to gestational age and sex-specific weight percentile distribution: SGA and LGA if <10th and ≥90th percentiles, respectively. Analyses were performed using linear mixed models with the ART centres as random effect. MAIN RESULTS AND THE ROLE OF CHANCE: Transfers led to, respectively, 19 006, 1798, and 9195 deliveries corresponding to delivery rates per transfer of 26.1%, 17.0%, and 23.5% after fresh ET, slow-freezing FET, and vitrification FET, respectively. FET cycles were performed in either ovulatory cycles (n = 21 704) or artificial cycles (n = 34 237), leading to 5910 and 10 322 pregnancies, respectively, and corresponding to pregnancy rates per transfer of 31.6% and 33.3%. A significantly higher rate of spontaneous miscarriage was observed in artificial cycles when compared with ovulatory cycles (33.3% versus 21.4%, P < 0.001, in slow freezing groups and 31.6% versus 21.8%, P < 0.001 in vitrification groups). Consequently, a lower delivery rate per transfer was observed in artificial cycles compared with ovulatory cycles both in slow freezing and vitrification groups (15.5% versus 18.9%, P < 0.001 and 22.8% versus 24.9%, P < 0.001, respectively). Among a total of 26 585 live-born singletons, 16 413 babies were born from fresh ET, 1644 from slow-freezing FET, and 8528 from vitrification FET. Birthweight was significantly higher in the FET groups than in the fresh ET group, with no difference between the two freezing techniques. Likewise, LGA rates were higher and SGA rates were lower in the FET groups compared with the fresh ET group whatever the method used for embryo freezing. In a multivariable analysis, the risk of LGA following FET was significantly increased in artificial compared with ovulatory cycles. In contrast, the risk of LGA was not associated with either the freezing procedure (vitrification versus slow freezing) or the embryo stage (cleaved embryo versus blastocyst) at freezing. Regarding the vitrification method, the risk of LGA was not associated with either the vitrification medium used or the embryo stage. LIMITATIONS, REASONS FOR CAUTION: No data were available on maternal context, such as parity, BMI, infertility cause, or maternal comorbidities, in the French national database. In particular, we cannot exclude that the increased risk of LGA observed following FET with artificial cycles may, at least partially, be associated with a confounding effect of some maternal factors. No information about embryo culture and incubation conditions was available. Most of the vitrification techniques were performed using the same device and with two main vitrification media, limiting the validity of a comparison of risk for LGA according to the device or vitrification media used. WIDER IMPLICATIONS OF THE FINDINGS: Our results seem reassuring, since no potential foetal growth disorders following embryo vitrification in comparison with slow freezing were observed. Even if other factors are involved, the endometrial preparation treatment seems to have the greatest impact on LGA risk following FET. FET during ovulatory cycles could minimize the risk for foetal growth disorders. STUDY FUNDING/COMPETING INTEREST(S): This work has received funding from the French Biomedicine Agency (Grant number: 19AMP002). None of the authors has any conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Efficient pathway for men fertility preservation in testicular cancer or lymphoma: a cross-sectional study of national 2018 data.

BACKGROUND: In 15-49 years-old men, the main cancers are testicular cancer (TC) and lymphomas (L): freezing of ejaculated sperm is primarily used for male fertility preservation (FP) before cancer treatment. Our objective was to analyze the French FP rate in 15-49 years-old men diagnosed with TC or L in 2018. We designed a national descriptive cross-sectional study of sperm banking rate in men with a diagnosis of TC, Hodgkin L (HL) or non-Hodgkin L (NHL). From the French National Cancer Institute (INCa) 2018 data, we extracted the estimated incidence of TC and L in metropolitan France. From the 2018 activity report of CECOS network (Centers for Study and Banking of Eggs and Sperm), we extracted the number of men with TC or L who banked ejaculated sperm. We estimated the proportion of 15-49 years-old men diagnosed with TC or L who banked sperm. RESULTS: Among 15-49 years-old men, INCa estimated 38,048 new cancer diagnoses in metropolitan France in 2018: 2,630 TC and 3,913 L (943 HL and 2,970 NHL). The CECOS network provided data from 26/27 metropolitan centers (96% response rate): 1,079 sperm banking for men with TC, 375 for HL and 211 for NHL. We estimated that the 2018 sperm banking rate in France was 41% for TC, 40% for HL, and 7% for NHL. CONCLUSIONS: To our knowledge, our paper is the first cross-sectional study with multicenter and national data analyzing FP rate in cancer men: it suggests an efficient pathway for men to FP before cancer treatment, compared to previously published studies. Although sperm banking rate in 15-49 years-old men could definitely be improved, further studies should evaluate the information given to patients before gonadotoxic treatments, the factors associated with the absence of sperm banking and whether this lack of referral induces a loss of chance for these men.

