Analysis of mtDNA variant segregation during early human embryonic development: a tool for successful NARP preimplantation diagnosis.

BACKGROUND: Diseases arising from mitochondrial DNA (mtDNA) mutations are usually serious pleiotropic disorders with maternal inheritance. Owing to the high recurrence risk in the progeny of carrier females, "at-risk" couples often ask for prenatal diagnosis. However, reliability of such practices remains under debate. Preimplantation diagnosis (PGD), a theoretical alternative to conventional prenatal diagnosis, requires that the mutant load measured in a single cell from an eight cell embryo accurately reflects the overall heteroplasmy of the whole embryo, but this is not known to be the case. OBJECTIVE: To investigate the segregation of an mtDNA length polymorphism in blastomeres of 15 control embryos from four unrelated couples, the NARP mutation in blastomeres of three embryos from a carrier of this mutation. RESULTS: Variability of the mtDNA polymorphism heteroplasmy among blastomeres from each embryo was limited, ranging from zero to 19%, with a mean of 7%. PGD for the neurogenic ataxia retinitis pigmentosa (NARP) mtDNA mutation (8993T-->G) was therefore carried out in the carrier mother of an affected child. One of three embryos was shown to carry 100% of mutant mtDNA species while the remaining two were mutation-free. These two embryos were transferred, resulting in a singleton pregnancy with delivery of a healthy child. CONCLUSIONS: This PGD, the first reported for a mtDNA mutation, illustrates the skewed meiotic segregation of the NARP mtDNA mutation in early human development. However, discrepancies between the segregation patterns of the NARP mutation and the HV2 polymorphism indicate that a particular mtDNA nucleotide variant might differentially influenced the mtDNA segregation, precluding any assumption on feasibility of PGD for other mtDNA mutations.

[Preimplantation genetic diagnosis (PGD): results from a Parisian center]. / Indications et résultats du diagnostic pré-implantatoire (DPI).

OBJECTIVE: To report the results of preimplantation genetic diagnosis (PGD) cycles performed in our unit from 2000 to 2004. Materials and methods. One hundred and seventy-one couples were enrolled in the PGD program over this period. The collected oocytes were inseminated by intracytoplasmic sperm injection (ICSI). The resulting embryos were biopsied on the third day of development and the genetic analysis was performed on the same day. Embryo transfers were carried out on the fourth day. RESULTS: The 416 stimulation cycles started yielded 280 oocyte pick-ups, 3506 oocytes retrieved, of which 2966 were suitable for ICSI. Among the 1982 embryos obtained, 1337 embryos were biopsied and genetic diagnosis was performed for 1083 (81%) of them. 381 embryos were transferred during the course of 189 transfer procedures. There were 51 clinical and 46 ongoing (35 single, 11 twin) pregnancies. In addition, 25 frozen embryo replacement cycles were initiated, leading to 6 embryo transfers and 1 ongoing pregnancy. A total of 58 unaffected children were born. CONCLUSION: PGD has gained a place among the choices offered to couples at risk of transmission of a serious and incurable genetic disease. It might be a realistic alternative to prenatal diagnosis for patients carrier of chromosomal rearrangements, single gene defects, X-linked disesases or mitochondrial DNA disorders.

[Pre-implantation genetic diagnosis and spontaneous pregnancies: an unexpected acting out]. / Diagnostic génétique préimplantatoire et grossesses spontanées: un passage à l'acte inattendu.

OBJECTIVES: To understand the psychological motives that led these couples to take risks involved in genetic transmission and the possibility of another spontaneous pregnancy. To report our psychoanalytic experience to explain why after a request of pre-implantation genetic diagnosis (PGD), some women prefer to be spontaneously pregnant. PATIENTS AND METHOD: Between 2000 and 2004, out of 643 requests of PGD, 541 have been admitted, among which 123 spontaneous pregnancies appeared during the process of PGD. RESULTS: Some encounters with the patients have shown some reasons to these spontaneous pregnancies - denial of IVF, ambivalence of this desire for a child, denial of the risk, control of the procreation, family loyalty and fertility affirmation. DISCUSSION AND CONCLUSION: The resort to PGD is more ambivalent than the thought of it because it means, beyond the request, conflict with transmission, culture, and history origins.

[Extending preimplantation genetic diagnosis to HLA typing: the Paris experience]. / Le diagnostic préimplantatoire couplé au typage HLA: l'expérience parisienne.

