When to cryopreserve ovarian tissue: Determining the effects of chemotherapy on the ovarian reserve by studying follicular density and apoptosis.

OBJECTIVES: Patients scheduled to receive chemotherapy should be counselled on fertility preservation. Known gonadotoxic chemotherapies such as alkylating agents have a high risk of altering ovarian reserve. In some cases, the urgency of treatment requires the use of chemotherapy before fertility preservation, which will be carried out at a later stage. Usually the ovarian tissue is cryopreserved. The aim of our study is to investigate the impact of chemotherapies on follicular density and the apoptosis of reserve follicles. METHODS: We included 140 patients: 63 patients, mean age 18.8 years, were included in the group "no chemotherapy" (group A) and 77 patients, mean age 17.1 years, in the group "received chemotherapy before ovarian conservation" (group B). None of the patients had had pelvic radiotherapy prior to ovarian cryopreservation. The histological parameters studied were follicular density and the presence of malignant cells. We selected 12 patients from group A and 15 patients from group B, comparable in age and pathology, for whom we evaluated follicle apoptosis by immunostaining cleaved caspase-3. RESULTS: We demonstrated an inverse relationship between follicular density and age (p < 0.0001), as well as a lack of effect of chemotherapy on follicular density (p = 0.87). We showed the impact of various chemotherapies, especially with alkylating agents, on the apoptosis of ovarian follicles (p < 0.0001). Three patients had ovarian tissue infiltration, two of which were malignant. CONCLUSION: This work underlines the fact that conservation of ovarian tissue after chemotherapy remains possible.

Human ovarian cryopreservation: vitrification versus slow freezing from histology to gene expression.

Cryopreservation of ovarian tissue is one of the strategies offered to girls and women needing gonadotoxic treatment to preserve their fertility. The reference method to cryopreserve is slow freezing; vitrification is an alternative method. The aim was to evaluate which of the two is the best method for human ovarian tissue cryopreservation. Each ovary was divided into three groups: (i) fresh; (ii) slow freezing; and (iii) vitrification. An evaluation of the follicular density, quality and the expression six genes (CYP11A, STAR, GDF9, ZP3, CDK2, CDKN1A) were performed. We observed no significant difference in follicular density within these three groups. Slow freezing altered the primordial follicles compared to the fresh tissue (31.8% vs 55.9%, p = 0.046). The expression of genes involved in steroidogenesis varied after cryopreservation compared to the fresh group; CYP11A was under-expressed in slow freezing group (p = 0.01), STAR was under-expressed in the vitrification group (p = 0.01). Regarding the expression of genes involved in cell cycle regulation, CDKN1A was significantly under-expressed in both freezing groups (slow freezing: p = 0.0008; vitrification: p = 0.03). Vitrification had no effect on the histological quality of the follicles at any stage of development compared to fresh tissue. There was no significant difference in gene expression between the two techniques.

Development and Survival of Human Ovarian Cells in Chitosan Hydrogel Micro-Bioreactor.

Background and Objectives: To test the long-term ability of human ovarian cortex cells to develop in unconventional culture conditions. Materials and Methods. Ovarian cortex cells from fetuses aged 23 to 39 weeks gestation were cultured for 90 days in hollow chitosan hydrogel micro-bioreactors and concurrently in traditional wells. Various cell-type counts were considered. Results: With intact follicles as a denominator, the percentage of growing intact follicles at Day 0 varied widely between ovaries (0 to 31.7%). This percentage tended to increase or stay relatively constant in bioreactor as in control cultures; it tended more toward an increase over time in bioreactor vs. control cultures. Modeled percentages showed differences (though not significant) in favor of bioreactor cultures (16.12% difference at D50 but only 0.12% difference at D90). With all follicles present as a denominator, the percentage of growing primary and secondary follicles at D0 varied widely between ovaries (0 to 29.3%). This percentage tended to increase over time in bioreactor cultures but to decrease in control cultures. Modeled percentages showed significant differences in favor of bioreactor cultures (8.9% difference at D50 and 11.1% difference at D90). At D50 and D90, there were only few and sparse apoptotic cells in bioreactor cultures vs. no apoptotic cells in control cultures. Conclusions: Over three months, bioreactor folliculogenesis outperformed slightly traditional culture. This is an interesting perspective for follicle preservation and long-term toxicological studies.