RéSUMé: CONTEXTE: Chez les hommes de 15 à 49 ans, les principaux cancers sont le cancer du testicule (CT) et les lymhomes (L): la congélation de spermatozoïdes éjaculés est utilisée en première intention pour leur préservation de fertilité (PF) avant traitement du cancer. Notre objectif était d'analyser le taux de PF chez les hommes de 15 à 49 ans diagnostiqués avec un CT ou un L en 2018 en France. Nous avons réalisé une étude nationale transversale descriptive du taux de congelation de spermatozoïdes chez les hommes âgés de 15 à 49 ans diagnostiqués avec un CT, un L de Hodgkin (LH) ou un L non-Hodgkinien (LNH). A partir des données de l'Institut National du Cancer (INCa) de 2018, nous avons extrait l'incidence estimée de CT et de L en France métropolitaine. A partir des données du bilan d'activité 2018 de la Federation Française des CECOS (Centre d'Etude et de Conservation des Oeufs et du Sperme), nous avons extrait le nombre d'hommes avec un CT ou un L qui ont congelé leurs spermatozoïdes. Nous avons enfin estimé la proportion d'hommes de 15 à 49 ans diagnostiqués avec un CT ou un L qui ont congelé leurs spermatozoïdes. RéSULTATS: Chez les hommes de 15 à 49 ans, l'INCa a estimé en 2018 38 048 nouveaux cas de cancers diagnostiqués en France métropolitaine en 2018: 2 630 CT et 3 913 L (943 LH et 2 970 LNH). Le réseau des CECOS a produit les résultats issus de 26/27 centres métropolitains (taux de réponse de 96%): 1 079 congélations de sperme pour des hommes atteints de CT, 375 pour LH et 211 pour LNH. Nous avons estimé que le taux de congelation de spermatozoïdes de 2018 en France était de 41% pour le CT, 40% pour le LH et 7% pour le LNH. CONCLUSIONS: A notre connaissance, notre travail est la première étude transversale multicentrique de données nationales analysant le taux de PF chez les hommes atteints de cancer: il suggère un parcours patient efficace pour la PF des hommes avant traitement d'un cancer, par rapport aux études précédemment publiées. Bien que le taux de PF chez les hommes puisse certainemen être amélioré, des études futures devraient évaluer l'information donnée aux patients avant traitement gonadotoxique, les facteurs associés à l'absence de PF et si le défaut d'adressage au CECOS induit un perte de chance pour ces hommes. MOTS-CLéS: Chimiothérapie, Radiothérapie, Oncofertiité, Azoospermia, Paternité.

Impact of low- or moderate-risk gonadotoxic chemotherapy prior to testicular tissue freezing on spermatogonia quantity in human (pre)pubertal testicular tissue.

STUDY QUESTION: What is the impact of low- or moderate-risk gonadotoxic chemotherapy received prior to testicular tissue freezing (TTF), and of the cancer itself, on spermatogonia quantity in testicular tissue from (pre)pubertal boys? SUMMARY ANSWER: Vincristine, when associated with alkylating agents, has an additional adverse effect on spermatogonia quantity, while carboplatin has no individual contribution to spermatogonia quantity, in testicular tissue of (pre)pubertal boys, when compared to patients who have received non-alkylating chemotherapy. WHAT IS KNOWN ALREADY: The improved survival rates after cancer treatment necessitate the inclusion of fertility preservation procedures as part of the comprehensive care for patients, taking into consideration their age. Sperm cryopreservation is an established procedure in post-pubertal males while the TTF proposed for (pre)pubertal boys remains experimental. Several studies exploring testicular tissue of (pre)pubertal boys after TTF have examined the tubular fertility index (TFI, percentage of seminiferous tubule cross-sections containing spermatogonia) and the number of spermatogonia per seminiferous tubule cross-section (S/T). All studies have demonstrated that TFI and S/T always decrease after the introduction of chemotherapeutic agents, especially those which carry high gonadotoxic risks such as alkylating agents. STUDY DESIGN, SIZE, DURATION: Testicular tissue samples from 79 (pre)pubertal boys diagnosed with cancer (from 6 months to 16 years of age) were cryopreserved between May 2009 and June 2014. Their medical diagnoses and previous chemotherapy exposures were recorded. We examined histological sections of (pre)pubertal testicular tissue to elucidate whether the chemotherapy or the primary diagnosis affects mainly TFI and S/T. PARTICIPANTS/MATERIALS, SETTING, METHODS: (Pre)pubertal boys with cancer diagnosis who had been offered TTF prior to conditioning treatment for hematopoietic stem cell transplantation were included in the study. All the patients had previously received chemotherapy with low- or moderate-risk for future fertility. We have selected patients for whom the information on the chemotherapy received was complete. The quantity of spermatogonia and quality of testicular tissue were assessed by both morphological and immunohistochemical analyses. MAIN RESULTS AND THE ROLE OF CHANCE: A significant reduction in the number of spermatogonia was observed in boys treated with alkylating agents. The mean S/T values in boys exposed to alkylating agents were significantly lower compared to boys exposed to non-alkylating agents (P = 0.018). In contrast, no difference was observed for patients treated with carboplatin as the sole administered alkylating agent compared to the group of patients exposed to non-alkylating agents. We observed an increase of S/T with age in the group of patients who did not receive any alkylating agent and a decrease of S/T with age when patients received alkylating agents included in the cyclophosphamide equivalent dose (CED) formula (r = 0.6166, P = 0.0434; r = -0.3759, P = 0.0036, respectively). The TFI and S/T decreased further in the group of patients who received vincristine in combination with alkylating agents (decrease of 22.4%, P = 0.0049 and P < 0.0001, respectively), but in this group the CED was also increased significantly (P < 0.0001). Multivariate analysis, after CED adjustment, showed the persistence of a decrease in TFI correlated with vincristine administration (P = 0.02). LIMITATIONS, REASONS FOR CAUTION: This is a descriptive study of testicular tissues obtained from (pre)pubertal boys who were at risk of infertility. The study population is quite heterogeneous, with a small number of patients in each sub-group. Our results are based on comparisons between patients receiving alkylating agents compared to patients receiving non-alkylating agents rather than chemotherapy-naive patients. The French national guidelines for fertility preservation in cancer patients recommend TTF before highly gonadotoxic treatment. Therefore, all the patients had received low- or moderate-risk gonadotoxic chemotherapy before TTF. Access to testicular tissue samples from chemotherapy-naive patients with comparable histological types of cancer was not possible. The functionality of spermatogonia and somatic cells could not be tested by transplantation or in vitro maturation due to limited sample sizes. WIDER IMPLICATIONS OF THE FINDINGS: This study summarizes the spermatogonial quantity of (pre)pubertal boys prior to TTF. We confirmed a negative correlation between the cumulative exposure to alkylating agents and spermatogonial quantity. In addition, the synergistic use of vincristine in combination with alkylating agents showed a cumulative deleterious effect on the TFI. For patients for whom fertility preservation is indicated, TTF should be proposed for chemotherapy with a predicted CED above 4000 mg/m2. However, the data obtained from vincristine and carboplatin use should be confirmed in a subsequent study including more patients. STUDY FUNDING/COMPETING INTEREST(S): This study had financial support from a French national research grant PHRC No. 2008/071/HP obtained by the French Institute of Cancer and the French Healthcare Organization. The sponsors played no role in the study. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Freezing Does Not Alter Sperm Telomere Length despite Increasing DNA Oxidation and Fragmentation.

