[Low circulating anti-Müllerian hormone and normal follicle stimulating hormone levels: which prognosis in an IVF program?]. / Pronostic en fécondation in vitro des patientes ayant une AMH basse et une FSH normale.

OBJECTIVE: To evaluate the results of controlled ovarian hyperstimulation (COH) for IVF in patients with low anti-Müllerian hormone (AMH) and normal basal follicle stimulating hormone (FSH) and Estradiol levels (≤50 pg/mL). PATIENTS AND METHODS: A retrospective cohort study including 704 patients for whom AMH and FSH levels (measured between days 3 and 5 of the menstrual cycle) were available, is performed in the IVF center at the Sèvres Hospital (France). Three groups are designed and analyzed: group 1 with AMH less or equal to 2 ng/mL and FSH less or equal to 10 mUI/mL (study group), Group 2 with AMH greater than 2 ng/mL and FSH less or equal to 10 mUI/mL (control group) and Group 3 with AMH less or equal to 2 ng/mL and FSH greater than 10 mUI/mL (group with decreased ovarian reserve). RESULTS: IVF outcome for patients from the study group is significantly worse than that of the second but not than that of the third group. In the first group, the number of retrieved oocytes, the number of total obtained embryos, the clinical pregnancy rate and the live birth rate are significantly lower than in the second group; moreover, there are more cancelled cycles because of poor response in the first group. There is no difference with the third group. DISCUSSION AND CONCLUSIONS: This study shows that women with a low baseline AMH have a similar response to COH to the poor responders patients with a decreased ovarian reserve revealed by an elevated FSH level. Thus, when a woman undergoing IVF cycle presents a low AMH, she might be considered as a poor responder patient regardless of the FSH level and, although the clinical pregnancy rate is not so disappointing (18%), the couple should be informed of a higher risk of cycle cancellation.

[Flow Index Vascularization dynamics in the preovulatory phase of controlled ovarian hyperstimulation: profile and relationship with conception rate]. / Dynamique de l'index de vascularisation et de flux dans la phase préovulatoire de l'hyperstimulation ovarienne contrôlée. Relation avec le taux de conception.

OBJECTIVES: To study the evolution of subendometrial vascularization flow index (VFI) in controlled ovarian hyperstimulation (COH+IIU) cycles and compare its dynamic changes in pregnant and non pregnant women. PATIENTS AND METHODS: This is a retrospective study on 61 couples. To determine the profile of IVF just before ovulation, patients had 3D-Power Doppler angiography (3D-PDA) in this precise preovulatory phase. RESULTS: We observed a decreasing profile of VFI in 52% (32/61) of the main group cycles, in 45% (14/31) of the subgroup with spontaneous peak of LH (luteinizing hormone), and in 60% (18/30) of the hCG (human chorionic gonadotropin) trigger subgroup. There were ten conceptions, (10/61, 16.4%), including eight (8/32, 30%) that were associated with a decreasing profile of VFI versus two (2/29, 7%) that were associated with an increasing profile (NS). In the detection of LH peak subgroup, we observed five conceptions (5/14, 36%) associated with a decreasing profile versus one conception (1/17, 6%) associated with an increasing profile of VFI (OR: 8.3; confidence interval [CI] 95%: 0.8-444; P=0.06 in favor of a of decreasing VFI profile). In the hCG subgroup, there were three conceptions in the decreasing VFI profile (3/18, 17%) and one conception with increasing VFI (1/12, 17%, P=0.6). DISCUSSION AND CONCLUSION: In controlled ovarian hyperstimulation, a decreasing VFI profile is more common compared to an increasing one. Such au profile seems to be associated with a higher conception rate in cycles with spontaneous LH surge.

[Teratozoospermia, myth or reality? A 10-years retrospective study on 101404 consecutive sperm samples]. / Tératozoospermie: mythe ou réalité? Étude d'une cohorte de 101 404 examens de sperme.