Preimplantation genetic diagnosis (PGD) consists in the genetic analysis of one or two cells. These cells (blastomeres) are sampled from embryos, obtained by in vitro fertilization, at the third day of development. Since 1998, the bioethical laws (1994) and their decrees restricted PGD practices in France, strictly to the avoidance of the birth of a child affected with a genetic defect. In parallel, works on blood cord transplantation, taken at the birth of a compatible HLA sibling, showed very encouraging results, particularly for the treatment of Fanconi anemia. In 2001, Verlinsky et al., have reported the first PGD for Fanconi anaemia combined with HLA typing, allowing the birth of a healthy child, HLA-identical with his affected sister. The "designer baby" concept was born. The French law, which allowed PGD under specific conditions, i.e. when the genetic defect has been characterized in one parent at least, recently extended PGD to HLA typing when embryos are at risk of a genetic disorder. Article L.2131-4-1 (August 2004) allows the practice of HLA typing for PGD embryos when an elder sibling is affected with a genetic disorder and need stem cell transplantation. The HLA-matched offspring resulting from PGD can give cord blood at birth to supply the necessary therapy. This double selection give rise to serious ethical problems, but technical difficulties and legal restrictions will probably limit the development of such a procedure.

Follow-up of a cohort of 422 children aged 6 to 13 years conceived by in vitro fertilization.

OBJECTIVE: To contact the total cohort of children conceived by IVF-ET consecutively in our center between June 1981 and December 1988. DESIGN: Retrospective study. SETTING: Infertility unit of the department of Obstetrics and Gynecology, Antoine Béclère Hospital, Clamart, France. PATIENT(S): Complete information was obtained on 370 children. The percentage lost for follow-up was 9%. INTERVENTION(S): To assess the children's well-being, telephone interviews of the parents and questionnaires sent to the parents and/or pediatrician were used. MAIN OUTCOME MEASURE(S): Surgical procedures, malformation, height and weight, school performance. RESULT(S): The physical growth of these children showed no major pathological features, with only 2.2% of them being below 2 SD for weight and 0.3% for height. The rates of malformation were not significantly different between these children and the general population. School performance was good, with 92.2% presenting encouraging outcome. Fifty-eight percent of the parents of children aged 6 to 10 years old did not inform their children about the IVF nor did 34% of the parents of children aged 11 to 13. Subsequent to the birth of the IVF child, 30 patients (8.9%) had a spontaneous pregnancy. However, five of them (15.1%) were ectopic. CONCLUSION(S): This study reports, for the first time, reassuring data on the long-term assessment of a large group of older IVF-ET children conceived consecutively, with a low percentage of subjects lost for follow-up.

[Contribution of genotyping for fetal sex determination in maternal serum for preimplantation genetic diagnosis of X-linked diseases]. / Intérêt du génotypage du sexe foetal dans le sang maternel dans les indications de diagnostic génétique préimplantatoire de sexe.

OBJECTIVE: Couples with a risk of transmitting X-linked diseases included in a preimplantation genetic diagnosis (PGD) center need early and rapid fetal sex determination during pregnancy in two situations. The first situation corresponds to control of embryo sexing after PGD, the second one being that of couples in PGD program having a spontaneous pregnancy. Determination of fetal sex can be achieved by karyotyping using invasive procedures such as chorionic villus sampling (CVS), amniocentesis or cordocentesis and by non-invasive procedures such as ultrasound (US) examination. CVS is the earliest invasive procedure for fetal sex determination and molecular analysis of X-linked genetic disorders during the first trimester but it is associated with a risk of fetal loss. US allows reliable fetal sex determination only during the second trimester. Recently, reliable non-invasive fetal sex determination was realized by using SRY gene amplification in maternal serum. PATIENTS AND METHODS: We report the prospective use of fetal sex determination in maternal serum in our PGD center. Management of pregnancies was performed using this non-invasive procedure in four cases of embryo sexing control and nine cases of spontaneous pregnancies in couples included in PGD program for X-linked diseases. RESULTS: Fetal sex results using SRY gene amplification on maternal serum were in complete concordance with fetal sex observed by cytogenetic analysis or US examination, as well as at birth. DISCUSSION AND CONCLUSION: This new strategy allowed rapid sex determination during the first trimester and permitted to avoid performing invasive procedures in nine pregnancies.

