Patricia Fauque

Specific epigenetic alterations of IGF2 - H19 locus in spermatozoa from infertile men

DNA methylation marks, a key modification of imprinting, are erased in primordial germ cells and sex specifically re-established during gametogenesis. Abnormal epigenetic programming has been proposed as a possible mechanism compromising male fertility. We analysed by pyrosequencing the DNA methylation status of 47 CpGs located in differentially methylated regions (DMRs), the DMR0 and DMR2 of the IGF2 gene and in the 3rd and 6th CTCF-binding sites of the H19 DMR in human sperm from men with normal semen and patients with teratozoospermia (T) and/or oligo-astheno-teratozoospermia (OAT). All normal semen samples presented the expected high global methylation level for all CpGs analysed. In the teratozoospermia group, 11 of 19 patients presented a loss of methylation at variable CpG positions either in the IGF2 DMR2 or in both the IGF2 DMR2 and the 6th CTCF of the H19 DMR. In the OAT group, 16 of 22 patients presented a severe loss of methylation of the 6th CTCF, closely correlated with sperm concentration. The methylation state of DMR0 and of the 3rd CTCF was never affected by the pathological status of sperm samples. This study demonstrates that epigenetic perturbations of the 6th CTCF site of the H19 DMR might be a relevant biomarker for quantitative defects of spermatogenesis in humans. Moreover, we defined a methylation threshold sustaining the classification of patients in two groups, unmethylated and methylated. Using this new classification of patients, the observed intrinsic imprinting defects of spermatozoa appear not to impair significantly the outcome of assisted reproductive technologies.

Assisted Reproductive Technology affects developmental kinetics, H19 Imprinting Control Region methylation and H19 gene expression in individual mouse embryos

BackgroundIn the last few years, an increase in imprinting anomalies has been reported in children born from Assisted Reproductive Technology (ART). Various clinical and experimental studies also suggest alterations of embryo development after ART. Therefore, there is a need for studying early epigenetic anomalies which could result from ART manipulations, especially on single embryos. In this study, we evaluated the impact of superovulation, in vitro fertilization (IVF) and embryo culture conditions on proper genomic imprinting and blastocyst development in single mouse embryos.In this study, different experimental groups were established to obtain embryos from superovulated and non-superovulated females, either from in vivo or in vitro fertilized oocytes, themselves grown in vitro or not. The embryos were cultured either in M16 medium or in G1.2/G2.2 sequential medium. The methylation status of H19 Imprinting Control Region (ICR) and H19 promoter was assessed, as well as the gene expression level of H19, in individual blastocysts. In parallel, we have evaluated embryo cleavage kinetics and recorded morphological data.ResultsWe show that:1. The culture medium influences early embryo development with faster cleavage kinetics for culture in G1.2/G2.2 medium compared to M16 medium.2. Epigenetic alterations of the H19 ICR and H19 PP are influenced by the fertilization method since methylation anomalies were observed only in the in vitro fertilized subgroup, however to different degrees according to the culture medium.3. Superovulation clearly disrupted H19 gene expression in individual blastocysts. Moreover, when embryos were cultured in vitro after either in vivo or in vitro fertilization, the percentage of blastocysts which expressed H19 was higher in G1.2/G2.2 medium compared to M16.ConclusionCompared to previous reports utilizing pools of embryos, our study enables us to emphasize a high individual variability of blastocysts in the H19 ICR and H19 promoter methylation and H19 gene expression, with a striking effect of each manipulation associated to ART practices. Our results suggest that H19 could be used as a sensor of the epigenetic disturbance of the utilized techniques.

Modulation of imprinted gene network in placenta results in normal development of in vitro manipulated mouse embryos

Genomic imprinting regulates the expression of a group of genes monoallelically expressed in a parent-of-origin specific manner. Allele-specific DNA methylation occurs at differentially methylated regions (DMRs) of these genes. We have previously shown that in vitro fertilization and embryo culture result in methylation defects at the imprinted H19-Igf2 locus at the blastocyst stage. The current study was designed to evaluate the consequences of these manipulations on genomic imprinting after implantation in the mouse. Blastocysts were produced following three experimental conditions: (i) embryos maintained in culture medium after in vivo fertilization or (ii) in vitro fertilization and (iii) a control group with embryos obtained after in vivo fertilization and timed mating. Blastocysts were all transplanted into pseudopregnant females. Embryos and placentas were collected on day 10.5 of development. DNA methylation patterns of the H19, Igf2, Igf2r and Dlk1-Dio3 DMRs were analyzed by quantitative pyrosequencing. In contrast to blastocyst stage, methylation profiles were normal both in embryonic and placental tissues after in vitro fertilization and culture. Expression of a selected set of imprinting genes from the recently described imprinted gene network (IGN) (including Igf2 and H19) was analyzed in placental tissues by quantitative RT-PCR. Placentas obtained after in vitro fertilization and embryo culture displayed significantly disturbed levels of H19 and Igf2 mRNA, as well as of most other genes from the IGN. As embryos were phenotypically normal, we hypothesize that the modulation of a coordinated network of imprinted genes results in a compensatory process capable of correcting potential dysfunction of placenta.

