Search

STUDY QUESTION Does mitochondrial deficiency affect human embryonic preimplantation development? SUMMARY ANSWER The presence of a pathogenic mitochondrial variant triggers changes in the gene expression of preimplantation human embryos, compromising their development, cell differentiation, and survival. WHAT IS KNOWN ALREADY Quantitative and qualitative anomalies of mitochondrial DNA (mtDNA) are reportedly associated with impaired human embryonic development, but the underlying mechanisms remain unexplained. STUDY DESIGN, SIZE, DURATION Taking advantage of the preimplantation genetic testing for mitochondrial disorders in at-risk couples, we have compared gene expression of 9 human embryos carrying pathogenic variants in either mtDNA genes or nuclear genes encoding mitochondrial protein to 33 age-matched control embryos. PARTICIPANTS/MATERIALS, SETTING, METHODS Single-embryo transcriptomic analysis was performed on whole human blastocyst embryos donated to research. MAIN RESULTS AND THE ROLE OF CHANCE Specific pathogenic mitochondrial variants downregulate gene expression in preimplantation human embryos [566 genes in oxidative phosphorylation (OXPHOS)-deficient embryos], impacting transcriptional regulators, differentiation factors, and nuclear genes encoding mitochondrial proteins. These changes in gene expression primarily alter OXPHOS and cell survival pathways. LIMITATIONS, REASONS FOR CAUTION The number of OXPHOS-deficient embryos available for the study was limited owing to the rarity of this material. However, the molecular signature shared by all these embryos supports the relevance of the findings. WIDER IMPLICATIONS OF THE FINDINGS While identification of reliable markers of normal embryonic development is urgently needed in ART, our study prompts us to consider under-expression of the targeted genes reported here, as predictive biomarkers of mitochondrial dysfunction during preimplantation development. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the ‘Association Française contre les Myopathies (AFM-Téléthon)’ and the ‘La Fondation Maladies Rares’. No competing interests to declare. TRIAL REGISTRATION NUMBER N/A.

Data on preimplantation genetic testing (PGT-M) in patients with genetic susceptibility to cancer are scarce in the literature, while there is, in our experience, a growing familiarity with assisted reproduction techniques (ART) among pathogenic variant heterozygotes. We performed a retrospective multicenter study of PGT-M outcomes among French patients with genetic susceptibility to cancer. Our objectives were to collect data on this complex issue, and to help cancer geneticists counsel their patients of reproductive age. We also wanted to increase awareness regarding PGT-M among cancer genetics professionals. Patients from three university hospital cancer genetics clinics who had requested PGT-M between 2000 and 2019 were included retrospectively. Data were extracted from medical records. Patients were then contacted directly to collect missing and up-to-date information. Out of 41 eligible patients, 28 agreed explicitly to participate when contacted and were therefore included. They carried PV in VHL (n = 9), APC (n = 8), CDH1 (n = 5), STK11 (n = 2), AXIN2, BRCA1, MEN1, and FH (n = 1). Seven patients were denied PGT-M based on multidisciplinary team meetings or subsequently by the ART hospital teams, two changed their minds, and two were yet to start the process. PGT-M was successful in seven patients (25%), with a mean age at PGT-M request of 27. Most had von Hippel-Lindau. PGT-M failed in the remaining ten, with a mean age at PGT-M request of 32. The main reason for failure was non-implantation of the embryo. Of these, four patients were pursuing PGT-M at the time of last contact. PGT-M outcomes in patients with cancer susceptibility syndromes were satisfactory. These patients should be informed about PGT-M more systematically, which would imply greater awareness among cancer genetics professionals regarding ART. Our series was not representative of cancer susceptibility syndromes in general; the predominance of cases with syndromes characterized by early-onset, highly penetrant disease is explained by the restrictive French guidelines.

STUDY QUESTION Can ovarian tissue cryopreservation (OTC) be performed after controlled ovarian hyperstimulation (COH)? SUMMARY ANSWER Unilateral oophorectomy after transvaginal oocyte retrieval is feasible on stimulated ovaries during one surgical step. WHAT IS KNOWN ALREADY In the fertility preservation (FP) field, the timeframe between patient referral and start of curative treatment is limited. Combining oocyte pick-up with ovarian tissue (OT) extraction has been reported to improve FP but COH applied before OT extraction is not currently recommended. STUDY DESIGN, SIZE, DURATION This retrospective cohort-controlled study involved 58 patients who underwent oocyte cryopreservation immediately followed by OTC between September 2009 and November 2021. The exclusion criteria were a delay between oocyte retrieval and OTC of >24 h (n = 5) and IVM of oocytes obtained ex vivo in the ovarian cortex (n = 2). This FP strategy was performed either after COH (stimulated group, n = 18) or after IVM (unstimulated group, n = 33). PARTICIPANTS/MATERIALS, SETTING, METHODS Oocyte retrieval followed by OT extraction on the same day was performed either without previous stimulation or after COH. Adverse effects of surgery and ovarian stimulation, mature oocyte yield and pathology findings of fresh OT were retrospectively analysed. Thawed OTs were analysed prospectively, for vascularization and apoptosis using immunohistochemistry, when patient consent was obtained. MAIN RESULTS AND THE ROLE OF CHANCE No surgical complication occurred after OTC surgery in either group. In particular, no severe bleeding was associated with COH. The number of mature oocytes obtained increased after COH (median = 8.5 (25% = 5.3–75% = 12.0)) compared to the unstimulated group (2.0 (1.0–5.3), P LIMITATIONS, REASONS FOR CAUTION The study reports FP from a small number of women following OTC. Follicle density and other pathology findings are an estimate only. WIDER IMPLICATIONS OF THE FINDINGS Unilateral oophorectomy can be successfully performed after COH with limited bleeding risk and an absence of impact on thawed OT. This approach could be proposed to post pubertal patients when the number of mature oocytes expected is low or when the risk of residual pathology is high. The reduction of surgical steps for cancer patients also has positive implications for introducing this approach into clinical practice. STUDY FUNDING/COMPETING INTEREST(S) This work was made possible through the support of the reproductive department of Antoine-Béclère Hospital and of the pathological department of Bicêtre Hospital (Assistance Publique Hôpitaux de Paris, France). The authors have no conflict of interest to disclose in this study. TRIAL REGISTRATION NUMBER N/A.