[Growth discordance in dichorionic twin pregnancies: risk factors, diagnosis and management]. / Conflits d'intérêt prénatals entre les jumeaux. Discordance de croissance foetale des jumeaux bichoriaux: facteurs de risque, diagnostic et prise en charge.

OBJECTIVE: To analyse the risk factors and perinatal consequences of growth discordance among dichorionic twin pregnancies. Subjects and methods. A cohort of 346 dichorionic twin pregnancies delivered at one institution between January 1996 and December 1999 was analyzed. Two groups were compared, according to the presence or absence of growth discordance (n=72 and 274, respectively). Birth weight discordance was defined as a difference of 20% or more of the weight of the heavier twin, associated with an intra-uterine growth restriction (10(th) percentile) of at least one twin. Ultrasound discordance was defined as a difference of estimated fetal weight>20%. The two groups were compared by univariate and multivariate analysis. RESULTS: The main risk factors for birth weight discordance in multivariate analysis were ovulation induction (OR=1.6 [1.0-2.4]), multifetal pregnancy reduction (OR=2.3 [1.3-4.2]), and fetal malformations (OR=2.4 [1.0-5.4]). Ultrasound shows a poor performance in predicting discordance with a sensitivity of 55.6%, a specificity of 94.2%, a positive predictive value of 71.4% and a negative predictive value of 89.0%. Birth weight discordance was associated with a poor fetal and neonatal outcome: gestational age at delivery was 34.4 weeks versus 35.4 weeks, there were more caesarean deliveries (OR=1.9 [1.3-2.8]), a higher perinatal mortality (OR=3.7 [1.6-8.5]), and more neonatal intensive care unit admissions (OR=1.8 [1.2-2.7]). Multivariate analysis shows that growth discordance is an independent risk factor for mortality but not for cerebral adverse outcome or respiratory distress syndrome. CONCLUSION: Ovulation induction and multifetal pregnancy reduction are independent risk factors for twin birth weight discordance, which carries a poor perinatal prognosis.

[Preimplantation genetic diagnosis and its psychological effects]. / Aspects psychologiques du diagnostic génétique pré-implantatoire (DPI).

OBJECTIVE: Report our psychoanalyst experience in clinical practice of pre-implantation genetic diagnosis (PGD). METHODOLOGY: Between January 1999 and July 2001, 230 couples attended a multidisciplinary PGD consultation and the information meeting that preceded it. Eighty-six of these couples met the team's psychoanalyst during a private encounter (either as couples or individually, depending on their preference). RESULTS: The development of this technique was capital for couples at risk who, prior to PGD, had often suffered a termination of pregnancy and who may have a child who was seriously ill. Whether they were carriers or affected by the disease, PGD gave them the possibility to react to their condition. Those who have repeatedly been subjected to a painful experience were given the opportunity to play an active part in their own medical history. CONCLUSION: Beyond the scientific claims of this method, PGD contributes to the healing process of distressed couples and responds to their quest for recognition. We believe that this evolution in preventative medical practice, seen as a positive development, must necessarily lead to a number of ethical as well as psychoanalytical considerations, discussions and interrogations.

[First birth after preimplantation genetic diagnosis performed on thawed embryos]. / Première naissance après un diagnostic génétique pré-implantatoire (DPI) pratiqué sur embryons décongelés.

OBJECTIVE: To report the birth of the first infant conceived after preimplantation genetic diagnosis (PGD) performed on frozen-thawed embryos in our PGD center. PATIENTS AND METHODS: Three couples (C1, C2 and C3) who had frozen embryos from a previous in vitro fertilization attempt were enrolled in our PGD program. Embryos were thawed one day before the biopsy procedure for the couples C1 and C3 and the day of the biopsy for the couple C2. The single cell genetic analysis was performed by a multiplex PCR for the couple C1 and by fluorescent in situ hybridization for the couples C2 and C3. The embryos transfers were carried out on the third or fourth day. RESULTS: Out of ten thawed embryos, eight were biopsied and five were transferred during three embryos transfers. Two biochemical and one ongoing pregnancy were obtained yielded one birth. CONCLUSIONS: PGD may be offered to couples at risk of transmission of a serious and incurable genetic disease and having frozen embryos.

[Regionalization of perinatal care and in utero referrals]. / Régionalisation des soins périnatals et transfert in utero.