OBJECTIVES: Several studies have reported a time-related decline in semen quality. In this context, 101,404 spermograms-spermocytograms performed in a single andrology laboratory from January 2000 to December 2009 were investigated retrospectively. Variations of sperm parameters were analyzed over the years. PATIENTS AND METHODS: For each semen sample, age at semen collection, duration of sexual abstinence, volume of seminal fluid, pH, concentration of spermatozoa, percentages of overall motile and progressive motile sperm, percentage of morphologically abnormal spermatozoa (according to David's classification) and amplitude of lateral head displacement (ALH) were analyzed. For each parameter, the mean value per year was determined. To examine trends over time the statistical tests used were analyses of variance and correlation studies. RESULTS: Data showed an increasing age of patients from 36.5 years in 2000 to 37.2 years in 2009. The semen concentration was 63.5 millions/mL in 2000 and 63.6 millions/mL in 2009 with a slight fall from 2001 to 2008. The vitality continuously increased from 67.8% in 2000 to 73.5% in 2009 as well as the overall motility (from 37.7% to 39.2%) and the progressive motility (from 34.6% to 36.2%). The percentage of morphologically abnormal spermatozoa continuously increased from 64.5% to 84.2% (r=0.43, P<0.001). DISCUSSION AND CONCLUSION: Contrary to a variety of works, our very large study of semen analysis did not show a real decline in semen WHO parameters during a 10-year period, except for the percentage of morphologically abnormal spermatozoa, which dramatically increased over the years.

Effect of growth hormone on oocyte competence in patients with multiple IVF failures.

In a preliminary, unpublished randomized study conducted in 2000 on 39 patients, including a placebo group, it was observed that the addition of growth hormone (GH) during ovarian stimulation in patients with poor-quality oocytes increased the pregnancy rate. However, the results were not statistically significant due to the small number of patients in each group. A protocol with 8 IU GH was tested in 291 patients with three or more previous failures of embryo transfer for no clearly identifiable reasons. The analysis was restricted to patients receiving either recombinant FSH or human menopausal gonadotrophin (HMG) (n = 245). They were compared retrospectively to all patients with three or more failures during the same period of time but stimulated only with recombinant FSH or HMG, without GH, in an observational study design. Co-stimulation with GH gave better results in terms of number of oocytes collected and embryos obtained. Pregnancy rate per retrieval was higher than in the control group (25.7% versus 18.2%, P < 0.01) and reached a level similar to the one observed in the study centre for the whole population. Ovarian stimulation associated with GH can be proposed for patients with a history of repeated assisted reproduction failures. An improvement of cytoplasmic competence is proposed as an explanation.

[Teratozoospermia at the time of intracytoplasmic morphologically selected sperm injection (IMSI)]. / La tératozoospermie à l'heure de l'intracytoplasmic morphologically selected sperm injection (IMSI).

Until now, the morphological sperm analysis (spermocytogram) allows to define sperm normality, but the relationship between sperm morphology and fertility is not yet assessed. Although several studies do not report any relationship between abnormal sperm morphology and ICSI results, nevertheless, the success rate of ICSI sems to be dependent on injected sperm morphological aspect. Detailed morphological sperm examination (especially sperm head) at high magnification (from x 6600 to x 12500) (MSOME) in real time allows to select the best spermatozoa before oocyte injection (IMSI). In some cases, implantation and ongoing pregnancy rates were improved with this sperm selection method. Ultramorphologic criteria were established and the most predictive factor of sperm quality is the presence of vacuoles in the sperm head. Those vacuoles appear to be related to DNA damage (fragmentation and/or denaturation) and affect embryo development. To standardize those observations, several authors tried to establish sperm MSOME classifications in order to be used in routine and to replace the conventional spermocytogram in the next future.

[Causes and clinical implications of sperm DNA damages]. / Causes et implications cliniques des altérations de l'ADN des spermatozoïdes.

Numerous recent studies involve DNA damages associated with poor fertilization rates, early embryo development defect, and poor quality of conceptus following Assisted Reproductive Technologies (ART). The authors denounce a particularly high rate of miscarriages and childhood cancer or dominant genetic mutations such as achondroplasia, Apert syndrome or aberrant gene imprinting such as Angelman and Beckwith Wiedeman syndromes. Gametes DNA defects have numerous origins which are difficult to determine; they are known to involve hypomethylation, oxydative stress and environmental factors.(adducts formation). DNA defect is also linked to a more or less delayed apoptotic phenomenon. Exposure to radiations or radiofrequency electromagnetic emissions can also induce DNA alterations into the spermatozoa of infertile men. Although the underlying mechanisms are unclear, these DNA defects have obvious consequences on reproduction of mammalian species. Detection of such anomalies before ART, are an important step toward developing strategies for clinical management according to the aetiology.