Correlations were reported between sperm telomere length (STL) and male fertility, sperm DNA fragmentation, and oxidation. Sperm freezing is widely used for assisted reproductive techniques, fertility preservation, and sperm donation. However, its impact on STL remains unknown. For this study, semen surplus from patients who underwent routine semen analysis were used. The impact of slow freezing on STL was analyzed by performing qPCR before and after freezing. Sperm populations with different STL were evaluated using Q-FISH. The relationship between sperm DNA oxidation, DNA fragmentation, and STL was assessed in fresh and frozen sperm samples. No significant impact of slow freezing on STL was observed, neither measured by qPCR nor Q-FISH. However, Q-FISH allowed for the distinguishing of sperm populations with different STLs within individual sperm samples. Slow freezing induced different STL distributions for some of the analyzed sperm samples, but no correlation was found between STL and sperm DNA fragmentation or oxidation. Slow freezing does not alter STL despite increasing sperm DNA oxidation and fragmentation. As STL alterations could be transmitted to offspring, the lack of impact of the slow freezing method on STL ensures the safety of this procedure.

Validation of a SARS-CoV-2 RT-PCR assay: a requirement to evaluate viral contamination in human semen.

RESEARCH QUESTION: Is it possible to validate an accurate and reliable method for direct detection of SARS-CoV-2 by reverse transcription polymerase chain reaction (RT-PCR) in human semen fractions? DESIGN: This qualitative improvement study aimed to provide a prospective validation of SARS-CoV-2 detection in male semen. The SARS-CoV-2 genome was detected by multiplex real-time RT-PCR on patient samples that underwent routine semen analyses for infertility at the Center for Reproductive Medicine at the University Hospital of Clermont-Ferrand. Samples comprised surplus semen collected for treatment with assisted reproductive technology. Seminal fluid and spermatozoa fractions were isolated with density gradient centrifugation and cryopreserved. Positive samples were prepared with a standard of inactivated SARS-CoV-2 particles. RESULTS: The analytical method was validated in both seminal fluid and spermatozoa fractions. In both semen fractions, the assay was repeatable, reproducible and showed high sensitivity with a limit of detection of 0.33 SARS-CoV-2 genome copies/µl. The limit of quantification was 1 copy of the SARS-CoV-2 genome/µl. The method was effective regardless of semen quality (normal and altered sperm parameters), number of spermatozoa or the cryoprotectant media used to freeze spermatozoa. CONCLUSION: This validated RT-PCR assay provided accurate and reliable screening of SARS-CoV-2 in seminal fluid and spermatozoa fractions. This method is essential to ensure protection against viral contamination in the cryobanking process.

Assessment of the architecture and integrity of frozen-thawed testicular tissue from (pre)pubertal boys with cancer.