Further improvement in perinatal morbidity and mortality figures implies in utero referral so high risk infants, particularly very early maturity infants, can be managed immediately in specialized centers. The success of such referrals depends directly on the coherence of the regional perinatal health care network. It has been demonstrated that the chances of survival and of sequelae-free survival are greater for very premature infants born in a center with a neonatal intensive care unit. The situation in France leaves room for improvement. Only 15% of the infants born before 33 months gestation and with birth weight under 1500 g are born in level III maternity wards equipped with a neonatal intensive care unit. The goal is 80%. We have tried to analyze the reasons behind this situation and propose ways to better organize perinatal health care.

[Preimplantation diagnosis with HLA typing: birth of the first double hope child in France]. / Diagnostic préimplantatoire avec typage HLA : naissance du premier enfant du double espoir en France.

Preimplantation genetic diagnosis (PGD) is authorized in France since 1999. After 10 years, technical results are encouraging. With the development of new technologies, our team is able to diagnosis the large majority of chromosome translocations and 75 monogenic diseases. However, PGD remains limited because of the growing augmentation of demands causing an increasing delay for the first procedure of more than 18 months. Since 2006, 19 couples asked for a PGD with HLA typing. In January 2011, 11 couples have already been included in our PGD program. The birth of the first child after PGD with HLA typing offers new perspectives of treatment for these couples.

Preimplantation genetic diagnosis for autosomal recessive polycystic kidney disease.

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.

Myotonic dystrophy: does it affect ovarian follicular status and responsiveness to controlled ovarian stimulation?

BACKGROUND: Myotonic dystrophy (MD) is characterized by myotonia, multisystemic lesions and hypogonadism. In women, the relationship between MD and infertility remains controversial. This study investigated the ovarian status and response to controlled ovarian stimulation (COS) in MD women entering our preimplantation genetic diagnosis programme. METHODS: We elected to compare MD patients with X-linked disorders (XLD) carriers, given that XLD have not been shown to affect ovarian status. On the one hand, we analysed all the cycles performed and, on the other hand, we conducted a subanalysis based on only first cycles. RESULTS: MD and XLD groups were similar with regard to women's ages, day 3 parameters, number of oocytes retrieved, embryos obtained and prevalence of top quality embryos. The day of HCG was significantly delayed and the prevalence of poor quality embryos was higher in the MD group. The subanalysis on first cycles only also showed significantly fewer mature follicles on the day of HCG in MD population. Implantation and pregnancy rates were similar in both groups; however, no pregnancy occurred at the first cycle in MD (0 out of 4), whereas 77% of pregnancies (10/13) occurred at the first attempt in XLD carriers. CONCLUSIONS: These results indicate that the responsiveness to COS was moderately hindered in MD women as compared to controls. Reassuring data about implantation and pregnancy rates support the feasibility of PGD in selected mildly affected MD women.

Multiple displacement amplification improves PGD for fragile X syndrome.

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.

[Caffey's disease with antenatal onset]. / Maladie de Caffey à début anténatal.

The authors report one case of Caffey's syndrome with antenatal onset. X-ray of the uterus, that was required because of an hydramnios, led to the diagnosis. Ultrasonography showed the fetal anomalies. Data in the literature concerning case reports of Caffey's syndrome with antenatal onset show a moderate incidence of familial cases.

Perinatal outcome and follow-up of 82 children aged 1-9 years old conceived from cryopreserved embryos.

Embryo cryopreservation is routinely used in in-vitro fertilization (IVF)-embryo transfer programmes. Yet very few studies have reported the follow-up of children conceived with frozen/thawed embryos. This study was designed to follow up the total cohort of children conceived with cryopreserved embryos in A. Béclère Hospital in Clamart, France between 1986 and 1994. We were able to study 89 children, aged 1-9 years old, out of the 93 conceived during this period (lost to follow-up: 4.3%). The prematurity rate was 14.7% for the singleton and 85.7% for the twins. Half of these premature deliveries occurred during the 36th week of amenorrhea. In all, 8% of the singleton and 28.6% of the twins were small for gestational age. At the time of the study, only three children aged 1 and 2 years old were below the 10th percentile. The total malformation rate was 3.4% when two abortions performed during the study period were added to the one (short ureter) found in our study group. The medical and surgical illness as well as principal acquisitions for children <5 years old and scholastic performance for older children did not show pathological features.