[Sperm quality and selection]. / Qualité et sélection du spermatozoïde.

Selection of a live and morphologically normal spermatozoa is a fundamental stage in ICSI success because of its potential effects on early and late embryo development. In addition to the routine tests such as the spermogram and the spermocytogram, a number of tests have been developed for this purpose : the hyaluronic acid test, which measures the rate of DNA fragmentation using TUNEL (% of fragmented DNA) or electrophoresis separation of SPZ, SPZ evaluation using the FISH method, MSOME (motile sperm organelle morphology examination) techniques, et IMSI (intracytoplasmic morphologically selected sperm injection), which can evaluate the nuclear vacuoles, etc.

[Quality of follow-up of children]. / Qualité du suivi des enfants.

A nonmedical follow-up of the children born through ART is recommended by guidelines to evaluate any consequences, whether beneficial or harmful, of their mode of conception. Governed by medical confidentiality, according to the CNIL, this follow-up should not be done only through a questionnaire but by a direct relation with a healthcare professional. FOLLOW UP, used in 27 ART centers in France, is an example of this follow-up.

[Indications for IMSI]. / Les indications de l'IMSI.

ICSI failures (essentially fertilization and implantation failures) can be due to the quality of the oocytes, the spermatozoon injected and its manipulation, uterine factors and paternal factors, such as DNA fragmentation of the spermatozoa. Pre-selection of the spermatozoon is the crucial phase for ensuring successful ICSI. The characteristics of the sperm must be checked under the microscope at a magnification of x 6600 as certain abnormalities, in particular nuclear vacuoles, cannot be seen at x 300. In our centre, we conducted a study on 72 patients which showed that with more than 30% of fragmented DNA, neither implantation or birth was achieved using ICSI. Performing IMSI on the same patients allowed us to obtain implantation rates of 17.4% with 30 to 40 % of fragmented DNA and of 33.3% above 40%. Births were achieved in 17.4% and 28.6% of cases respectively. IMSI has the advantage of allowing extremely careful selection of the spermatozoon microinjected, so that it is of normal shape, with the least vacuoles possible and a low DNA fragmentation rate. It is, however, a complicated technique that cannot be routinely performed.

Embryos with high implantation potential after intracytoplasmic sperm injection can be recognized by a simple, non-invasive examination of pronuclear morphology.

Embryos are conventionally selected for transfer based on the evaluation of the cleavage speed and extent of blastomere fragmentation. Here we examined whether the predictive value of these criteria, as indicators of the chance of embryo implantation, can be further potentiated by adding previously described criteria reflecting the regularity of pronuclear development. In a group of embryos selected for transfer in 380 fresh embryo transfer cycles according to the conventional criteria, the transfer of only those embryos that developed from zygotes judged normal at the pronuclear stage (pattern 0) gave significantly higher pregnancy (44.8%) and implantation (30.2%) rates compared with the pregnancy (22.1%; P < 0. 05) and implantation rates (11.2%; P < 0.001) for the transfers of only those embryos that developed from zygotes judged abnormal (non-pattern 0). The transfer of only one pattern 0 embryo was sufficient for the optimal chance of pregnancy (no differences in pregnancy rates after transfer of one, two or three pattern 0 embryos), whereas the transfer of two pattern 0 embryos mostly resulted in a twin pregnancy. The inclusion of the criteria based on pronuclear morphology can thus lead to the application of a single embryo transfer policy and optimize the selection of embryos for transfer and cryopreservation.

Differentiation of spermatogenic cells during in-vitro culture of testicular biopsy samples from patients with obstructive azoospermia: effect of recombinant follicle stimulating hormone.