Research question What is the impact of radiation exposure on oocyte quality and female fertility? Design Prepubertal mice underwent whole-body irradiation with a single dose (0.02, 0.1, 0.5, 2, 8 Gy) of gamma- or X-rays. Oocytes were quantified in irradiated (n = 36) and sham-treated (n = 8) mice. After a single exposure to 2 Gy, formation of DNA double-strand breaks (n = 10), activation of checkpoint kinase (Chk2) (n = 10) and dynamics of follicular growth (n = 18) were analysed. Fertility assessment was performed in adult irradiated mice and controls from the number of pups per mouse (n = 28) and the fetal abortion rate (n = 24). Ploidy of mature oocytes (n = 20) was analysed after CREST immunostaining, and uterine sections were examined. Results Radiation exposure induced a massive loss of primordial follicles with LD50 below 50 mGy for both gamma and X-rays. Growing follicles survived doses up to 8 Gy. This difference in radiosensitivity was not due to a different amount of radio-induced DNA damage, and Chk2 was activated in all oocytes. Exposure to a 2 Gy dose abolished the long-term fertility of females due to depletion of the ovarian reserve. Detailed analysis indicates that surviving oocytes were able to complete folliculogenesis and could be fertilized. This transient fertility allowed irradiated females to produce a single litter albeit with a high rate of fetal abortion (23%, P = 0.0096), related to altered ploidy in the surviving oocytes (25.5%, P = 0.0035). Conclusions The effects of radiation on surviving oocyte quality question natural conception as a first-line approach in cancer survivors. Together, the data emphasize the need for fertility preservation before radiation exposure and call for reassessment of the use of cryopreserved oocytes.

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

Objective The aims of this study were to follow up the monitoring, health and anxiety from women who became pregnant after an embryo transfer or a intrauterine insemination during the COVID-19 epidemic in France Study Design This is a single centre, retrospective study from December 2019 to March 2020 based on a phone call interview using a specific questionnaire sheet specially developed for this study. Questionnaires from 104 pregnant women were completed and descriptive data are then analyzed. Results Women with ongoing pregnancies (n = 88) did not change their physician visits. The COVID-19 outbreak has created no or few additional stresses for 77 % of pregnant women since the lockdown started. We report a miscarriage rate of 14.4 % (n = 15) and documented 10 patients (11.3 %) who had symptoms related to COVID-19. No severe symptoms and no hospitalization in intensive care unit were identified. Conclusion The epidemic context did not disrupt the medical monitoring of pregnancies and we did not recover an increased rate of miscarriage after ART. None of the patients who had COVID-related symptoms presented with severe clinical manifestations. Surprisingly, pregnant women were psychologically able to experience the lockdown.

In this study, we aimed to evaluate the pregnancy outcomes for embryos biopsied twice at cleavage and blastocyst stage for preimplantation genetic testing (PGT). This retrospective monocentric study, conducted between January 2016 and March 2021, described all PGT results on one hand and the PGT results for undiagnosed embryos submitted to a second biopsy on the other hand. Among the 5865 embryos biopsied during the study period, 510 embryos were genetic undiagnosed after the first embryo biopsy at cleavage stage (8.7%). The rate of undiagnosed embryos was significantly higher for PGT for structural rearrangement (PGT-SR) than PGT for monogenic disease (PGT-M) (10.2% vs 7.4% respectively, p0.001). Thirty-three embryos were compatible with a second biopsy at blastocyst stage before being directly frozen. Among them 17 were diagnosed as healthy for the researched pathology (51.5%). At the time of our study, 11 of the 17 preserved embryos were thawed and transferred. Embryo survival at thawing was 100% and 5 pregnancies were obtained (clinical pregnancy rate of 45.5% per transfer), including 3 live births. A second biopsy for inconclusive embryos after PGT does not seem to have an impact on thaw survival and implantation rate. For couple, this strategy avoids to discard transferable embryos.

Context Myotonic dystrophy (DM) is an autosomal dominant disorder characterized mainly by myotonia but also by primary hypogonadism. No study has reported on fertility management of patients affected by DM type 1 (DM1). Objective This study investigates the impact of CTG repeats in the DMPK gene on semen quality and preimplantation genetic diagnosis (PGD) outcome. Design This is a monocentric retrospective observational study conducted from January 2003 to January 2019. Setting Antoine Béclère University Hospital, Clamart, France. Patients Three groups were compared in this study: male DM1 patients (Group A, n = 18), unaffected partners of DM1 female patients (Group B, n = 30), and proven fertile men (Group C, n = 33). Reproductive outcomes after PGD were compared between groups A and B. Results Sperm volume was reduced in group A (2.0 mL) when compared with groups B (3.0 mL; P < 0.01) and C (3.5 mL; P < 0.01). Progressive motility in raw sperm was also decreased in group A (30%) as compared to group C (40%; P < 0.01). The median number of progressive spermatozoa retrieved after sperm preparation was 2.7 million (M) in group A, which was significantly less than those of groups B (10.0 M; P < 0.01) and C (62.2 M; P < 0.01). Sperm motility was inversely correlated to the number of CTG repeats (Spearman r2 = 0.48, Pearson r2 = 0.35). Cumulative live birth rate per transfer was similar between groups, with 32.2% in group A versus 26.8% in group B. Conclusions As a precautionary measure, we advise physicians to perform regular monitoring of semen quality in affected males, which would allow sperm cryopreservation should semen parameters fall. PGD allows good reproductive outcomes without disease transmission.