Clinical data and parenthood of 63 infertile and Y-microdeleted men

OBJECTIVE To collect follow-up data for infertile men with Y microdeletion. DESIGN Retrospective, observational survey. SETTING Multicenter IVF units associated with genetics laboratories. PATIENT(S) Sixty-three patients with Y microdeletion. INTERVENTION(S) Karyotype analysis, Y microdeletion screening, and assisted reproductive technology. MAIN OUTCOME MEASURES Medical history, karyotype, nature of the AZF deletion, semen parameters, testis biopsy results, choice of assisted reproductive technology, and results of intracytoplasmic sperm injection (ICSI). RESULTS Abnormal karyotypes were found in 8 men (12.7%), who were azoospermic except 1. Of these 8 men, 5 presented a combined AZFb+c deletion, and 3 had a deletion in AZFc only. Most men (39 of 63) were azoospermic, 3 were cryptoazoospermic, and 19 had extreme oligozoospermia (sperm concentration </=1.10(6)/mL). Sperm concentration above 1.10(6)/mL was found for 2 men (3.1%). A testis biopsy was performed in 27 azoospermic men, resulting in positive sperm extraction in 6 cases. To date, 42 ICSI cycles with either testicular (n = 5) or ejaculated spermatozoa (n = 37) have been carried out in 23 couples with male partners with AZFc deletion. Eighteen clinical pregnancies were obtained, leading to the birth of 14 babies. Donor insemination had been chosen by 28 couples, leading to the birth of 9 children. CONCLUSION Karyotype analysis should be systematically performed in Y microdeleted men. Intracytoplasmic sperm injection can be offered to half of AZFc-deleted patients, providing real opportunities to have a child.

Artificial versus stimulated cycles for endometrial preparation prior to frozen–thawed embryo transfer

The objective of this study was to compare the implantation rate, pregnancy rate and endometrial thickness of frozen-thawed embryo transfers using endometrial preparation with either an artificial cycle or stimulated cycle. This was a prospective randomized trial at a single academic IVF centre. Seventy-seven patients undergoing artificial cycles received oral oestradiol; patients with endometrium < 7 mm on day 9-10 were switched to vaginal oestradiol. Eighty-six patients undergoing stimulated cycles received recombinant FSH followed by human gonadotrophin hormone injection. Vaginal progesterone was begun 2 or 3 days prior to embryo transfer. There was no difference in implantation rate (8.5% versus 7.3%), pregnancy rate (16% versus 13%), cancellation rate (both 23%) or endometrium thickness (8.7 +/- 1.1 mm versus 8.7 +/- 1.0 mm) between artificial and stimulated cycles. Stimulated cycles had a higher incidence of thin endometrium (27% versus 5%, P < 0.01). In artificial cycles, patients switched to vaginal oestradiol had improved pregnancy rate (31%) versus patients who received oral oestradiol alone (13%) (P = 0.05). It is concluded that artificial and stimulated cycles produce comparable pregnancy rates, implantation rates, cancellation rates and endometrial thickness, although stimulated cycles have a higher incidence of thin endometrium. Vaginal oestradiol supplementation improved implantation rates.

HIV-positive patients undertaking ART have longer infertility histories than age-matched control subjects

OBJECTIVE To review 5 years of assisted reproductive treatments (ART) provided to couples affected by human immunodeficiency virus (HIV). DESIGN Age-matched cohort study. SETTING University-based tertiary center. PATIENT(S) Couples in whom the male (n = 87), female (n = 57), or both (n = 17) partners were HIV infected. The first ART cycle was compared with three sets of age-matched control subjects (3-to-1) which included 261, 171, and 51 couples, respectively. INTERVENTION(S) ART in HIV-infected couples and age-matched controls. MAIN OUTCOME MEASURE(S) Infertility duration and ART outcome. RESULT(S) When initiating ART, all three HIV-infected groups had longer infertility histories, computed from when conception was attempted or infertility diagnosed, compared with noninfected age-matched control subjects. Outcome, however, was not different when only the male or female partner was infected, though with a trend toward higher cancellation and lower pregnancy rates. When both partners were HIV infected, cancellation were higher and pregnancy rates lower (12% versus 41.2%), than in age-matched control subjects. CONCLUSION(S) Our data showed longer infertility histories in all HIV-infected couples when undertaking their first ART. Outcome, however, was not altered when only one partner--male or female--was HIV infected. Efforts should therefore aim at assuring that HIV-infected couples access ART as promptly as their noninfected counterparts.

