Catherine Poirot

Induction of puberty by autograft of cryopreserved ovarian tissue.

Summary of results of oestradiol, FSH, LH, inhibin B, and AMH from the day of transplantation to day 4 (D4) of the second spontaneous menstrual period future pubic hair, between the abdominal skin and the abdominal muscle. An abdominal pocket was opened and three thawed ovarian fragments were deposited inside. Hormonal investigations (fi gure) on the fi rst day of the ovarian transplantation showed FSH 89 IU/L, LH 36 IU/L, oestradiol less than 37 pmol/L, and AMH less than 0∙18 pmol/L. 2 months after the graft, breast growth started bilaterally, and she had reached Tanner stage S2 4 months after the graft. At this time, pubic and axillary hair appeared. H er fi rst menstruation was 8 months af ter transplantation, lasting 4 days. Breast development reached Tanner stage S3. At 3 years and 3 months after ovarian autotransplantation, our patient was 172 cm tall and weighed 52 kg. Her menstruation was regular for 2 years after the graft and irregular thereafter. Her breasts were completely developed with normal shape.Cryopreservation of ovarian tissue before potentially sterilising treatment can preserve ovarian function. 13 children have been born worldwide after auto-transplantation of ovarian fragments.

In vitro fertilization without ovarian stimulation: a simplified protocol applied in 80 cycles

Ovulation induction with various hormonal agents has become a standard component of in vitro fertilization (IVF) cycles to obtain multiple oocytes. Failure to anticipate the retrieval of more than two oocytes often results in cancellation of the cycle. In this study, we report our results in 80 unstimulated IVF cycles. Serum estradiol (E2) and pelvic ultra-sound monitoring were begun on day 9 of the cycle. Human chorionic gonadotropin (hCG) was administered when the E2 level exceeded 180 pg/mL and the dominant follicle was greater than 18 mm. Eighteen pregnancies were obtained (22.5%/cycle), and 14 (17.5%/cycle) are ongoing. We conclude that favorable results can be obtained from unstimulated IVF cycles, despite replacement of a single embryo.

A new in vitro fertilization technique: intravaginal culture.

Intravaginal culture (IVC) is a new technique elaborated by the authors for the fertilization and culture of human oocytes. Its principle consists of fertilization and early development of the eggs in a closed, air-free milieu without the addition of CO2. One to five ovocytes are deposited in a tube completely filled with 3 ml of culture medium less than 1 hour after their recovery, with 10,000 to 20,000 spermatozoa per ml previously prepared. The tube is then hermetically closed and it is placed in the maternal vagina and held by a diaphragm for incubation for 44 to 50 hours. After this time, the content of the tube is examined and embryos are transferred to the uterus. In the first 100 consecutive punctures, 22 clinical pregnancies were obtained: 17 deliveries, 3 spontaneous abortions, and 2 tubal pregnancies. Also, a randomized study comparing IVC to in vitro fertilization (IVF) was done (160 cycles) and no statistically different cleavage, transfer, or pregnancy rate was seen between IVC and IVF. By simplifying the laboratory manipulations, this technique decreases the cost of IVF and permits its standardization and diffusion. It creates a psychologic comfort permitting active participation of the mother in this stage of embryo development. Also, the use of this technique may give greater knowledge of human gamete metabolism and of the physiology of reproduction.

Identification of a new recurrent Aurora kinase C mutation in both European and African men with macrozoospermia