STUDY QUESTION What are the chances of obtaining a healthy transferable cleavage-stage embryo according to the number of mature oocytes in fragile X mental retardation 1 (FMR1)-mutated or premutated females undergoing preimplantation genetic testing (PGT)? SUMMARY ANSWER In our population, a cycle with seven or more mature oocytes has an 83% chance of obtaining one or more healthy embryos. WHAT IS KNOWN ALREADY PGT may be an option to achieve a pregnancy with a healthy baby for FMR1 mutation carriers. In addition, FMR1 premutation is associated with a higher risk of diminished ovarian reserve and premature ovarian failure. The number of metaphase II (MII) oocytes needed to allow the transfer of a healthy embryo following PGT has never been investigated. STUDY DESIGN, SIZE, DURATION The study is a monocentric retrospective observational study carried out from January 2006 to January 2020 that is associated with a case-control study and that analyzes 38 FMR1 mutation female carriers who are candidates for PGT; 16 carried the FMR1 premutation and 22 had the full FMR1 mutation. PARTICIPANTS/MATERIALS, SETTING, METHODS A total of 95 controlled ovarian stimulation (COS) cycles for PGT for fragile X syndrome were analyzed, 49 in premutated patients and 46 in fully mutated women. Only patients aged ≤38 years with anti-Müllerian hormone (AMH) >1 ng/ml and antral follicle count (AFC) >10 follicles were eligible for the PGT procedure. Each COS cycle of the FMR1-PGT group was matched with the COS cycles of partners of males carrying any type of translocation (ratio 1:3). Conditional logistic regression was performed to compare the COS outcomes. We then estimated the number of mature oocytes needed to obtain at least one healthy embryo after PGT using receiver operating characteristic curve analysis. MAIN RESULTS AND THE ROLE OF CHANCE Overall, in the FMR1-PGT group, the median number of retrieved and mature oocytes per cycle was 11 (interquartile range 7–15) and 9 (6–12), respectively. The COS outcomes of FMR1 premutation or full mutation female carriers were not altered compared with the matched COS cycles in partners of males carrying a balanced translocation in their karyotype. Among the 6 (4–10) Day 3 embryos obtained in the FMR1-PGT group, a median number of 3 (1–6) embryos were morphologically eligible for biopsy, leading to 1 (1–3) healthy embryo. A cutoff value of seven MII oocytes yielded a sensitivity of 82% and a specificity of 61% of having at least one healthy embryo, whereas a cutoff value of 10 MII oocytes led to a specificity of 85% and improved positive predictive value. LIMITATIONS, REASONS FOR CAUTION This study is retrospective, analyzing a limited number of cycles. Moreover, the patients who were included in a fresh PGT cycle were selected on ovarian reserve parameters and show high values in ovarian reserve tests. This information could influence our conclusion. WIDER IMPLICATIONS OF THE FINDINGS The results relate only to the target population of this study, with a correct ovarian reserve of AMH >1 and AFC >10. However, the information provided herein extends knowledge about the current state of COS for FMR1 mutation carriers in order to provide patients with proper counseling regarding the optimal number of oocytes needed to have a chance of transferring an unaffected embryo following PGT. STUDY FUNDING/COMPETING INTEREST(S) None. TRIAL REGISTRATION NUMBER N/A.

PURPOSE: The aim of this study was to evaluate the outcomes of frozen oocytes or embryos cryopreserved after controlled ovarian stimulation (COS) or in vitro maturation (IVM) for female cancer patients who underwent a fertility preservation (FP) prior to gonadotoxic therapy. METHODS: A retrospective cohort study from 2009 to December 2017 was conducted. Among the 667 female cancer patients who underwent oocytes or embryos cryopreservation for FP, 40 (6%) have returned to the fertility clinic between 2011 and 2019 to use their frozen material after being cured. We compared these thaw cycles outcomes according to the techniques used at the time of cryopreservation. RESULTS: Among the 40 women cancer survivors who used their cryopreserved material, thirty patients have benefited from at least one embryo transfer. Ten patients did not have an embryo transfer since the oocytes did not survive after the thawing process or because no embryo was obtained after fertilization. We related three live births following FP using IVM (two from frozen oocytes and one after embryo cryopreservation). Five live births were obtained when COS was performed at the time of FP (one from frozen oocytes and four after embryo cryopreservation). CONCLUSIONS: Our preliminary results, although they are obtained in a small sample, are encouraging and show that different FP techniques can be used in female cancer patients and lead to live births. IVM is one of the options available that does not delay the start of chemotherapy or if ovarian stimulation using gonadotropins is contraindicated. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-021-02168-3.

Mitochondrial DNA (mtDNA) mutations cause severe maternally inherited disorders, although mechanisms regulating mother-to-offspring transmission have not yet been elucidated. To investigate if mtDNA mutations affect embryonic development, we compared morphology, viability and mtDNA content in control (n = 165) and mitochondrial (n = 16) human embryos at the cleavage-stage. mtDNA copy number (CN) was assessed in one or two embryonic cells, by real-time PCR. The presence of a maternal or embryonic mtDNA mutation did not impact on either embryonic quality or viability. mtDNA CN was not altered by mtDNA mutations, suggesting that mtDNA defects do not modify mtDNA metabolism at this early stage.