Shallow artificial networks with morphokinetic time-lapse parameters coupled to ART data allow to predict live birth.

Purpose: The purpose of this work was to construct shallow neural networks (SNN) using time-lapse technology (TLT) from morphokinetic parameters coupled to assisted reproductive technology (ART) parameters in order to assist the choice of embryo(s) to be transferred with the highest probability of achieving a live birth (LB). Methods: A retrospective observational single-center study was performed, 654 cycles were included. Three SNN: multilayers perceptron (MLP), simple recurrent neuronal network (simple RNN) and long short term memory RNN (LSTM-RNN) were trained with K-fold cross-validation to avoid sampling bias. The predictive power of SNNs was measured using performance scores as AUC (area under curve), accuracy, precision, Recall and F1 score. Results: In the training data group, MLP and simple RNN provide the best performance scores; however, all AUCs were above 0.8. In the validating data group, all networks were equivalent with no performance scores difference and all AUC values were above 0.8. Conclusion: Coupling morphokinetic parameters with ART parameters allows to SNNs to predict the probability of LB, and all SNNs seems to be efficient according to the performance scores. An automatic time recognition system coupled to one of these SNNs could allow a complete automation to choose the blastocyst(s) to be transferred.

Delaying testicular sperm extraction in 47,XXY Klinefelter patients does not impair the sperm retrieval rate, and AMH levels are higher when TESE is positive.

STUDY QUESTION: Should testicular sperm extraction (TESE) in non-mosaic 47,XXY Klinefelter syndrome (KS) patients be performed soon after puberty or could it be delayed until adulthood? SUMMARY ANSWER: The difference in sperm retrieval rate (SRR) in TESE was not significant between the 'Young' (15-22 years old) cohort and the 'Adult' (23-43 years old) cohort of non-mosaic KS patients recruited prospectively in parallel. WHAT IS KNOWN ALREADY: Several studies have tried to define predictive factors for TESE outcome in non-mosaic KS patients, with very heterogeneous results. Some authors have found that age was a pejorative factor and recommended performing TESE soon after puberty. To date, no predictive factors have been unanimously recognized to guide clinicians in deciding to perform TESE in azoospermic KS patients. STUDY DESIGN, SIZE, DURATION: Two cohorts (Young: 15-22 years old; Adult: 23-43 years old) were included prospectively in parallel. A total of 157 non-mosaic 47,XXY KS patients were included between 2010 and 2020 in the reproductive medicine department of the University Hospital of Lyon, France. However 31 patients gave up before TESE, four had cryptozoospermia and three did not have a valid hormone assessment; these were excluded from this study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Data for 119 patients (61 Young and 58 Adult) were analyzed. All of these patients had clinical, hormonal and seminal evaluation before conventional TESE (c-TESE). MAIN RESULTS AND THE ROLE OF CHANCE: The global SRR was 45.4%. SRRs were not significantly different between the two age groups: Young SRR=49.2%, Adult SRR = 41.4%; P = 0.393. Anti-Müllerian hormone (AMH) and inhibin B were significantly higher in the Young group (AMH: P = 0.001, Inhibin B: P < 0.001), and also higher in patients with a positive TESE than in those with a negative TESE (AMH: P = 0.001, Inhibin B: P = 0.036). The other factors did not differ between age groups or according to TESE outcome. AMH had a better predictive value than inhibin B. SRRs were significantly higher in the upper quartile of AMH plasma levels than in the lower quartile (or in cases with AMH plasma level below the quantification limit): 67.7% versus 28.9% in the whole population (P = 0.001), 60% versus 20% in the Young group (P = 0.025) and 71.4% versus 33.3% in the Adult group (P = 0.018). LIMITATIONS, REASONS FOR CAUTION: c-TESE was performed in the whole study; we cannot rule out the possibility of different results if microsurgical TESE had been performed. Because of the limited sensitivity of inhibin B and AMH assays, a large number of patients had values lower than the quantification limits, preventing the definition a threshold below which negative TESE can be predicted. WIDER IMPLICATIONS OF THE FINDINGS: In contrast to some studies, age did not appear as a pejorative factor when comparing patients 15-22 and 23-44 years of age. Improved accuracy of inhibin B and AMH assays in the future might still allow discrimination of patients with persistent foci of spermatogenesis and guide clinician decision-making and patient information. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by a grant from the French Ministry of Health D50621 (Programme Hospitalier de Recherche Clinical Régional 2008). The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER: NCT01918280.