STUDY QUESTIONnCan we identify new sequence variants in the aurora kinase C gene (AURKC) of patients with macrozoospermia and establish a genotype-phenotype correlation?nnnSUMMARY ANSWERnWe identified a new non-sense mutation, p.Y248*, that represents 13% of all mutant alleles. There was no difference in the phenotype of individuals carrying this new mutation versus the initially described and main mutation c.144delC.nnnWHAT IS KNOWN ALREADYnThe absence of a functional AURKC gene causes primary infertility in men by blocking the first meiotic division and leading to the production of tetraploid large-headed spermatozoa. We previously demonstrated that most affected men were of North African origin and carried a homozygous truncating mutation (c.144delC).nnnSTUDY DESIGN, SIZE, DURATIONnThis is a retrospective study carried out on patients consulting for infertility and described as having >5% large-headed spermatozoa. A total of 87 patients are presented here, 43 patients were published previously and 44 are new patients recruited between January 2008 and December 2011.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnAll patients consulted for primary infertility in fertility clinics in France (n = 44), Tunisia (n = 30), Morocco (n = 9) or Algeria (n = 4). Sperm analysis was carried out in the recruiting fertility clinics and all molecular analyses were performed at Grenoble teaching hospital. DNA was extracted from blood or saliva and the seven AURKC exons were sequenced. RT-PCR was carried out on transcripts extracted from leukocytes from one patient homozygous for p.Y248*. Microsatellite analysis was performed on all p.Y248* patients to evaluate the age of this new mutation.nnnMAIN RESULTS AND THE ROLE OF CHANCEnWe identified a new non-sense mutation, p.Y248*, in 10 unrelated individuals of European (n = 4) and North African origin (n = 6). We show that this new variant represents 13% of all mutant alleles and that the initially described c.144delC variant accounts for almost all of the remaining mutated alleles (85.5%). No mutated transcripts could be detected by RT-PCR suggesting a specific degradation of the mutant transcripts by non-sense mediated mRNA decay. A rare variant located in the 3 untranslated region was found to strictly co-segregate with p.Y248*, demonstrating a founding effect. Microsatellite analysis confirmed this linkage and allowed us to estimate a mutational age of between 925 and 1325 years, predating the c.144delC variant predicted by the same method to have arisen 250-650 years ago. Patients with no identified AURKC mutation (n = 15) have significantly improved parameters in terms of vitality and concentration of normal spermatozoa, and a decreased rate of spermatozoa with a large head and multiple flagella (P < 0.001).nnnLIMITATIONS, REASONS FOR CAUTIONnDespite adherence to the World Health Organization guidelines, large variations in most characteristic sperm parameters were observed, even for patients with the same homozygous mutation. We believe that is mainly related to inter-laboratory variability in sperm parameter scoring. This prevented us from establishing clear-cut values to indicate a need for molecular analysis of patients with macrozoospermia.nnnWIDER IMPLICATIONS OF THE FINDINGSnThis study confirms yet again the importance of AURKC mutations in the aetiology of macrozoospermia. Although a large majority of patients are of North African origin, we have now identified European patients carrying a new non-sense mutation indicating that a diagnosis of absence of a functional AURKC gene should not be ruled out for non-Magrebian individuals. Indirect evidence indicates that AURKC might be playing a role in the meiotic spindle assembly checkpoint (SAC) during meiosis. We postulate that heterozygous men might have a more relaxed SAC leading to a more abundant sperm production and a reproductive advantage. This could be the reason for the rapid accumulation of the two AURKC mutations we observe in North African individuals.nnnSTUDY FUNDING/COMPETING INTEREST(S)nNone of the authors have any competing interest. This work is part of the project Identification and Characterization of Genes Involved in Infertility (ICG2I) funded by the programme GENOPAT 2009 from the French Research Agency (ANR).

Fertility preservation in prepubertal children

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.