Research question Chromosomal translocations are known genetic causes of premature ovarian insufficiency syndrome. Are certain translocations associated with decreased capacity of small antral follicles to respond to exogenous FSH? Does the prognosis after preimplantation genetic testing for structural rearrangements differ in couples with female or male translocation carriers and according to the type of translocation? Design A single-centre, retrospective, observational study covering a 10-year period. One hundred and thirty-nine females carrying a translocation were compared with 192 partners of male translocation carriers. To evaluate ovarian response to FSH, the follicular output rate was used, defined by ratio between the pre-ovulatory follicle count on day of HCG x 100/antral follicle count (AFC). To determine a cut-off of metaphase II oocytes and biopsied embryos as predictor of obtaining a balanced embryo transfer, receiver operator characteristic curves were plotted. Result A decreased capacity of small antral follicles to respond to exogenous FSH in female translocation carriers was found. The number of metaphase II oocytes in both groups was weakly informative as a predictor of obtaining an embryo transfer. The number of biopsied embryos had some clinical value, however, and allowed a cut-off of 6.5 to be determined for female translocation carriers versus 5.5 for the partners of male translocation carriers. Live birth rates, however, were not different between female and male translocations carriers. Conclusions Female translocation carriers may respond poorly to ovarian stimulation, and present a higher rate of unbalanced embryos, which means that higher gonadotrophin doses may be required to increase the number of biopsied embryos.

Resume Le diagnostic preimplantatoire (DPI) est une alternative au diagnostic prenatal classique pour les couples a risque de transmettre une maladie genetique severe, comme les maladies liees a des mutations de l'ADN mitochondrial (ADNmt). Le diagnostic preimplantatoire consiste en l'analyse genetique d'une ou quelques cellules embryonnaires, prelevees a partir d'embryons issus d'une fecondation in vitro (FIV) entre le troisieme et le cinquieme jour de leur developpement. Dans le cas de mutation de l'ADNmt, l'analyse genetique consiste a quantifier le taux de mutation sur ces cellules afin de determiner le risque pour cet embryon de developper, ou non, une maladie grave. Notre experience de 15 annees illustre la fiabilite de cette procedure. Nous avons pris en charge 15 patientes heteroplasmiques, 26 cycles de stimulation ovarienne ont ete debutes, 25 ponctions ovariennes, et 16 transferts d'embryons ont pu etre realises, aboutissant a 3 grossesses et a la naissance de 3 enfants. Le taux de mutation mesure sur une cellule prelevee sur 94 embryons s'est avere tres semblable aux taux de mutation des autres cellules pour toutes les mutations testees (m.8344A > G, m.3243A > G, m.8993T > G, m.8993T > C, m.9185T > C, m.10197G > A). La plupart des embryons transferes (17/25) et des enfants nes (2/3) de cette procedure sont porteurs de la mutation maternelle, et la question du pronostic a long terme de ces enfants reste posee. Le DPI reste une procedure lourde avec de faibles chances de succes, et ne peut s'appliquer aux patientes homoplasmiques ou quasi-homoplasmiques. Le developpement de procedures alternatives, comme le transfert nucleaire, est donc une necessite.

L’annonce recente de la naissance d’un enfant issu d’un transfert du fuseau meiotique d’un ovocyte d’une patiente porteuse d’une mutation de l’ADN mitochondrial vers un ovocyte d’une patiente non atteinte (« donneuse de cytoplasme »), a ravive le debat sur l’innocuite de ces procedures. Les reserves emises concernent principalement la possibilite de reversion genetique, les incertitudes liees a la perturbation du dialogue entre le noyau et les mitochondries et aux effets secondaires d’un etat heteroplasmique induit par ces techniques. La possibilite d’etendre les indications de transfert de noyaux aux patientes en echec de fecondation in vitro , souligne la necessite de repondre de facon urgente a ces questions.

Research question Chromosomal translocations are known genetic causes of male infertility. Are certain translocations or chromosomal regions more directly associated with sperm defects? Is there a threshold of sperm impairment that can be relevant for detection of translocations? Design This is a monocentric retrospective observational study covering a 10-year period. Eighty-one patients carrying a reciprocal translocation (RCT) and 63 carrying a Robertsonian translocation (ROBT) were compared with 105 fertile patients. Semen quality before and after sperm migration was compared. The aims were to define whether a threshold based on sperm analysis could be proposed for detection of translocations and to identify whether some redundant chromosomal regions might be associated with sperm quality defects. Results The number of progressive spermatozoa retrieved after sperm preparation (NPS-ASP) was altered in both RCT and ROBT carriers compared with controls, with a stronger alteration in ROBT. Based on the NPS-ASP results in this large group of translocation carriers, a relatively robust threshold, fixed at less than 5 million, may be proposed for detection of translocations. The alteration of NPS-ASP was independent of the chromosome involved in ROBT, while in RCT, four redundant chromosomal regions (1q21, 6p21, 16q21, 17q11.2) were associated with poor or very poor NPS-ASP. Conclusions The NPS-ASP appears to be a good parameter to assess sperm function and would be a useful tool to detect chromosomal translocations. Four redundant regions have been identified on four chromosomes, suggesting that they may contain genes of interest to study sperm functions.

The recent birth from a mitochondrial DNA mutation carrier of a child, conceived after transfer in a donor oocyte of the meiotic spindle, taken from the maternal oocyte, revived the debate on the safety of these procedures. The doubts concern mainly the possibility of genetic reversion, the uncertainties about potential disturbances of the dialogue between nuclear and mitochondrial genomes and the side effects of a heteroplasmic state induced by these techniques. The possibility to expand nuclear transfer applications to patients experiencing in vitro fertilization failure, urges us to answer these questions rapidly.