An Update on In Vitro Folliculogenesis: A New Technique for Post-Cancer Fertility.

INTRODUCTION: Obtaining in vitro mature oocytes from ovarian tissue to preserve women's fertility is still a challenge. At present, there is a therapeutic deadlock for girls and women who need emergency fertility preservation in case of a high risk of ovary invasion by malignant cells. In such a case, ovarian tissue cannot be engrafted; an alternative could be in vitro folliculogenesis. METHODS: This review focuses on the progress of in vitro folliculogenesis in humans. PubMed and Embase databases were used to search for original English-language articles. RESULTS: The first phase of in vitro folliculogenesis is carried out in the original ovarian tissue. The addition of one (or more) initiation activator(s) is not essential but allows better yields and the use of a 3D culture system at this stage provides no added value. The second stage requires a mechanical and/or enzymatic isolation of the secondary follicles. The use of an activator and/or a 3D culture system is then necessary. CONCLUSION: The current results are promising but there is still a long way to go. Obtaining live births in large animals is an essential step in validating this in vitro folliculogenesis technique.

Anti-müllerian hormone levels and antral follicle count in women with a BRCA1 or BRCA2 germline pathogenic variant: A retrospective cohort study.

BACKGROUND: Some studies suggested a decreased ovarian reserve among BRCA1/2 pathogenic variant carriers, with conflicting results. METHODS: We conducted a retrospective single-center observational study of ovarian reserve and spontaneous fertility comparing BRCA1/2 pathogenic variant carriers to controls (women who attended consultations to discuss fertility preservation before gonadotoxic treatment). Measures of associations between plasma AMH concentration, AFC and BRCA1/2 status were modelled by nonlinear generalized additive regression models and logistic regressions adjusted for age at plasma storage, oral contraceptive use, body mass index, cigarette smoking, and the AMH assay technique. RESULTS: The whole population comprised 119 BRCA1/2 pathogenic variant carriers and 92 controls. A total of 110 women (42 carriers, among whom 30 were cancer-free, and 68 controls) underwent an ovarian reserve evaluation. Spontaneous fertility analysis included all women who previously attempted to become pregnant (134 women). We observed a tendency towards a premature decrease in ovarian reserve in BRCA1/2 pathogenic variant carriers, but no difference in mean AMH or AFC levels was found between BRCA1/2 pathogenic variant carriers and controls. An analysis of the extreme levels of AMH (≤5 pmol/l) and AFC (≤7 follicles) by logistic regression suggested a higher risk of low ovarian reserve among BRCA1/2 pathogenic variant carriers (adjusted odds ratio (OR) = 3.57, 95% CI = 1.00-12.8, p = 0.05; and adjusted OR = 4.99, 95% CI = 1.10-22.62, p = 0.04, respectively). DISCUSSION: Attention should be paid to BRCA1/2 pathogenic variant carriers' ovarian reserve, considering this potential risk of premature alteration.

A machine learning system with reinforcement capacity for predicting the fate of an ART embryo.

The aim of this work was to construct a score issued from a machine learning system with self-improvement capacity able to predict the fate of an ART embryo incubated in a time lapse monitoring (TLM) system. A retrospective study was performed. For the training data group, 110 couples were included and, 891 embryos were cultured. For the global setting data group, 201 couples were included, and 1186 embryos were cultured. No image analysis was used; morphokinetic parameters from the first three days of embryo culture were used to perform a logistic regression between the cell number and time. A score named DynScore was constructed, the prediction power of the DynScore on blastocyst formation and the baby delivery were tested via the area under the curve (AUC) obtained from the receiver operating characteristic (ROC). In the training data group, the DynScore allowed the blastocyst formation prediction (AUC = 0.634, p < 0.001), this approach was the higher among the set of the tested scores. Similar results were found with the global setting data group (AUC = 0.638, p < 0.001) and it was possible to increase the AUC of the DynScore with a regular update of the prediction system by reinforcement, with an AUC able to reach a value above 0.9. As only the best blastocysts were transferred, none of the tested scores was able to predict delivery. In conclusion, the DynScore seems to be able to predict the fate of an embryo. The reinforcement of the prediction system allows maintaining the predictive capacity of DynScore irrespective of the various events that may occur during the ART process. The DynScore could be implemented in any TLM system and adapted by itself to the data of any ART center.Abbreviations: ART: assisted reproduction technology; TLM: time lapse monitoring system; AUC: area under the curve; ROC: receiver operating characteristic; eSET: elective single embryo transfer; AIS: artificial intelligence system; KID: known implantation data; AMH: anti-Müllerian hormone; BMI: body mass index; WHO: World Health Organization; c-IVF: conventional in-vitro fertilization; ICSI: intracytoplasmic sperm injection; PNf: pronuclear formation; D3: day 3; D5: day 5; D6: day 6; GnRH: gonadotrophin releasing hormone; FSH: follicle stimulating hormone; LH: luteinizing hormone; hCG: human chorionic gonadotropin; PVP: polyvinyl pyrrolidone; PNf: time of pronuclear fading; tx: time of cleavage to x blastomeres embryo; ICM: inner cell mass; TE: trophectoderm; NbCellt: number of cells at t time; FIFO: first in first out; TD: training data group; SD: setting data group; R: real world.