BACKGROUND: Testicular tissue freezing is proposed for fertility preservation to (pre)pubertal boys with cancer before highly gonadotoxic treatment. Studies accurately comparing human (pre)pubertal testicular tissue quality before freezing and after thawing are exceptional. No study has reported this approach in a systematic manner and routine care. OBJECTIVES: To assess the impact of a control slow freezing protocol on testicular tissue architecture and integrity of (pre)pubertal boys after thawing. MATERIALS AND METHODS: (Pre)pubertal boys (n = 87) with cancer from 8 Reproductive Biology Laboratories of the French CECOS network benefited from testicular tissue freezing before hematopoietic stem cell transplantation. Seminiferous tubule cryodamage was determined histologically by scoring morphological alterations and by quantifying intratubular spermatogonia and the expression of DNA replication and repair marker in frozen-thawed testicular fragments. RESULTS: A significant increase in nuclear and epithelial score alterations was observed after thawing (p < 0.0001). The global lesional score remained lower than 1.5 and comparable to fresh testicular tissue. The number of intratubular spermatogonia and the expression of DNA replication and repair marker in spermatogonia and Sertoli cells did not vary significantly after thawing. These data showed the good preservation of the seminiferous tubule integrity and architecture after thawing, as previously reported in our studies performed in prepubertal mice and rats. DISCUSSION: The current study reports, for the first time, the development of a semi-quantitative analysis of cryodamage in human (pre)pubertal testicular tissue, using a rapid and useful tool that can be proposed in routine care to develop an internal and external quality control for testicular tissue freezing. This tool can also be used when changing one or several parameters of the freezing-thawing procedure. CONCLUSION: Control slow freezing protocol without seeding maintains the seminiferous tubule architecture and integrity, the concentration of spermatogonia and the expression of DNA replication and repair marker in spermatogonia and Sertoli cells after thawing.

Beneficial effects of hypotaurine supplementation in preparation and freezing media on human sperm cryo-capacitation and DNA quality.

BACKGROUND: Although widely used, slow freezing considerably modifies the functions of human spermatozoa. Cryopreservation induces nuclear sperm alterations and cryo-capacitation, reducing the chances of pregnancy. Hypotaurine is naturally present in the male and female genital tracts and has capacitating, osmolytic and anti-oxidant properties. The analysis were performed on surplus semen of men with normal (n = 19) or abnormal (n = 14) sperm parameters. Spermatozoa were selected by density gradient centrifugation before slow freezing. For each sample, these steps were performed in parallel with ("H+" arm) or without ("H-" arm) hypotaurine supplementation. After thawing, we measured total and progressive mobility, vitality, acrosome integrity, markers of capacitation signaling pathway and nuclear quality. For the latter, we focused on sperm chromatin packaging, DNA fragmentation and the presence of vacuoles in the sperm nucleus. RESULTS: Post-thaw spermatozoa selected and frozen in the presence of hypotaurine had a higher vitality (+ 16.7%, p < 0.001), progressive and total motility (+ 39.9% and +  21.6% respectively, p < 0.005) than spermatozoa from the control "H-" arm. Hypotaurine also reduced the non-specific phosphorylation of the capacitation protein markers P110 and P80 (p < 0.01), indicating a decrease in cryo-capacitation. Hypotaurine supplementation reduced chromatin decondensation, measured by chromomycin A3 (- 16.1%, p < 0.05), DNA fragmentation (- 18.7%, p < 0.05) and nuclear vacuolization (- 20.8%, p < 0.05). CONCLUSION: Our study is the first to demonstrate beneficial effects of hypotaurine supplementation in preparation and freezing procedures on human spermatozoa sperm fertilization capacity and nucleus quality. Hypotaurine supplementation limited cryo-capacitation, increased the proportion of live and progressively motile spermatozoa and reduces the percentage of spermatozoa showing chromatin decondensation, DNA fragmentation and nuclear vacuolation. TRIAL REGISTRATION: Clinical Trial, NCT04011813 . Registered 19 May 2019 - Retrospectively registered.

RéSUMé: CONTEXTE: Bien que largement utilisée, la congélation lente modifie considérablement les fonctions des spermatozoïdes humains. La cryoconservation induit des altérations nucléaires du sperme et une cryocapacitation, réduisant les chances de grossesse. L'hypotaurine est. naturellement présente dans les voies génitales masculines et féminines et possède des propriétés capacitantes, osmotiques et anti-oxydantes. Les mesures ont été réalisées sur le reliquat de sperme d'hommes avec des paramètres spermatiques normaux (n = 19) ou anormaux (n = 14). Les spermatozoïdes ont été sélectionnés par centrifugation sur gradient de densité (test de migration survie) avant congélation lente. Pour chaque prélèvement, ces étapes ont été réalisées en parallèle avec des milieux supplémentés en hypotaurine (bras « H+ ¼) ou sans hypotaurine (bras « H- ¼). Après décongélation, nous avons mesuré la mobilité totale et progressive, la vitalité, l'intégrité de l'acrosome, des marqueurs de la voie de signalisation de la capacitation et la qualité nucléaire. Pour cette dernière, nous nous sommes concentrés sur la condensation de la chromatine, la fragmentation de l'ADN et la présence de vacuoles dans le noyau du sperme. RéSULTATS: Post-décongélation, les spermatozoïdes sélectionnés et congelés en présence d'hypotaurine avaient une vitalité plus élevée (+ 16,7%, p < 0,001), une motilité progressive et totale (+ 39,9% et + 21,6% respectivement, p < 0,005) que les spermatozoïdes du bras « H- ¼ sans suplémentation. L'hypotaurine a également réduit la phosphorylation non spécifique des marqueurs protéiques de capacitation P110 et P80 (p < 0,01), indiquant une diminution de la cryocapacitation. La supplémentation en hypotaurine a réduit la décondensation de la chromatine, mesurée par la chromomycine A3 (− 16,1%, p < 0,05), la fragmentation de l'ADN (− 18,7%, p < 0,05) et la vacuolisation nucléaire (− 20,8%, p < 0,05). CONCLUSION: Notre étude est. la première à démontrer les effets bénéfiques de la supplémentation en hypotaurine dans les milieux de préparation et de congélation sur la capacité de fécondation des spermatozoïdes humains et leur qualité nucléaire. La supplémentation en hypotaurine a limité la cryocapacitation, augmenté la proportion de spermatozoïdes vivants et progressivement mobiles et réduit le pourcentage de spermatozoïdes présentant une décondensation de la chromatine, une fragmentation de l'ADN et une vacuolisation nucléaire. ENREGISTREMENT DE L'ESSAI: essai clinique, NCT04011813 . Enregistré le 19 mai 2019 - Enregistré rétrospectivement.