Short gamete co-incubation (SGCO) consists in decreasing the duration of contact between oocytes and sperm from the standard overnight insemination (SOI) toward 2 hours. However, the effectiveness of this technique to improve in vitro fertilization and embryo transfer (IVF-ET) outcomes remains controversial. Our study was designed to evaluate the efficiency of SGCO in a poor prognosis population with a history of fragmented embryos defined by the presence of at least 50% of the embryos with more than 25% of cytoplasmic fragments. From January 2010 to January 2014, 97 couples were included in a SGCO protocol. We separated women into 2 subgroups: younger and older than 35 years. Compared to SOI, after SGCO, 2-cell stage embryos were higher in all women (p0.001) and less fragmented in women over 35 years (p0.05). On day 2, top quality embryos obtained and transferred were higher with SCGO than with SOI, independently of the age of the women (p0.001). Moreover, the number of embryos with less than 25% of fragmentation was higher after SGCO than SOI (p0.001) whereas the number of multinucleated embryos was lower (p0.001). We observed that after fresh ET, independently of the age of the women, the clinical pregnancy rate was 3 times higher after SGCO than after SOI. However, the live-birth rate was 4 times higher with SGCO than with SOI in women above 35 years but 3 times higher with SGCO than with SOI in women younger than 35 years. The present results indicate that for a particular indication, reducing the time of oocytes and sperm co-incubation may improve IVF-ET outcomes in terms of live-birth rate.AMH: anti mullerian hormone; COC: cumulus-oocytes complex; E2: estradiol; ET: embryo transfer; FET: frozen embryo transfer; FSH: follicle stimulating hormone; GnRH: gonadotrophin releasing hormone; hCG: human chorionic gonadotropin hormone; hMG: human menopausal gonadotropin hormone; IRB: institutional review board; IVF: in vitro fertilization; IVF-ET: in vitro fertilization and embryo transfer; MNB: multinucleated blastomere; mRNA: messanger ribonucleic acid; OC: oocyte retrieval; O2: oxygen; ROS: reactive oxygen species; SGCO: short gamete co-incubation; SOI: standard overnight insemination.

Mitochondrial DNA (mtDNA) content is thought to remain stable over the preimplantation period of human embryogenesis that is, therefore, suggested to be entirely dependent on ooplasm mtDNA capital. We have explored the impact of two disease-causing mutations [m.3243A>G myopathy, encephalopathy, lactic acidosis and stroke-like syndrome (MELAS) and m.8344A>G myoclonic epilepsy associated with ragged-red fibers (MERRF)] on mtDNA amounts in human oocytes and day 4-5 preimplantation embryos. The mtDNA amount was stable in MERRF and control materials, whereas gradually increasing from the germinal vesicle of oogenesis to the blastocyst stage of embryogenesis in MELAS cells, MELAS embryos carrying ∼3-fold higher mtDNA amount than control embryos (P = 0.0003). A correlation between mtDNA copy numbers and mutant loads was observed in MELAS embryos (R(2) = 0.42, P < 0.0013), suggestive of a compensation for the respiratory chain defect resulting from high mutation levels. These results suggest that mtDNA can replicate in early embryos and emphasize the need for sufficient amount of wild-type mtDNA to sustain embryonic development in humans.

Context:The 21-hydroxylase deficiency is the most common cause of congenital adrenal hyperplasia. Pregnant women presenting a risk of genetic transmission may be treated with synthetic glucocorticoids such as dexamethasone (DEX) to prevent female fetus virilization.Objective:The aim of this study was to assess the potential deleterious effects of DEX exposure on fetal ovarian development.Settings:Human fetal ovaries, ranging from 8–11 weeks after fertilization, were harvested from material available after legally induced abortions. They were cultured in the absence or presence of DEX (2, 10, or 50 μm) over 14 d, and histological analyses were performed.Results:The glucocorticoid receptor NR3C1 was present and the signaling pathway active in the fetal ovary as demonstrated by the expression of NR3C1 target genes, such as PLZF and FKBP5, in response to DEX exposure. DEX decreased germ cell density at the 10 and 50 μm doses. Exposure to DEX, even at the highest dose, did not change oogonial proliferation as monitored by 5-bromo-2′-deoxyuridine incorporation and significantly increased the apoptotic rate, detected with cleaved caspase 3 staining. Interestingly, the expression of the prosurvival gene KIT was significantly decreased in the presence of DEX during the course of the culture.Conclusion:We have demonstrated for the first time that in vitro exposure to high doses of DEX impairs human fetal oogenesis through an increase in apoptosis. These data are of high importance, and additional epidemiological studies are required to investigate the female fertility of those women who have been exposed to DEX during fetal life.

It has been two decades since pregnancies have been obtained through in vitro maturation (IVM) of germinal vesicle-stage oocytes retrieved from non-stimulated ovaries. This technique first offered in PCOS cannot be recommended today in this indication because the results do not match those of regular ART, and new GnRH antagonist and agonist-trigger protocols reliably prevent OHSS.

Journal de Gynecologie Obstetrique et Biologie de la Reproduction - Vol. 41 - N° 1 - p. 41-47

Looking for a qualitative marker of ovarian function, we aimed to verify whether responsiveness of antral follicles to FSH administration, as reflected by the Follicular Output RaTe (FORT), is related to their reproductive competence.We studied 322 IVF-ET candidates aged 25-43 years who underwent controlled ovarian hyperstimulation with similar initial FSH doses. Antral follicle (3-8 mm) count (AFC) and pre-ovulatory follicle (16-22 mm) count (PFC) were performed, respectively, at the achievement of pituitary suppression (before FSH treatment) and on the day of hCG administration. The FORT was calculated by PFC × 100/AFC. FORT groups were set according to tercile values: low (42%; n= 102), average (42-58%; n= 123) and high (58%; n= 97).The average FORT was 50.6% (range, 16.7-100.0%). Clinical pregnancy rates per oocyte retrieval increased progressively from the low to the high FORT groups (33.3, 51.2 and 55.7%, respectively, P0.003) and such a relationship assessed by logistic regression was independent of the confounding covariates, women's ages, AFC and PFC.The observed relationship between IVF-ET outcome and the percentage of antral follicles that effectively respond to FSH administration reaching pre-ovulatory maturation suggests that FORT may be a qualitative reflector of ovarian follicular competence. Further studies with broader inclusion criteria and more personalized protocols are needed to validate these results.