Cellular and Molecular Impact of Vitrification Versus Slow Freezing on Ovarian Tissue.

Objective: To evaluate a vitrification protocol from histology to gene expression to slow freezing. Methods: Ovaries from 12 prepubertal ewes. The same ovary was cut into fragments, studied fresh, frozen, and vitrified. Follicle morphology by hematoxylin-eosin-safran staining, vitality by Trypan Blue, and apoptosis by marking cleaved caspase-3 were studied. The expression of gene: anti-Müllerian hormone (AMH), cytochrome p450 family 11 subfamily A member 1 (CYP11A), and steroidogenic acute regulatory protein (STAR; granulosa cells); growth differentiation factor 9 (GDF9) and zona pellucida glycoprotein 3 (ZP3; oocytes); and cyclin D2 (CCND2) and cyclin-dependent kinase inhibitor 1A (CDKN1A; cell cycle regulation), was evaluated by reverse transcription quantitative polymerase chain reaction. Results: The slow freezing protocol had a significant negative impact on intact primordial follicles compared with fresh tissue (37.6% vs. 62.5%, p = 0.003). More intact follicles after vitrification were observed compared with slow freezing (p = 0.037). The apoptotic primordial follicles were similar after slow freezing and vitrification (12.6% vs. 13.9%). Concerning granulosa cell genes, slow freezing led to a trend toward overexpression of AMH messenger RNA (mRNA; p = 0.07); while vitrification led to a significant overexpression of CYP11A mRNA (p = 0.003), and a trend toward an overexpression of STAR mRNA (p = 0.06). Concerning oocyte genes, both techniques did not lead to a difference of GDF9 and ZP3 mRNA. Concerning cell cycle genes, slow freezing led to a significant underexpression of CCND2 (p = 0.04); while vitrification did not lead to a difference for CCND2 and CDKN1A mRNA. Conclusion: Vitrification preserved follicular morphology better than slow freezing and led to gene overexpressed, while slow freezing led to gene underexpressed. Impact statement The preservation of female fertility and in particular the cryopreservation of ovarian tissue (OT) is a major public health issue aimed at improving the quality of life of patients after gonadotoxic treatments. The use of slow freezing of this OT, which is the reference technique, is not optimal due to tissue alteration. The alternative would be vitrification. This study compares these two techniques. We have highlighted that vitrification preserved follicular morphology better than slow freezing and led to gene overexpressed, while slow freezing led to gene underexpressed.

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.

An ART score to note objectively the quality of an ART procedure.

OBJECTIVE: To construct an ART score to evaluate an ART procedure before the result (pregnancy or not), and to provide objective data in discussions with couples in the decision to discontinue further attempts. STUDY DESIGN: A retrospective multicentrique study was performed. The ART score was constructed using data from the MediFirst© database used in our center. The development of the score was conducted on a sample of 507 in vitro fertilization cycles carried out between January 2011 and July 2011. Model calibration and determination of the discrimination capacity of the ART score were performed with 4463 cycles in our center and 1369 cycles from an external ART center. The ART score was validated temporally and geographically with clinical pregnancy and take home baby rate. RESULTS: The ART score was obtained from data from both partners and ART procedure. The ART score was segmented into four classes depending on the clinical pregnancy rate. There was a linear relationship between the ART score and clinical pregnancy rate (r = 1.0, p < 0.001). The ART score was validated temporally and geographically. CONCLUSION: An objective ART score has been constructed and validated. It will be of help to ART teams and it is an objective tool to explain to a couple the choices for the next ART attempt.