Domestic use of pesticides during early periods of development and risk of testicular germ cell tumors in adulthood: a French nationwide case-control study.

BACKGROUND: Testicular germ cell tumours (TGCT) are the most frequent cancers in young men in developed countries and their incidence rate has doubled worldwide over the past 40 years. Early life exposures to pesticides are suspected to increase TGCT risk. Our research aimed at estimating adult TGCT risk associated with parental domestic use of pesticides during early periods of child development. METHODS: We conducted a case-control study of 304 TGCT cases, aged 18-45 years old, recruited in 20 French university hospitals, and 274 controls frequency-matched on hospital and birth year. Participants' mothers provided information on their domestic use of pesticides from 1 year before start of pregnancy to 1 year after their son's birth, for gardening activities, treatment of indoor plants, pets, wood and mold, and pest control. Odds ratios (OR) for TGCT (overall and by histological subtype) and 95% confidence intervals (CI) were estimated using conditional logistic regression. RESULTS: Prevalence of reported domestic use of pesticides was 77.3% for insecticides, 15.9% for fungicides and 12.1% for herbicides. While no association was found for any use of insecticides (OR = 1.27, CI = 0.80-2.01) or herbicides (OR = 1.15, CI = 0.67-2.00), elevated risks of TGCT overall (OR = 1.73, CI = 1.04-2.87) and non-seminoma subtype (OR = 2.44, CI = 1.26-4.74) were observed for any use of fungicides. When specific purposes were examined, using fungicides and/or insecticides for woodwork (OR = 2.35, CI = 1.06-5.20) and using insecticides on cats and dogs (OR = 1.95, CI = 1.12-3.40) were associated with increased risk of non-seminoma subtype. We found no association for seminoma subtype. CONCLUSIONS: Although recall bias may partially explain the elevated ORs, our study provides some evidence of a positive association between domestic use of pesticides during early periods of development, particularly fungicides and risk of adult TGCT and non-seminoma. Given the common domestic use of pesticides in France, further research on TGCT risk is warranted.

Fertility preservation and sperm donation in transgender individuals: The current situation within the French CECOS network.

BACKGROUND: Many studies reported that reproductive desire could be high among transgender individuals. In France, fertility preservation and sperm donation were very little proposed to transgender individuals until recently, mainly because the Bioethics Law allows the use of assisted reproductive technologies only in infertile couples and prohibits surrogacy. OBJECTIVES: To evaluate the distribution of care on the French territory concerning fertility preservation and sperm donation in transgender individuals. MATERIALS AND METHODS: A multicentric national survey was carried out between January 2019 and October 2020 in 28 assisted reproductive technology centres of the French CECOS (Centres d'Etudes et de Conservation des Oeufs et du Sperme) network. Each centre was questioned to find out how many transgender individuals came, were informed and cared for fertility preservation and sperm donation. RESULTS: Concerning fertility preservation, 71.4% of centres received transgender individuals and performed gamete cryopreservation; 581 transgender individuals consulted for fertility preservation. Transgender women were more likely to desire (p < 0.0001) and achieve (p < 0.0001) fertility preservation than transgender men. Concerning sperm donation in couples including a transgender man, 68% of centres offer the complete course from the first consultation to the completion of the assisted reproductive technology cycles; 122 offsprings have been conceived with sperm donation in couples including a transgender man since 1999. DISCUSSION: Our results showed that even if all centres do not propose fertility preservation or sperm donation in transgender individuals, these assisted reproductive technologies are present throughout the French territory. The major point is that both fertility preservation and sperm donation in transgender individuals have grown significantly and that the care of these patients is improving year after year. CONCLUSION: In France, most of CECOS centres can take care of transgender individuals for fertility preservation and sperm donation. The French Bioethics Law allows these latter, and transgender individuals can benefit from a financial support of the national health care insurance for fertility preservation and sperm donation.

Impact of disease activity and treatments on ovarian reserve in patients with rheumatoid arthritis in the ESPOIR cohort.

