Growth development of French children born after in vitro maturation.

BACKGROUND: Several lines of evidence indicate that immature oocyte retrieval and subsequent in vitro maturation (IVM) without ovarian stimulation may be a reliable option in assisted reproductive technologies (ART). However, few outcome data are available for children born following this technique. OBJECTIVE: We assessed height and weight development of French children conceived after IVM. METHODS: All children conceived after IVM at Antoine Beclere Hospital (Clamart, France) and born between June 2003 and October 2008 (n = 38) were included in a prospective cohort study and compared with a control group of children conceived by ICSI without IVM, matched for maternal age, gestational age and singleton/twin pregnancies. Follow-up included clinical examination at one year and a questionnaire completed by parents when the children were two years old (97% follow-up rate). RESULTS: No statistical differences between IVM and control groups were found for boys. Mean weight, height and head circumference at birth were significantly greater for IVM than for ICSI girls (3.236 kg vs 2.701 kg (p = 0.03); 49 cm vs 47 cm (p = 0.01) and 34 cm vs 33 cm (p = 0.04), respectively). At one year, IVM girls remained heavier (mean weight 10.2 kg vs 8.6 kg (p = 0.001)) and taller (76 cm vs 73 cm (p = 0.03)), and there was a two-point difference in BMI between the two groups of girls (18 vs 16 (p = 0.01)). CONCLUSION: Our results in girls born after IVM should be interpreted with caution. It remains unclear whether the observed sexual dimorphism is due to IVM technology or to maternal characteristics such as underlying infertility in patients with polycystic ovary syndrome (PCOS). Further monitoring of the outcomes of these infants is required.

Mutation dependance of the mitochondrial DNA copy number in the first stages of human embryogenesis.

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.

Poor correlations in the levels of pathogenic mitochondrial DNA mutations in polar bodies versus oocytes and blastomeres in humans.

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 (R(2) = 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.

[Extending preimplantation genetic diagnosis to HLA typing: the French exception]. / Le diagnostic pré-implantatoire couplé au typage HLA: l'expérience française.

Umut-Talha, a "sibling savior", was born on 26 January 2011 at Beclère 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).

[Ten years' experience of preimplantation genetic diagnosis in Paris: remaining obstacles and potential solutions]. / Diagnostic pré-implantatoire: dix ans d'expérience en région parisienne: impasse actuelle et solutions à venir.

Preimplantation genetic diagnosis (PGD) has been authorized in France since 1999. Encouraging results have been obtained during the past 10 years in our Paris center, where 832 patients have undergone 1056 IVF-PGD procedures. With the advent of new techniques for the identification of genetic disease markers, our center can now offer PGD procedures for aneuploidy and 75 single-gene diseases. New indications for PGD have also been developed, such as mitochondrial DNA diseases, amyloid neuropathy, pulmonary arterial hypertension, and HLA typing The implantation rate is currently 29,6% and, by 31 December 2009, 151 healthy babies had been born. Unfortunately, demand for PGD procedures far outstrips available technical capacity, and the waiting period is longer than 18 months. Increased funding is urgently needed

Segregation of mtDNA throughout human embryofetal development: m.3243A>G as a model system.

Mitochondrial DNA (mtDNA) mutations cause a wide range of serious diseases with high transmission risk and maternal inheritance. Tissue heterogeneity of the heteroplasmy rate ("mutant load") accounts for the wide phenotypic spectrum observed in carriers. Owing to the absence of therapy, couples at risk to transmit such disorders commonly ask for prenatal (PND) or preimplantation diagnosis (PGD). The lack of data regarding heteroplasmy distribution throughout intrauterine development, however, hampers the implementation of such procedures. We tracked the segregation of the m.3243A>G mutation (MT-TL1 gene) responsible for the MELAS syndrome in the developing embryo/fetus, using tissues and cells from eight carrier females, their 38 embryos and 12 fetuses. Mutant mtDNA segregation was found to be governed by random genetic drift, during oogenesis and somatic tissue development. The size of the bottleneck operating for m.3243A>G during oogenesis was shown to be individual-dependent. Comparison with data we achieved for the m.8993T>G mutation (MT-ATP6 gene), responsible for the NARP/Leigh syndrome, indicates that these mutations differentially influence mtDNA segregation during oogenesis, while their impact is similar in developing somatic tissues. These data have major consequences for PND and PGD procedures in mtDNA inherited disorders.

Five years' experience of preimplantation genetic diagnosis in the Parisian Center: outcome of the first 441 started cycles.