Safeguarding Fertility With Whole Ovary Cryopreservation and Microvascular Transplantation: Higher Follicular Survival With Vitrification Than With Slow Freezing in a Ewe Model.

BACKGROUND: In young women, ovarian cortex cryopreservation before gonadotoxic chemotherapy and its avascular grafting after cancer healing permitted fertility restoration. However, ischemia reduced the grafts' lifespan. Microvascular transplantation of cryopreserved whole ovary may allow immediate revascularization, ensuring better fertility preservation, but the best cryopreservation method is unknown. We aimed to compare slow freezing and vitrification of whole ovary for fertility preservation purposes, in an ewe model. METHODS: Twelve ewes were allocated at random to slow freezing (n = 6) or vitrification group (n = 6). Ewes' left ovary was removed and cryopreserved. Dimethyl sulfoxide 2 M was used as cryoprotector for slow freezing. Vitrification was obtained using increasing concentrations of a vitrification solution of the latest generation (VM3) and gradual temperature lowering to minimize toxicity. After a month, the right ovary was removed, the left ovary was thawed/warmed, and its vessels were anastomosed to the right pedicle. Fertility and ovarian function were assessed for 3 years. Ovarian follicles in native and transplanted ovaries were counted and compared at study completion. RESULTS: Hormonal secretion resumed in all ewes of both groups. One ewe of the slow-freezing group delivered healthy twins 1 year 9 months and 12 days after transplantation. Estimated whole follicle survival was very low in both groups but significantly higher after vitrification than after slow freezing (0.3% ± 0.5% vs 0.017% ± 0.019%, respectively; p < 0.05). CONCLUSIONS: Further progress is needed before whole-ovary cryopreservation can be considered an option for safeguarding fertility. Whole ovary vitrification provides better follicular survival compared to slow freezing and may be a valuable cryopreservation option.

[Gonadal function after treatment for a childhood or adolescent cancer]. / Fonction gonadique et fertilité après traitement pour un cancer dans l'enfance ou l'adolescence : évaluation et prise en charge.

Due to high cure rate in childhood and adolescent cancer, fertility preservation is a major concern. Chemotherapy, radiotherapy and surgery may alter gonadal function, and uterine cavity in women. In women, combined toxicity affecting both endocrine function and ovulation are observed leading to premature ovarian insufficiency. In men, spermatogenesis is frequently affected whereas endocrine function is almost always preserved. The current article focuses on investigations concerning gonadal function after treatment for a cancer during childhood or adolescence and treatment of subsequent infertility or hypogonadism. Nevertheless, those therapeutic are still limited and pretherapeutic preservation of fertility is preferred when possible.

Preliminary results of a prospective study of testicular sperm extraction in young versus adult patients with nonmosaic 47,XXY Klinefelter syndrome.

CONTEXT: Testicular sperm extraction (TESE) in adult patients with nonmosaic 47,XXY provides a sperm retrieval rate (SRR) of approximately 50%. Age is the only significant prognostic factor. Whether TESE should be performed in adolescent patients for sperm cryopreservation remains to be determined. OBJECTIVE: The objective of the study was to compare SRR between young (15-23 y) and adult (> 23 y) patients with 47,XXY, and to determine whether previous androgenic treatment had a deleterious effect. DESIGN: We designed a prospective comparative study between two groups enrolled in parallel from September 2010 onward. SETTING: University hospital. PATIENTS: Forty one patients with nonmosaic 47,XXY karyotype and azoospermia were included. Twenty five patients from 15-22 years of age were assigned to the "Young" group, and 16 patients age 23 years or more, to the "Adult" group. INTERVENTION: A bilateral testicular open biopsy was performed by a single surgeon. The reproductive biologist who performed TESE was blind to the patient's age. Principal Outcome Measure: The main outcome measure was the SRR. The TESE procedure was considered positive if at least 20 sperm cells could be cryopreserved for intracytoplasmic sperm injection. RESULTS: SRR was 13/25 = 52% in the Young group and 10/16 = 62.5% in the Adult group, the difference being nonsignificant (P = .73). Ages were 24.3 ± 7.4 years in the 23 cases of positive TESE, and 23.7 ± 7.4 in the 18 cases of negative TESE, the difference being nonsignificant (P = .42). SRR was 9/17 = 52.9% for patients with and 14/24 = 59.1% for patients without previous testosterone (T) treatment, the difference being nonsignificant (P = .98). CONCLUSIONS: According to the present results, performing TESE at a younger age (15-23 y) in patients with azoospermic nonmosaic 47,XXY Klinefelter did not increase SRR relative to adult patients (25-39 y). Previous replacement treatment with moderate doses of T did not seem to be deleterious for the recovery of sperm cells by TESE.