Gene Therapy in Patients with Transfusion-Dependent β-Thalassemia

Background Donor availability and transplantation‐related risks limit the broad use of allogeneic hematopoietic‐cell transplantation in patients with transfusion‐dependent β‐thalassemia. After previously establishing that lentiviral transfer of a marked β‐globin (βA‐T87Q) gene could substitute for long‐term red‐cell transfusions in a patient with β‐thalassemia, we wanted to evaluate the safety and efficacy of such gene therapy in patients with transfusion‐dependent β‐thalassemia. Methods In two phase 1–2 studies, we obtained mobilized autologous CD34+ cells from 22 patients (12 to 35 years of age) with transfusion‐dependent β‐thalassemia and transduced the cells ex vivo with LentiGlobin BB305 vector, which encodes adult hemoglobin (HbA) with a T87Q amino acid substitution (HbAT87Q). The cells were then reinfused after the patients had undergone myeloablative busulfan conditioning. We subsequently monitored adverse events, vector integration, and levels of replication‐competent lentivirus. Efficacy assessments included levels of total hemoglobin and HbAT87Q, transfusion requirements, and average vector copy number. Results At a median of 26 months (range, 15 to 42) after infusion of the gene‐modified cells, all but 1 of the 13 patients who had a non–β0/β0 genotype had stopped receiving red‐cell transfusions; the levels of HbAT87Q ranged from 3.4 to 10.0 g per deciliter, and the levels of total hemoglobin ranged from 8.2 to 13.7 g per deciliter. Correction of biologic markers of dyserythropoiesis was achieved in evaluated patients with hemoglobin levels near normal ranges. In 9 patients with a β0/β0 genotype or two copies of the IVS1‐110 mutation, the median annualized transfusion volume was decreased by 73%, and red‐cell transfusions were discontinued in 3 patients. Treatment‐related adverse events were typical of those associated with autologous stem‐cell transplantation. No clonal dominance related to vector integration was observed. Conclusions Gene therapy with autologous CD34+ cells transduced with the BB305 vector reduced or eliminated the need for long‐term red‐cell transfusions in 22 patients with severe β‐thalassemia without serious adverse events related to the drug product. (Funded by Bluebird Bio and others; HGB‐204 and HGB‐205 ClinicalTrials.gov numbers, NCT01745120 and NCT02151526.)

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 (nu2009=u200922) was associated with a higher percentage of progressively motile spermatozoa (Pu2009<u20090.0001) versus efavirenz (nu2009=u200938) as well as vitality (Pu2009=u20090.0004). 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. J. Med. Virol. 83:1391–1394, 2011.

Intracytoplasmic sperm injection with microsurgically retrieved spermatozoa in azoospermic men infected with human immunodeficiency virus 1 or hepatitis C virus: the EP43 AZONECO ANRS study

OBJECTIVEnTo evaluate the viral contamination of sperm obtained after testicular sperm extraction (TESE) and microsurgical epididymal sperm aspiration (MESA) in men with azoospermia and human immunodeficiency virus (HIV) or hepatitis C virus infection.nnnDESIGNnProspective study.nnnSETTINGnFertility clinic, and reproductive technology and virology laboratories.nnnPATIENT(S)nSix men with azoospermia: two HIV-1 infected with undetectable blood viral load and four HCV infected with detectable blood viral load.nnnINTERVENTION(S)nProcessing by gradients density centrifugation and washing of surgically recovered sperm (TESE and MESA); virological analysis; inxa0vitro fertilization with intracytoplasmic sperm injection (ICSI).nnnMAIN OUTCOME MEASURE(S)nDetection of HIV-1 RNA or HCV RNA in gradient supernatants, testis tissues and final processed spermatozoa, and of HIV-1 DNA in testis tissues.nnnRESULT(S)nGradient supernatants and testis tissues tested HCV RNA positive in all cases while processed spermatozoa always tested negative. Gradient supernatants, testis tissues, and processed spermatozoa tested HIV-1 RNA negative. HIV-1 DNA was detectable in one testis tissue. All female partners tested HCV or HIV negative after ICSI.nnnCONCLUSION(S)nDensity gradient and washing suppressed virus detection in final suspensions of testicular and epididymal spermatozoa. ICSI after MESA or TESE appears to be feasible and could be offered in azoospermic men infected by HCV or HIV.

Fertilité et cancer

Information about chemo and/or radiotherapy gonadotoxicity and about fertility preservation is essential. Sperm cryopreservation has to be systematically offered before gonadotoxic treatments. Efficiency of ovarian function preservation with GnRH agonists is still debated. A controlled ovarian stimulation is necessary before oocyte or embryo cryopreservation. It is only feasible if the treatment is not urgent and if the tumor is not hormone-sensitive. If the treatment is highly gonadotoxic, an ovarian tissue cryopreservation may be appropriate. It is the only fertility preservation technique feasible for prepubertal girls. It is now possible to preserve the fertility of prepubertal boys by cryopreservation of testicular tissue. It is essential to send patients and/or their parents to a specialized fertility preservation center.