Objective To review and summarize data from the scientific literature on the use of androgens to improve ovarian function. Design Review of pertinent literature. Setting University hospital. Patient(s) Women exposed to androgens. Intervention(s) None. Main Outcome Measure(s) Critical review of the literature. Result(s) The artificial increase of intra-ovarian androgen concentrations constitutes an attractive concept for improving the deficient ovarian function of poor responders. Data from studies conducted in animals treated with high-dose androgens, together with observations made in hyperandrogenic women or female-to-male transsexuals receiving virilizing androgen doses, indicate that androgens may increase follicle responsiveness to FSH and/or the number of growing follicles in the ovary. Yet, definite clinical demonstration of such a concept still is not available. Conclusion(s) Current clinical approaches aiming at increasing androgen availability in the ovary showed conflicting results. Therefore, additional studies using proper strategies to achieving higher intra-ovarian androgen concentrations for longer intervals are required to define the clinical efficiency of androgens in poor responders.

Umut-Talha, a "sibling savior", was born on 26 January 2011 at Beclere Hospital after embryo selection at the Paris preimplantation genetic diagnosis (PGD) center. His birth revived the controversy over "double PGD". This procedure, authorized in France since 2006, allows couples who already have a child with a serious, incurable genetic disease, to opt for PGD in order to select a healthy embryo that is HLA-matched to the affected sibling and who may thus serve as an ombilical cord blood donor. The procedure is particularly complex and the baby take-home rate is still very low. Double PGD is strictly regulated in France, and candidate couples must first receive individual authorization from the Biomedicine Agency. In our experience, these couples have a strong desire to have children, as reflected by the large number of prior spontaneous pregnancies (25% of couples). Likewise, most of these couples request embryo transfer even when there is no HLA-matched embryo, which accounts for more than half of embryo transfers. The controversy surrounding this practice has flared up again in recent weeks, over the concepts of "designer babies" and "double savior siblings" (the baby is selected to be free of the hereditary disease, and may also serve as a stem cell donor for the affected sibling).

Because the mtDNA amount remains stable in the early embryo until uterine implantation, early human development is completely dependent on the mtDNA pool of the mature oocyte. Both quantitative and qualitative mtDNA defects therefore may negatively impact oocyte competence or early embryonic development. However, nothing is known about segregation of mutant and wild-type mtDNA molecules during human meiosis. To investigate this point, we compared the mutant levels in 51 first polar bodies (PBs) and their counterpart (oocytes, blastomeres, or whole embryos), at risk of having (1) the “MELAS” m.3243A>G mutation in MT-TL1 (n = 30), (2) the “MERRF” m.8344A>G mutation in MT-TK (n = 15), and (3) the m.9185T>G mutation located in MT-ATP6 (n = 6). Seven out of 51 of the PBs were mutation free and had homoplasmic wild-type counterparts. In the heteroplasmic PBs, measurement of the mutant load was a rough estimate of the counterpart mutation level (R2 = 0.52), and high mutant-load differentials between the two populations were occasionally observed (ranging from –34% to +34%). The mutant-load differentials between the PB and its counterpart were higher in highly mutated PBs, suggestive of a selection process acting against highly mutated cells during gametogenesis or early embryonic development. Finally, individual discrepancies in mutant loads between PBs and their counterparts make PB-based preconception diagnosis unreliable for the prevention of mtDNA disorder transmission. Such differences were not observed in animal models, and they emphasize the need to conduct thorough studies on mtDNA segregation in humans.

BACKGROUND: The initiation of meiosis is crucial to fertility. While extensive studies in rodents have enhanced our understanding of this process, studies in human fetal ovary are lacking. METHODS: We used RT-PCR and immunohistochemistry to investigate expression of meiotic factors in human fetal ovaries from 6 to 15 weeks post fertilization (wpf) and developed an organ culture model to study the initiation of human meiosis. RESULTS: We observed the first meiotic cells at 11 wpf, when STRA8, SPO11 and DMCI are first expressed. In culture, meiosis initiation is observed in 10 and 11 wpf ovaries and meiosis is maintained by addition of fetal calf serum. Meiosis is stimulated, compared with control, by retinoic acid (RA) (P < 0.05). No major change occurred in mRNA for CYP26B1, the RA-degrading enzyme proposed to control the timing of meiosis in mice. We did, however, observe increased mRNA levels for ALDH IAIin human ovary when meiosis began, and evidence for a requirement to synthesize RA and thus sustain meiosis. Indeed, ALDH inhibition by citral prevented the appearance of meiotic cells. Finally, 8 wpf ovaries (and earlier stages) were unable to initiate meiosis whatever the length of culture, even in the presence of RA and serum. However, when human germ cells from 8 wpf ovaries were placed in a mouse ovarian environment, some did initiate meiosis. CONCLUSIONS: Our data indicate that meiosis initiation in the human ovary relies partially on RA, but that the progression and regulation of this process appears to differ in many aspects from that described in mice.

Resume But Evaluer l’impact d’une demarche qualite pour un centre d’assistance medicale a la procreation dans l’optique d’une stabilite des resultats. Materiels et methodes Collecte prospective de tous les cycles de fecondation in vitro au centre d’assistance medicale a la procreation de l’hopital Antoine-Beclere (Clamart), ainsi que des issues de grossesse, pour les annees 2002 a 2006. Resultats 44,3 % de grossesse clinique et 38,0 % d’accouchement par ponction en cycle stimule, ces taux sont stables au cours des cinq annees etudiees. Le taux de grossesse multiple est encore de 30,7 %, avec une diminution progressive, parallele a la diminution du nombre d’embryons transferes. Conclusion Les resultats sont stables sur une duree de cinq ans. La demarche qualite semble incontournable a l’heure actuelle.