Abnormal methylation of KCNQ1OT1 and differential methylation of H19 imprinting control regions in human ICSI embryos.

Summary To evaluate the integrity of genomic imprinting in embryos that failed to develop normally following intracytoplasmic sperm injection (ICSI), we analysed the methylation profile of H19 and KCNQ1OT1 imprinting control regions, H19DMR and KvDMR1 respectively, in high-grade blastocysts and in embryos that exhibited developmental anomalies. Significant hypomethylation of KvDMR1 was specifically observed in 5/5 atypical blastocysts graded BC, which probably reflected the vulnerability of the imprint in the inner cell mass during the methylation remodelling phase in the early embryo. In addition, KvDMR1 was hypermethylated in 2/5 CC graded atypical blastocysts and in 2/8 embryos that exhibited developmental delay. H19DMR appeared differentially methylated in all groups of embryos. DNA methyltransfersase 1 (DNMT1) expression was similar in most of the tested embryos and could not account for the abnormal methylation patterns of KvDMR1 observed.

[Restoring fertility with frozen ovarian tissue; experimental results]. / Restauration de l'infertilité après congélation de tissu ovarien, données expérimentales.

In recent decades, reproductive surgery and medicine have entered a new era of prevention. Oocyte freezing is at the heart of this new era, along with the preservation offemale fertility. Several options are being evaluated, and time will tell which one will prove most suitable for routine use. Currently, only the freezing and transplantation of ovarian tissue seems to have entered clinical practice. Animal studies demonstrated its effectiveness, with pregnancies and births in several species. These studies showed that both slow and rapid freezing (vitrification) allows the survival of more than 80 % of primordial follicles after thawing. Similar results have since been obtained in humans. Freezing allows satisfactory follicular survival Reimplantation of frozen ovarian tissue has helped to restore menstrual cycling and, more importantly, to obtain pregnancies and births, either spontaneously or by IVF This restoration of fertility offers great hopefor female patients having to confront not only their disease but also the prospect of permanent infertility. Related research has led to major advances in biology and reproductive medicine.

Persistence of fertility despite semen alterations in a pinealectomised patient treated with melatonin.

OBJECTIVES: Little is known about the effect of chronic melatonin treatment on human reproductive function. We report here on the effect of 10 months treatment with a controlled-release melatonin preparation (Circadin®, 2 mg) on spermatogenesis and gonadotropic hormone status in a pinealectomised patient whose melatonin secretion was abolished. METHODS: Semen analysis, hormone (Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH), inhibin B, prolactin, testosterone and estradiol) and Sex Hormone-Binding Globulin (SHB G) concentrations were determined before and at the end of 4 and 10 months of, treatment. RESULTS: At the end of treatment, testosterone, sex hormone-binding globulin, prolactin and inhibin B levels did not display significant variation with time, whereas FSH and LH levels showed a tendency to a decrease, but remained in the normal range. Sperm concentration and total spermatozoa count dropped below the lower limit of the reference range during melatonin treatment, whereas motility and normal form percentages remained in the normal range. Fertility was preserved, since the patient's wife became pregnant during month 10 of melatonin treatment and gave birth to a healthy female baby. CONCLUSIONS: this isolated clinical observation shows that more investigations in large patient series are needed to document possible side-effects of melatonin administration on male reproductive function. One should therefore be cautious about melatonin prescription for circadian rhythm sleep disorders in young males.

Examination of viability and quality of ovarian tissue after cryopreservation using simple laboratory methods in ewe.