OBJECTIVES: Patients with RA have a higher prevalence of infertility than the general population. This study sought to examine the impact of RA disease activity and treatments on ovarian reserve measured by serum anti-Müllerian hormone (AMH) levels in the ESPOIR cohort. We sought to better define the indications for fertility preservation. METHODS: Patients and serum analysis data were derived from the French national cohort ESPOIR. Enrolled patients (n = 102; 18-37-year-olds) fulfilled ACR/EULAR 2010 criteria for RA. Serum AMH levels were measured at T0, T6, T12, T24 and T36 months post-diagnosis. The impacts of RA activity (DAS28 and CRP level) and treatments (MTX only or with other medications) were evaluated at each study visit. RESULTS: A gradual decrease in patients' serum AMH levels was observed over time, in line with the descending curve described for healthy women. Serum AMH levels of RA patients in comparison with the values considered normal for age did not reveal any significant differences (P > 0.05). We did not observe any impact of RA treatments. We demonstrated an inverse correlation between AMH variation and disease activity (DAS28: r = -0.27, P = 0.003; CRP: r = -0.16, P = 0.06). CONCLUSION: This is the first study to determine serum AMH levels of a large cohort of RA patients over 36 months. Rapid disease activity control appears to be required to limit changes in the ovarian reserve. Fertility preservation is not likely to be necessary if inflammation is promptly controlled. CLINICALTRIALS.GOV IDENTIFIER: NCT03666091.

Idiopathic central precocious puberty in a Klinefelter patient: highlights on gonadotropin levels and pathophysiology.

BACKGROUND: Idiopathic central precocious puberty (ICPP) is supposed to be non-existent in a context of testicular destruction that is typically present in Klinefelter syndrome (KS). Herein, we describe a rare case of ICPP in a Klinefelter patient (47,XXY) with 2 maternal X chromosomes. Moreover, we highlight the differences in gonadotropin levels in comparison to males with ICPP and a normal karyotype. CASE PRESENTATION: An 8 years old boy with a history of cryptorchidism was evaluated for precocious puberty (Tanner staging: P2/G3). Both testes measured 25x35mm. His hormonal profile confirmed a central origin of precocious puberty with high serum testosterone (4.3 ng/ml), luteinizing hormone [LH (3.5 UI/l)] and follicle stimulating hormone [FSH (7.7 UI/l)] levels. Luteinizing hormone-releasing hormone (LHRH) test amplified LH and FSH secretion to 24 and 14 UI/l respectively. Brain magnetic resonance imaging (MRI) was normal. No MKRN3 mutation was detected. He was treated for ICPP for two years. During puberty, he suffered from hypergonadotropic hypogonadism leading to the diagnosis of KS (47,XXY karyotype). Chromosomal analysis by fluorescent multiplex polymerase chain reaction (PCR) using X chromosome microsatellite markers identified 2 maternal X chromosomes. Analysing 8 cases of KS developing ICPP (our reported case and 7 other published cases) revealed that these KS patients with ICPP have higher LH and FSH levels during ICPP episode than in ICPP patients with a normal karyotype (ICPP with KS vs ICPP with a normal karyotype: LH levels 9.4 ± 12 vs 1.1 ± 0.6 UI/l; FSH levels 23.1 ± 38.5 vs 2.7 ± 1.5 UI/l). Furthermore, their response to gonadotropin-releasing hormone (GnRH) stimulation is characterized by excessive LH and FSH secretion (LH levels post-GnRH: 58 ± 48 vs 15.5 ± 0.8 UI/l; FSH levels post-GnRH: 49.1 ± 62.1 vs 5.7 ± 3.9 UI/l). CONCLUSIONS: ICPP in boys is extremely rare. The pathophysiology of ICPP in KS is unknown. However, maternal X supplementary chromosome and early testicular destruction may play a significant role in the initiation of ICPP, in part explaining the relative "overrepresentation of ICPP in KS. Thus, karyotype analysis could be considered for boys suffering from ICPP, especially if testicular size is smaller or gonadotropins are significantly elevated.

Sperm cryostorage in a dry tank: An accurate alternative.

This prospective study aimed to determine the effects of dry nitrogen cryostorage on human sperm characteristics in comparison with liquid nitrogen cryostorage. For this purpose, 42 men undergoing routine semen analysis (21 normozoospermia and 21 with altered semen parameters) were analyzed. After slow freezing, half of the straws of each sample were randomly stored in liquid and dry tanks, at the top and bottom levels of the latter. After 6 months storage, thawed samples were treated by density gradient centrifugation and sperm characteristics were compared. There was no difference in sperm progressive motility (15.1% ± 14.2% vs. 15.1% ± 12.7%; p = 0.76), sperm vitality (25.5% ± 17.7% vs. 26.2% ± 19%; p = 0.71), percentages of acrosome-reacted spermatozoa (38% ± 8.5% vs. 38.5% ± 7.4%; p = 0.53) and DNA fragmentation spermatozoa (27.3% ± 12.4% vs. 28.5% ± 12.9%, p = 0.47) after cryostorage in the dry or the liquid nitrogen tank. Moreover, we did not observe differences between either cryostorage system for normal and altered sperm samples. This lack of difference was also observed whatever the floor level of cryostorage in the dry tank. The temperature measurement of the dry tank showed a stable temperature at -194 °C throughout storage whatever the storage floor level, guaranteeing the stability of the low temperatures suitable for human sperm storage. Because of its greater safety, dry storage without contact with the liquid phase should be preferred and can be a useful alternative for the cryostorage of human sperm samples.