OBJECTIVE: To investigate the evolution of techniques and strategies and to evaluate the results of preimplantation genetic diagnosis (PGD) from January 2000 to December 2004 in chromosomal, monogenic and mitochondrial DNA disorders treated at our institution. DESIGN: Retrospective study. SETTING: Single French Parisian PGD center. PATIENT(S): Patients at risk of transmitting a serious genetic disorder to their offspring. INTERVENTION(S): 171 couples enrolled in the program undergoing stimulated and frozen embryo replacement cycles with PGD. MAIN OUTCOME MEASURE(S): Results of the 441 first PGD cycles performed for various genetic conditions. RESULT(S): During 5 years, 416 stimulation and 25 frozen embryo replacement cycles were started, among which 52 clinical and 47 ongoing pregnancies occurred. In stimulation cycles, the overall ongoing pregnancy rate was 24% per embryo transfer, 11% per started cycle, and 27% per couple. The implantation rate was 16%. CONCLUSION(S): These encouraging results demonstrate that PGD might be considered as a valid alternative to prenatal diagnosis. Nevertheless, couples referred for PGD must be selected and counseled appropriately, considering the complexity of the treatment and the relatively low take-home baby rate.

[Prenatal and preimplantation genetic diagnosis: decision tree, new practices?]. / Diagnostic prénatal et diagnostic pré-implantatoire: arbre décisionnel, nouvelles pratiques?

Preimplantation genetic diagnosis (PGD) purpose is to assess the genetic status of 3 day-old embryos. PGD offers thus to couples "at-risk" of a genetic disorder an earlier option to prenatal diagnosis (PND). At the beginning, PGD's indications, patients and law were very closed to PND, but PGD specificities are gradually raising. Particularly, indications vary considerably in countries where the absence of law authorizes all the practices. Some of these applications are moreover raising serious ethical issues. Even in France, where this activity is particularly supervised, the recent modification to the law marks this evolution.

Improved single-cell protocol for preimplantation genetic diagnosis of spinal muscular atrophy.

OBJECTIVE: To develop and validate a simple and reliable single-cell analysis protocol for the preimplantation genetic diagnosis (PGD) of spinal muscular atrophy (SMA). DESIGN: Molecular tests based on specific enzymatic digestion have already been described for SMA diagnosis. We modified the amplified DNA fragments so as to introduce a novel restriction site that provides an internal control for the completeness of the digestion. SETTING: The genetics and reproduction departments of two teaching hospitals. PATIENT(S): Six informed couples at risk of transmitting SMA. INTERVENTION(S): All patients underwent standard procedures associated with intracytoplasmic sperm injection. MAIN OUTCOME MEASURE(S): Improvement of SMA diagnostic efficiency and accuracy on single cell. RESULT(S): One hundred fifty lymphocytes were analyzed with our protocol. One hundred percent diagnostic accuracy was achieved from both homozygous normal and SMN1-deleted leukocytes. Successful molecular analysis was achieved for 36 of 42 biopsied embryos (86%). Twenty-five normal embryos were transferred, but no pregnancy was achieved. CONCLUSION(S): We developed an improved protocol for PGD of SMA that is simple, robust, and accurate; unfortunately, no pregnancies were achieved for any of the six patients who have undergone PGD in the program thus far.

Single cell co-amplification of polymorphic markers for the indirect preimplantation genetic diagnosis of hemophilia A, X-linked adrenoleukodystrophy, X-linked hydrocephalus and incontinentia pigmenti loci on Xq28.

Preimplantation genetic diagnosis (PGD) first consisted of the selection of female embryos for patients at risk of transmitting X-linked recessive diseases. Advances in molecular biology now allow the specific diagnosis of almost any Mendelian disease. For families with an identified X-linked recessive disease-causing mutation, non-specific diagnosis by sex identification can be considered as a sub-standard method, since it involves the unnecessary disposal of healthy male embryos and reduces success rate by diminishing the pool of embryos eligible for transfer. The most telomeric part of the X-chromosome long arm is a highly gene-rich region encompassing disease genes such as haemophilia A, X-linked adrenoleukodystrophy, X-linked hydrocephalus and incontinentia pigmenti. We developed five single-cell triplex amplification protocols with microsatellite markers DXS1073, DXS9901 (BGN), G6PD, DXS1108, DXS8087 and F8C-IVS13 located in this Xq terminal region. These tests allow the diagnosis of all diseases previously mentioned providing that the genetic material allowing the identification of the morbid allele can be obtained. The choice of the microsatellite set to use depends on the localisation of the gene responsible for the diagnosed pathology and on the informativity of the markers in particular families. Single-cell amplification efficiency was assessed on single lymphocytes. Amplification rate of the different markers ranged from 89-97% with an allele drop out rate of 2-19%. So far PGD has been carried out for three carrier females at risk of transmitting X-linked adrenoleukodystrophy, X-linked hydrocephalus and hemophilia A. The latter one is now pregnant.

Embryo reduction and birth weight discordance in dichorionic twins.