BACKGROUND: The objective of the present study is to assess viability tests and to evaluate follicle ovarian tissue quality after freezing-thawing procedures. METHODS: Ewe's ovaries were harvested at the slaughterhouse, after dissection each ovarian specimen was divided into two groups: fresh tissue (control group) and frozen tissue.In the first part of the study, the follicles viability was assessed by trypan blue staining, calcein AM/ethidium homodimer-1 staining (LIVE/DEAD viability/cytotoxicity kit, Molecular Probes) and morphology in the two groups. In the second part of the study the quality of the whole ovarian tissue was evaluated by the quantification of the release of lactate dehydrogenase measurement (Cytotoxicity Detection kit ROCHE), DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) in primordial and primary follicles (ApopDETEK Kit system Enzo) and morphology in the two groups. 100 Follicles (primordial and primary) were counted on both fresh and frozen hemiovary to assess this various tests. RESULTS: Ovarian follicle viability assessment was similar using trypan blue or calcein/ethidium staining. Follicles showed a decreased viability after freezing-thawing.After cryopreservation, a significant correlation between the percentage of normal follicles and viability rate was found using trypan blue (r=0.82, p<0.05) or calcein AM/ethidium homodimer-1 staining (r=0.76, p<0.05). Increased cytotoxicity showed by enhancement of LDH release was found after cryopreservation (21.60+/-1.1% vs 52.2+/-7.7%). A significant negative correlation between the percentage of morphologically normal follicles and cytotoxicity was observed. No significant difference in DNA fragmentation rate between frozen and control groups was found (26±8.2% vs 38±4.5%). CONCLUSION: We suggest the use of trypan blue staining for the histological assessment of viability, the use of LDH assay for the cytotoxicity assessement and finally the use of DNA fragmentation assessment to valid different freezing-thawing protocols.

Vitrification at the germinal vesicle stage does not affect the methylation profile of H19 and KCNQ1OT1 imprinting centers in human oocytes subsequently matured in vitro.

OBJECTIVE: To evaluate the integrity of genomic imprinting in oocytes vitrified at the germinal vesicle (GV) stage and in vitro matured (IVM) after thawing. DESIGN: Clinical research and application. SETTING: University-based fertility center. PATIENT(S): Immature oocytes were donated for research by patients who were included in an intracytoplasmic sperm injection program. INTERVENTION(S): Immature oocyte retrieval after ovarian stimulation, followed by oocyte vitrification, thawing, and IVM. MAIN OUTCOME MEASURE(S): Methylation profile of H19 and KCNQ1OT1 imprinting control regions, H19DMR and KvDMR1, respectively. RESULT(S): Among 184 vitrified GV oocytes, 102 survived thawing (55.4%), 77 (75.5%) of which reached the meiosis II (MII) stage after IVM. One hundred twenty control GV oocytes were only subjected to IVM; 70.8% reached the MII stage. GV vitrified as well as control oocytes acquired full imprint at KvDMR1 after IVM and generally retained the unmethylated state of H19DMR. CONCLUSION(S): For the first time, we show that oocyte vitrification does not affect the methylation profile of H19DMR and KvDMR1: during their IVM, vitrified GV oocytes acquire DNA methylation in the maternally imprinted KCNQ1OT1 gene with the same efficiency as fresh GV oocytes; the vitrification process does not alter the unmethylated state of the paternally imprinted H19 gene.

The Aurora Kinase C c.144delC mutation causes meiosis I arrest in men and is frequent in the North African population.

Infertility concerns a minimum of 70 million couples worldwide. An important proportion of cases is believed to have a genetic component, yet few causal genes have been identified so far. In a previous study, we demonstrated that a homozygous mutation (c.144delC) in the Aurora Kinase C (AURKC) gene led to the production of large-headed polyploid multi-flagellar spermatozoa, a primary infertility phenotype mainly observed in North Africans. We now want to estimate the prevalence of the defect, to improve our understanding of AURKC physiopathology in spermatogenesis and assess its implication in oogenesis. A carrier frequency of 1/50 was established from individuals from the Maghrebian general population, comparable to that of Y-microdeletions, thus far the only known recurrent genetic event altering spermatogenesis. A total of 62 patients were genotyped, all who had a typical phenotype with close to 100% large-headed spermatozoa were homozygously mutated (n = 32), whereas no AURKC mutations were detected in the others. Two homozygous females were identified; both were fertile indicating that AURKC is not indispensible in oogenesis. Previous FISH results had showed a great chromosomal heterogeneity in these patient's spermatozoa. We demonstrate here by flow cytometry that all spermatozoa have in fact a homogeneous 4C DNA content and are thus all blocked before the first meiotic division. Our data thus indicate that a functional AURKC protein is necessary for male meiotic cytokinesis while its absence does not impair oogenesis.