Zona pellucida from fertilised human oocytes induces a voltage-dependent calcium influx and the acrosome reaction in spermatozoa, but cannot be penetrated by sperm

BackgroundThe functions of three zona glycoproteins, ZP1, ZP2 and ZP3 during the sperm-zona pellucida (ZP) interaction are now well established in mice. The expression of an additional zona glycoprotein, ZPB/4, in humans, led us to reconsider the classical mouse model of gamete interaction. We investigated the various functions of human ZP (hZP) during the interaction of spermatozoa with fertilised and unfertilised oocytes.ResultsThe hZP of fertilised oocytes retained their ability to bind sperm (albeit less strongly than that from unfertilised oocytes), to induce an intraspermatic calcium influx through voltage-dependent channels similar to that observed with hZP from unfertilised oocytes and to promote the acrosome reaction at a rate similar to that induced by the ZP of unfertilised oocytes (61.6 ± 6.2% vs60.7 ± 9.1% respectively). Conversely, the rate of hZP penetrated by sperm was much lower for fertilised than for unfertilised oocytes (19% vs 57% respectively, p < 0.01). We investigated the status of ZP2 in the oocytes used in the functional tests, and demonstrated that sperm binding and acrosome reaction induction, but not ZP penetration, occurred whether or not ZP2 was cleaved.ConclusionThe change in ZP function induced by fertilisation could be different in human and mouse species. Our results suggest a zona blocking to polyspermy based at the sperm penetration level in humans.

Multiplying recipients paired with oocyte donors optimizes the use of donated oocytes

OBJECTIVE To review donor-egg assisted reproductive technology (ART) activity using young fertile donors (<37 years of age) paired with multiple recipients. DESIGN Age-matched cohort study. SETTING Tertiary ART center at Cochin Hospital, Paris. PATIENT(S) A total of 125 oocyte donors and 361 age-matched control subjects. Donated oocytes were attributed to 163 different recipients undertaking 258 transfer cycles. INTERVENTION(S) Donor-egg and regular ART. MAIN OUTCOME MEASURE(S) Controlled ovarian hyperstimulation (COH) outcome-oocytes provided-was compared in donors and control subjects. Clinical pregnancy (cPR), ongoing pregnancy (oPR), and implantation (IR) rates per transfer in recipients were compared with age-matched controls. IRs were analyzed in the various recipients as a function of the number of oocytes harvested. RESULT(S) COH outcome was similar in donors and control subjects. cPR (37.5%), oPR (28.4%), and IR (24.4%) were slightly but significantly lower in donor-egg recipients compared with control subjects (44.9%, 37.4%, and 31.8%, respectively). More embryos (average +2.06) were transferred fresh and fewer frozen. In recipients, IRs were independent from the number of oocytes received in the donor. CONCLUSION(S) Multiplying recipients paired with oocyte donors slightly lowered per-transfer outcome, but enabled more (average +2.06) embryos to be transferred fresh.

Optimal timing for oocyte denudation and intracytoplasmic sperm injection.

Objectives. To analyze the impact of oocyte denudation and microinjection timings on intracytoplasmic sperm injection (ICSI) outcomes. Study Design. We included ICSI cycles with the following parameters: rank 1 or 2, female age <36 years, male factor infertility, long protocol using GnRH agonist and rFSH for ovarian stimulation, and use of freshly ejaculated sperm (n = 110). Several ICSI parameters were analyzed according to the time between oocyte retrieval and denudation (T 1) and the time between denudation and ICSI (T 2) using a statistical logistic regression analysis. Results. Neither T 1 nor T 2 had a significant influence on the Metaphase II (MII) rate but the fertilisation rate (FR) showed a significant improvement when T 1 was longer (optimal results at T 1 = 3 hours) while FR significantly decreased with the increase of T 2. Optimal implantation (IR) and pregnancy (PR) rates were obtained when T 1 was around 2 hours. Conclusion. Incubation of oocytes around 2 hours between retrieval and denudation may not increase MII rate but appears to lead to the optimal combination of FR and IR.

From ultrastructural flagellar sperm defects to the health of babies conceived by ICSI

The objective of this retrospective study was to describe a population of patients displaying impaired sperm motility due to ultrastructural flagellar defects and to analyse the intracytoplasmic sperm injection (ICSI) results and neonatal outcomes in this population. The fertilization rate, embryo quality, clinical pregnancy rate, implantation rate, birth rate and perinatal health of babies were determined. Patients (n = 20) were divided into seven categories according to ultrastructural flagellar abnormalities. The type of flagellar abnormality significantly affected the fertilization rate (P <0.025). Two types of flagellar abnormalities showed slower early embryo cleavage kinetics (P <0.001) when axonemal central structures and periaxonemmal columns were abnormal or absent. Of 53 ICSI attempts, 14 resulted in clinical pregnancies (26.4% per cycle) after fresh and frozen embryo transfer. Three (21.4%) of these pregnancies ended in miscarriages and, in the remaining, 12 infants were born (7.2% of transferred embryos). The outcomes differed according to the ultrastructural defect. This study demonstrates that a high proportion of patients could father a child (45.0%). However, flagellar abnormalities appear to influence ICSI results and fetal development.