BACKGROUND: Twin birth weight discordance is associated with a poor perinatal outcome. The aim of this study was to analyse the risk factors of growth discordance among dichorionic twin pregnancies. METHODS: A cohort of 346 dichorionic twin pregnancies delivered at one perinatal centre between January 1996 and December 1999 was analysed. Two groups were created, according to the presence or absence of intra-pair birth weight discordance (n = 75 and 271 respectively). Birth weight discordance was defined as a difference of >/=20% of the weight of the heavier twin. The two groups were compared by uni- and multivariate analysis, with regard to the woman's characteristics, risk factors for growth restriction or discordance, and outcome of pregnancy. RESULTS: Pregnancies with birth weight discordance had a poor outcome compared with pregnancies without discordance, with a 4-fold increase in neonatal mortality. The rate of iatrogenic embryo reduction was significantly higher in discordant pregnancies (14.7 versus 6.6%, P = 0.03). The risk of birth weight discordance was increased with a larger starting number of embryos before reduction [20.2% (64/317), 28.6% (6/21), 57.1% (4/7) and 100% (1/1) respectively, for an initial number of two (no reduction), three, four, and five embryos, P = 0.02]. In multivariate analysis, embryo reduction was the only significant risk factor for the occurrence of birth weight discordance [adjusted odds ratio (OR) = 2.3 (1.0-5.2)]. CONCLUSIONS: Birth weight discordance carries a poor perinatal outcome. Embryo reduction is an independent risk factor for birth weight discordance in dichorionic twins. This finding emphasises the need for better control of assisted reproductive technology in order to avoid high-order multiple pregnancies.

[Preimplantation genetic diagnosis: update of the Parisian group]. / Le diagnostic génétique préimplantatoire: premier bilan du groupe parisien.

To report the birth of the first fourteen infants conceived after preimplantation genetic diagnosis (PGD) in our unit. Fifty-nine couples were enrolled between January 2000 and July 2001. They had a total of 71 oocyte pick-up cycles. The collected oocytes were inseminated by intracytoplasmic sperm injection. The resulting embryos were biopsied on the third day of development and the genetic analysis was performed on the same day. Most of the embryo transfers were carried out on the fourth day. The 71 oocyte pick-up cycles yielded 872 oocytes of which 731 were suitable for intracytoplasmic sperm injection. Among the 505 embryos obtained, 421 embryos were biopsied and genetic diagnosis was performed for 312 (74%) of these 127 embryos were transferred during the course of 58 transfer procedures. There were 18 biochemical and 12 ongoing (7 singles, 4 twins and 1 triple) pregnancies. Sixteen infants have been born and 2 are expected. PGD has gained a place among the choices offered to couples at risk of transmission of a serious and incurable genetic disease.

Clinical applications of fetal sex determination in maternal blood in a preimplantation genetic diagnosis centre.

BACKGROUND: Couples with a risk of transmitting X-linked diseases who are included in a preimplantation genetic diagnosis (PGD) programme need early and rapid fetal sex determination in two situations. The first situation is for the control of embryo sexing after PGD and the second situation is for those couples having a spontaneous pregnancy before the start of their PGD cycle. Among invasive techniques, chorionic villus sampling is the earliest 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. Non-invasive procedures such as ultrasound examination allow reliable fetal sex determination only during the second trimester. Reliable fetal sex determination can now be realised by using SRY gene amplification in maternal blood. METHODS AND RESULTS: We report the use of fetal sex determination from maternal serum as a diagnostic tool for the control of embryo sexing (two cases) and to manage spontaneous pregnancies in couples included in a PGD programme for X-linked diseases (five cases). Fetal sex determination using SRY gene amplification in maternal serum were in complete concordance with fetal sex observed by cytogenetic analysis or ultrasound examination and at birth. This novel strategy allowed the PGD results to be controlled precociously and avoided the performance of invasive procedures in four cases of female fetus. CONCLUSIONS: This rapid fetal sex determination during the first trimester provides advantages to both clinicians and patients in a PGD centre.

Birth of healthy female twins after preimplantation genetic diagnosis of cystic fibrosis combined with gender determination.

Two healthy sisters with a familial history of mental retardation were referred to our centre for preimplantation genetic diagnosis (PGD). Their two brothers showed severe mental retardation. The molecular basis for their disorder could not be identified, but one of the sisters and the mother presented a highly skewed pattern of X-inactivation reinforcing the likelihood of an X-linked mode of inheritance. Both sisters requested PGD to avoid the abortion of potentially affected male fetuses. PGD for sex by fluorescent in-situ hybridization was carried out for the first sister and resulted in the birth of a female child. The second sister and her partner, whose niece had cystic fibrosis (CF), were tested for CF mutations, and were both found to be deltaF508 heterozygous. We developed an efficient single cell PCR protocol for the simultaneous amplification of the CF (deltadeltaF508) locus as well as the X-linked amelogenin gene and its highly homologous pseudogene on the Y chromosome. Two PGD cycles were carried out to screen against male and deltaF508 homozygous deleted embryos. In each case several embryos could be selected for transfer and the second cycle resulted in a twin pregnancy followed by the birth of two healthy female infants.