[Access to fertility preservation for adolescents and young adults aged 15 to 24 years with cancers in Auvergne, France]. / Accès à la préservation de la fertilité des adolescents et jeunes adultes de 15 à 24 ans atteints de cancers en Auvergne, France.

INTRODUCTION: Cancers of adolescents and young adults have particular epidemiological specificities. The improvement in their survival should be accompanied by an increased consideration of the treatments' side effects, among which the potential decrease in fertility. The objective of the study was to describe the access to fertility preservation of these patients at the University Hospital of Clermont-Ferrand over a period of 3 years. METHODS: During this retrospective descriptive study, various socio-demographic and clinical data were collected. RESULTS: One hundred and fifty new cases of cancers were diagnosed in patients aged 15 to 24 years. Forty-four percent received at least one fertility consultation, 29 % for girls and 58 % for boys (P<0.001). The number of cases that did not result in fertility preservation was significantly higher for girls than boys (P=0.005). Fertility preservation was mainly achieved by cryopreservation of ovarian tissue in female adolescents, ovocytes in young women and sperm in boys. DISCUSSION: We observed sex disparities in access to fertility preservation. Despite the existence of recommendations, progress remains to be made. The establishment of clinico-biological platforms should allow a better awareness of patients and professionals, and thus promote access to fertility preservation techniques for young patients with cancer.

Sensitive and Specific Detection of Ewing Sarcoma Minimal Residual Disease in Ovarian and Testicular Tissues in an In Vitro Model.

Ewing sarcoma (EWS) is a common pediatric solid tumor with high metastatic potential. Due to toxic effects of treatments on reproductive functions, the cryopreservation of ovarian tissue (OT) or testicular tissue (TT) is recommended to preserve fertility. However, the risk of reintroducing residual metastatic tumor cells should be evaluated before fertility restoration. Our goal was to validate a sensitive and specific approach for EWS minimal residual disease (MRD) detection in frozen germinal tissues. Thawed OT (n = 12) and TT (n = 14) were contaminated with tumor RD-ES cells (10, 100, and 1000 cells) and EWS-FLI1 tumor-specific transcript was quantified with RT-qPCR. All contaminated samples were found to be positive, with a strong correlation between RD-ES cell numbers and EWS-FLI1 levels in OT (r = 0.93) and TT (r = 0.96) (p < 0.001). No transcript was detected in uncontaminated control samples. The invasive potential of Ewing cells was evaluated using co-culture techniques. After co-culturing, tumor cells were detected in OT/TT with histology, FISH, and RT-qPCR. In addition, four OT and four TT samples from children with metastatic EWS were tested, and no MRD was found using RT-qPCR and histology. We demonstrated the high sensitivity and specificity of RT-qPCR to detect EWS MRD in OT/TT samples. Clinical trial: NCT02400970.

Changes in total sperm count after gastric bypass and sleeve gastrectomy: the BARIASPERM prospective study.

BACKGROUND: The massive weight loss induced by bariatric surgery is associated with major benefits, but the effect on semen variables is still uncertain. OBJECTIVES: To explore semen modifications with gastric bypass and sleeve gastrectomy. SETTING: Five French University Hospitals. METHODS: Male candidates for bariatric surgery with no history of infertility were recruited in this controlled prospective study. Sperm characteristics were collected before surgery and then 6 months and up to 12 months after surgery. RESULTS: Forty-six adult men who underwent gastric bypass (n = 20) or sleeve gastrectomy (n = 26) were included. Total sperm count tended to be lower at 6 months and showed a significant decrease at 12 months in both surgery groups, at -69.5 million (-96.8 to -42.2 million; P = 0.0021). Total sperm count at 12 months relative to baseline was -41.4 million (P = .0391) after gastric bypass and -91.1 million (P = .0080) after sleeve gastrectomy. This was counterbalanced by an associated resolution of hypogonadism and decrease of DNA fragmentation in most patients with time after surgery. CONCLUSION: Improvement in some semen variables after bariatric surgery observed in 3 previous studies is in contrast to the lower mean total sperm count found in this study at 1 year. The possible reversibility of this effect in the long term and the impact of surgery on fertility both remain unknown.

Can the SCD test and terminal uridine nick-end labeling by flow cytometry technique (TUNEL/FCM) be used interchangeably to measure sperm DNA damage in routine laboratory practice?

BACKGROUND: Numerous tests have been proposed to evaluate sperm DNA integrity. To assess the sperm chromatin dispersion (SCD) test in an andrology laboratory, twenty-five men attending Clermont-Ferrand (France) University Hospital's Center for Reproductive Medicine were recruited. Sperm DNA damage was measured in the same semen samples using the SCD test and the Terminal Uridine Nick-end Labeling by flow cytometry technique (TUNEL/FCM) after density gradient centrifugation. RESULTS: SCD test reliability between readings, readers or slides was clearly established with very high agreement between measurements (Intraclass correlation coefficient (ICC) at 0.97, 0.95 and 0.98 respectively). Despite very good agreement between the SCD test and TUNEL/FCM (ICC at 0.94), the SCD test tended to slightly but significantly underestimate DNA damage compared with TUNEL (p = 0.0127). This systematic difference between the two techniques was - 3.39 ± 1.45% (mean ± SE). CONCLUSIONS: Andrology laboratories using the SCD test to measure sperm DNA damage need to know that it appears to give slightly underestimated measurements compared to TUNEL/FCM. However, this systematic underestimation is very small in amplitude. Both techniques give almost perfectly congruent results. Our study underlines the importance for each laboratory to validate its method to assess sperm DNA damage before implementing it in routine andrology lab practice.