Novel embryonic stem cell lines derived from embryos carrying structural chromosomal abnormalities obtained after preimplantation genetic diagnosis (PGD) are of interest to study in terms of the influence of abnormalities on further development. A total of 22 unbalanced blastocysts obtained after PGD were analysed for structural chromosomal defects. Morphological description and chromosomal status of these blastocysts was established and they were used to derive human embryonic stem cell (ESC) lines. An outgrowth of cells was observed for six blastocysts (6/22; 27%). For two blastocysts, the exact morphology was unknown since they were at early stage, and for four blastocysts, the inner cell mass was clearly visible. Fifteen blastocysts carried an unbalanced chromosomal defect linked to a reciprocal translocation, resulting in a positive outgrowth of cells for five blastocysts. One human ESC line was obtained from a blastocyst carrying a partial chromosome-21 monosomy and a partial chromosome-1 trisomy. Six blastocysts carried an unbalanced chromosomal defect linked to a Robertsonian translocation, and one showed a positive outgrowth of cells. One blastocyst carried an unbalanced chromosomal defect linked to an insertion and no outgrowth was observed. The efficiency of deriving human ESC lines with constitutional chromosomal disorders was low and probably depends on the initial morphological aspect of the blastocysts and/or the type of the chromosomal disorders.

BCL2-associated X protein (BAX) and B-cell leukaemia/lymphoma gene-2 (BCL2), which are, respectively, pro- and anti-apoptotic proteins of the BCL2 gene family, participate in the mitochondria-dependent apoptosis pathway. A correlation between low incidence of apoptosis in cumulus cells and oocyte maturation has previously been suggested in ovarian stimulation. However, little is known in unprimed ovaries. These authors have investigated whether BAX and BCL2 expression in cumulus cells affects the competency of in-vitro matured oocytes. We have studied 100 cumulus-oocyte-complexes (COC) recovered from unprimed ovaries of 13 women diagnosed with polycystic ovary syndrome (PCOS) and undergoing in-vitro maturation (IVM) with their informed consent. COC were matured for 24 h in a specific maturation medium and the cumulus was stripped from the oocyte. BAX and BCL2 mRNA content was measured in each COC using real-time polymerase chain reaction. We found that BCL2 mRNA expression was significantly higher in cumulus cells associated with mature oocytes than those associated with immature oocytes while BAX mRNA concentrations did not vary in cumulus cells. Regarding fertilization, higher BCL2 mRNA content was found in cumulus cells enclosing fertilized oocytes (0.140 versus 0.075; P = 0.03). These results suggest that BCL2 expression is strongly associated with the ability of oocytes to complete nuclear maturation and to be fertilized.

Le cancer est la deuxieme cause de deces chez les femmes de moins de 40 ans dans les pays occidentaux. Les cancers les plus frequents, chez les femmes entre 20 et 40 ans, sont le cancer de sein, le cancer du col de l’uterus et les leucemies (1).

Autosomal recessive polycystic kidney disease (ARPKD) is one of the most common hereditary renal cystic diseases, and is caused by mutations in the PKHD1 gene. Due to the poor prognosis, there is a strong demand for prenatal diagnosis. Preimplantation genetic diagnosis (PGD) represents an alternative because it avoids the physical and emotional trauma of a pregnancy termination in the case of an affected fetus. A standardized single-cell diagnostic procedure was developed, based on haplotype analysis, enabling PGD to be offered to couples at risk of transmitting ARPKD. Six linked markers within (D6S1714 and D6S243), or in close proximity to (D6S272, D6S436, KIAA0057, D6S1662) the PKHD1 gene were tested by multiplex nested-polymerase chain reaction (PCR), using a Qiagen multiplex PCR kit. PCR analyses were carried out on 50 single lymphocytes. The amplification rate was excellent (100%), with an allele drop-out (ADO) rate ranging from 0 to 8%. Five PGD cycles were performed and 23 embryos were biopsied and analysed using this test. Transferable embryos were obtained in 4 cycles, resulting in two pregnancies and the birth of a healthy boy. This standardized diagnostic procedure allowed the detection of recombination, contamination, and ADO events, providing high assay accuracy with wide applicability.

In vitro fertilization without stimulation, in natural or semi-natural cycles ("FIV-NAT") offers an interesting alternative to controlled ovarian hyperstimulation (COH) for some patients. This protocol may be proposed to patients suffering from altered ovarian status (AOS) with encouraging results. The second indication might be patients presenting repeated implantation failure (IMF). FIV-NAT allows avoiding possible adverse effects of HOC on the endometrium. Results are respectively 15.4 and 16.6% clinical pregnancy rate per oocyte retrieval for AOS and IMF, and 25.5 and 26.3% over 3 cumulative cycles. This treatment is less expensive and patient friendly.

Resume Objectifs Nous avons voulu savoir quelle etait la place de l'adoption dans le parcours des couples en cours d'Assistance medicale a la procreation, afin de repondre a une autre question : les couples infertiles suivis en traitement attendent-ils les conseils du gynecologue pour entreprendre une demarche d'adoption ou l'entreprennent-ils avant tout conseil ? Patientes et methode Nous avons interroge 90 couples, venus dans notre unite le premier jour de leur traitement, sur la place de l'adoption dans leur desir d'enfant : leur position en faveur ou non de l'adoption, leurs demarches, et son aboutissement ou non. Resultats Quarante-neuf pour cent des couples ne sont pas favorables a l'adoption, surtout lorsqu'ils ont deja eu des enfants par l'Assistance medicale a la procreation. Cependant, 84,5 % des couples interroges ont pense a l'adoption des l'annonce de l'infertilite. Soixante-neuf pour cent ont entrepris des recherches intellectuelles (livre, emission, consultation de site Internet, discussion avec adoptants et adoptes) sur le sujet. Au total dans la population etudiee, 29 % des couples ont commence une demarche d'adoption mais seuls 18 % ont l'intention de la mener vraiment a terme. Discussion et conclusion Les couples infertiles n'attendent pas tous les conseils du medecin pour s'interesser de pres a l'adoption mais peut-etre auront-ils besoin d'entendre les mots de la fin de la prise en charge medicale par leur medecin pour veritablement mener a terme ce projet. Certains prefereront de toute facon rester sans enfant ou sans autres enfants, ou bien passer au don de gametes.

Resume Objectifs Evaluer l'importance du rincage folliculaire lors des prelevements ovocytaires en cycle semi-naturel. Materiel et methode Nous avons compare prospectivement le devenir des ovocytes obtenus dans le liquide folliculaire (LF, n = 79) a ceux obtenus dans le liquide de rincage (R, n = 47) lors de 146 prelevements ovocytaires. Resultats Les groupes LF et R se sont averes similaires en ce qui concerne le taux de fecondation (79,7 versus 88,1 %, respectivement), la prevalence d'embryons de grade superieur (28,8 versus 37,8 %) et le taux d'implantation (24,1 versus 44,1 %). Conclusion La pratique du rincage folliculaire au cours du cycle semi-naturel ameliore les taux de grossesse. Les ovocytes recuperes dans le rincage ont le meme potentiel reproductif que les ovocytes preleves de premiere intention.

The objective of this retrospective analysis is to evaluate whether patients developing a dominant follicle in an in-vitro maturation (IVM) programme can be included in a natural cycle IVF (nIVF) programme. In a university teaching hospital, a total of 38 patients with polycystic ovarian syndrome were treated by metformin before undergoing an IVM cycle. Controlled nIVF was performed on seven patients with a dominant follicle. This paper reports three ongoing pregnancies from five embryo transfers in the latter group. Hence, nIVF can be a novel approach for women developing a dominant follicle during an IVM cycle.

Study question How can pre-meiotic germ cells persist in the human foetal ovary? Summary answer Numerous oogonia escaping meiotic entry were retrieved throughout human ovarian development simultaneously with the expression of signalling pathways preventing meiosis, typically described in the rodent embryonic testis. What is known already The transition from mitosis to meiosis is a key event in female germ cells that remains poorly documented in research on the human ovary. Previous reports described a strikingly asynchronous differentiation in the human female germ line during development, with the persistence of oogonia among oocytes and follicles during the second and third trimesters. The possible mechanisms allowing some cells to escape meiosis remain elusive. Study design size, duration In order to document the extent of this phenomenon, we detailed the expression profile of germ cell differentiation markers using 73 ovaries ranging from 6.4 to 35 weeks post-fertilization. Participants/materials setting, methods Pre-meiotic markers were detected by immunohistochemistry or qRT-PCR. The expression of the main meiosis-preventing factors identified in mice was analysed, and their functionality assessed using organ cultures. Main results and the role of chance Oogonia stained for AP2γ could be traced from the first trimester until the end of the third trimester. Female germ cell differentiation is organized both in time and space in a centripetal manner in the foetal human ovary. Unexpectedly, some features usually ascribed to rodent pre-spermatogonia could be observed in human foetal ovaries, such as NANOS2 expression and quiescence in some germ cells. The two main somatic signals known to inhibit meiosis in the mouse embryonic testis, CYP26B1 and FGF9, were detected in the human ovary and act simultaneously to repress STRA8 and meiosis in human foetal female germ cells. Large scale data N/A. Limitations reason for caution Our conclusions relied partly on in vitro experiments. Germ cells were not systematically identified with immunostaining and some may have thus escaped analysis. Wider implications of the findings We found evidence that a robust repression of meiotic entry is taking place in the human foetal ovary, possibly explaining the exceptional long-lasting presence of pre-meiotic germ cells until late gestational age. This result calls for a redefinition of the markers known as classical male markers, which may in fact characterize mammalian developing gonads irrespectively of their sex. Study funding/competing interest(s) This research was supported by the Universite Paris Diderot-Paris 7 and Universite Paris-Sud, CEA, INSERM, and Agence de la Biomedecine. The authors declare no conflict of interest.

We report an improvement in the PGD test for fragile X syndrome (FXS). Recently, multiple displacement amplification (MDA) has been reported to yield large amounts of DNA from single cells. Taking into account this technique, we developed a new PGD test for FXS, enabling combined analysis of linked polymorphic markers with the study of the non-expanded CGG repeat. Single cell amplification efficiency was first assessed on single lymphocytes. Amplification rate of the different markers ranged from 85 to 95% with an allele drop-out (ADO) rate comprised between 7 and 34%. Using this test, eight PGD cycles were carried out for six couples, and 37 embryos were analysed after preliminary MDA. Amplification rate was increased by this technique from 41 to 66% so that embryos with no results were rarer (14 versus 45% without MDA). Reliability of the test was considerably improved by combining direct with indirect genetic analysis. Furthermore, in cases of fully expanded alleles too large to be amplified by PCR, this test gives an internal amplification control. Embryonic transfers were carried out in all but one PGD cycles. One biochemical and one clinical pregnancy resulted, and a healthy child was born. This single diagnosis procedure could be suitable to most patients carrying FXS.

Journal de Gynecologie Obstetrique et Biologie de la Reproduction - Vol. 35 - N° 4 - p. 356-372