CONTEXTE: Plusieurs tests sont disponibles pour évaluer l'intégrité de l'ADN spermatique. Afin d'évaluer l'applicabilité de la technique de dispersion de la chromatine spermatique (SCD) dans un laboratoire d'andrologie, nous avons recruté 25 patients pris en charge au Centre de Médecine de la Reproduction du centre hospitalo-universitaire de Clermont-Ferrand (France). L'altération de l'ADN spermatique a été mesurée en ayant recours au test SCD et au test Terminal Uridine Nick-end Labeling en cytométrie en flux (TUNEL/CMF) dans les mêmes échantillons pour les deux techniques, après avoir réalisé un gradient de densité. RÉSULTATS: Pour le test SCD, la concordance entre les lectures, les lecteurs et les lames a été clairement établie avec un accord quasiment parfait entre les mesures (Coefficient de corrélation intra-classe (CCI) respectivement à 0,97, 0,95 et 0,98). Malgré une bonne concordance entre le test SCD et le test TUNEL/CMF (CCI à 0,94), le test SCD tend à sous-estimer légèrement mais de façon significative l'altération de l'ADN spermatique en comparaison avec le test TUNEL (p = 0,0127). Cette différence systématique entre les 2 techniques était de − 3.39 ± 1.45% (moyenne ± erreur standard). CONCLUSIONS: les laboratoires d'andrologie utilisant le test SCD pour mesurer l'altération de l'ADN spermatique doivent savoir qu'il donne apparemment des valeurs légèrement sous-estimées en comparaison du test TUNEL/CMF. Cependant, cette sous-estimation systématique est. de faible amplitude et les deux techniques donnent des résultats presque parfaitement concordants dans notre étude. Cette dernière montre bien que chaque laboratoire doit valider sa méthode sur site pour évaluer l'altération de l'ADN spermatique avant de le mettre en place en pratique quotidienne en andrologie.

Liver X Receptors: A Possible Link between Lipid Disorders and Female Infertility.

A close relationship exists between cholesterol and female reproductive physiology. Indeed, cholesterol is crucial for steroid synthesis by ovary and placenta, and primordial for cell structure during folliculogenesis. Furthermore, oxysterols, cholesterol-derived ligands, play a potential role in oocyte maturation. Anomalies of cholesterol metabolism are frequently linked to infertility. However, little is known about the molecular mechanisms. In parallel, increasing evidence describing the biological roles of liver X receptors (LXRs) in the regulation of steroid synthesis and inflammation, two processes necessary for follicle maturation and ovulation. Both of the isoforms of LXRs and their bona fide ligands are present in the ovary. LXR-deficient mice develop late sterility due to abnormal oocyte maturation and increased oocyte atresia. These mice also have an ovarian hyper stimulation syndrome in response to gonadotropin stimulation. Hence, further studies are necessary to explore their specific roles in oocyte, granulosa, and theca cells. LXRs also modulate estrogen signaling and this could explain the putative protective role of the LXRs in breast cancer growth. Altogether, clinical studies would be important for determining the physiological relevance of LXRs in reproductive disorders in women.

Sperm meiotic segregation of a balanced interchromosomal reciprocal insertion resulting in recurrent spontaneous miscarriage.

RESEARCH QUESTION: Is sperm fluorescence in-situ hybridization (FISH) useful to evaluate the risk of chromosomally unbalanced gametes in interchromosomal reciprocal insertion (IRI) carriers? How do these imbalances lead to recurrent miscarriages? DESIGN: This study reports a clinical and molecular study of a rare familial balanced IRI resulting in recurrent spontaneous miscarriage. Sperm FISH was performed to estimate the number of unbalanced gametes. RESULTS: A 31-year-old healthy male (proband) and his 28-year-old female partner were referred to the Genetics Department for three spontaneous miscarriages occurring during the first trimester of pregnancy. FISH analysis of the proband with the LSI TRA/D (14q11.2) and DiGeorge N25 (22q11.2) break-apart probes showed the presence of a balanced IRI between 14q11.2 and 22q11.2 chromosomal regions. This IRI was also identified in the proband's father. Sperm FISH with the same probes showed that more than 40% of gametes of the proband were unbalanced for either 14q11.2 or 22q11.2, despite normal sperm parameters. FISH analysis of a product of conception indicated that unbalanced gametes result in a non-viable fetus. CONCLUSIONS: This study shows the value of sperm FISH analysis in improving genetic reproductive advice for IRI carriers. Disruption of critical genes through this rearrangement and their consequent functional impairment could result in recurrent miscarriages. In this case, several genes located in the 14q11.2 region, particularly RNase 3, would be good candidates to explain the lethality of the imbalances.