In-vitro differentiation of spermatogenic cells is a potential approach to the treatment of male sterility due to spermatogenic arrest. This is a pilot study evaluating meiotic, morphogenetic and cytoplasmic maturation of spermatogenic cells from 18 patients with obstructive azoospermia, during in-vitro culture of partly disintegrated testicular biopsy samples in the presence or absence of recombinant follicle stimulating hormone (rFSH). Meiotic progression was detectable only in the presence of rFSH in culture medium. FSH-dependent condensation, peripheral migration and protrusion of spermatid nuclei, together with FSH-independent flagellar growth, were the main events indicating post-meiotic sperm cell differentiation. rFSH also promoted the progression of spermatid cytoplasmic maturation, reflected by acceleration of acrosomal development. These differentiation events appeared to be mediated by humoral activity of Sertoli cells, without the need for a direct Sertoli-sperm cell contact. These findings provide a background for similar studies in patients with non-obstructive azoospermia. If reproducible in the latter group, transmeiotic in-vitro differentiation of primary spermatocytes may be useful in cases of complete maturation arrest, whereas the development of culture-specific forms may help select viable spermatids in cases of complete spermiogenesis failure.

Germ cell apoptosis in men with complete and incomplete spermiogenesis failure.

Germ cell apoptosis was evaluated in 11 men suffering from nonobstructive azoospermia and enrolled in a spermatid conception programme. In six of these patients, round spermatids (Sa stage) were the most advanced spermatogenic cells recovered from testicular biopsy samples. This condition is referred to as complete spermiogenesis failure. In the remaining five men, a few late elongated spermatids (Sd stage) were unexpectedly found in the testicular biopsy samples on the day of treatment. This condition is referred to as incomplete spermiogenesis failure. Germ cell apoptosis in both groups of patients was examined by analysing cell smears prepared from mechanically disintegrated testicular tissues using terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), which detects apoptosis-specific DNA fragmentation, and annexin-V binding, detecting apoptosis-related translocation of plasma membrane phosphatidylserine to the membrane's outer surface. Both methods were combined, in double-fluorescence labelling preparations, with immunocytochemical detection of proacrosin, a specific germline marker. Patients with complete spermiogenesis failure had significantly higher frequencies of primary spermatocytes and round spermatids carrying the apoptosis-specific DNA damage in comparison with patients with incomplete spermiogenesis failure. Surprisingly, apoptosis-related phosphatidylserine externalization occurs rarely until the advanced stages of spermiogenesis. Since externalized phosphatidylserine is expected to be involved in the recognition of apoptotic cells by phagocytes, apoptotic spermatocytes and round spermatids may not be removed easily by phagocytosis. The high frequency of DNA damage in round spermatids from patients with complete spermiogenesis failure explains the low success rates of spermatid conception in these cases. The evaluation of apoptosis can help predict success rates of spermatid conception.

[Non-obstructive azoospermia and ICSI]. / Azoospermies sécrétoires et ICSI.

50 cases of non obstructive azoospermia required testicular sperm extraction and ICSI. Results are promising but ability to find spermatozoa remains questionable. Further studies are necessary to improve success of the method. Genetic research also need to be developed for better understanding the process.

Combined use of proacrosin immunocytochemistry and autosomal DNA in situ hybridisation for evaluation of human ejaculated germ cells.

The recently reported human pregnancies and births after fertilising oocytes with round spermatids recovered from the ejaculate of men with non-obstructive azoospermia have underscored the need for a more accurate evaluation of the nuclear and cytoplasmic maturation status of ejaculated germ cells. In this study we describe our first experience with a method combining the immunocytochemical visualisation of proacrosin with autosomal DNA fluorescence in situ hybridisation (FISH) to assess ejaculated germ cells from patients with a spermiogenesis defect. The proacrosin immunoreactivity, analysed with the use of the monoclonal antibody 4D4, has been detected in cells of round spermatid size presenting a haploid FISH figure as well as in larger cells whose ploidy corresponds to primary and secondary spermatocytes. These observations are in agreement with previously published results obtained, with the use of the same antibody, by immunocytochemical analysis of histological sections of testicular tissue. All the cells of round spermatid size possessing proacrosin immunoreactivity were found to be haploid by FISH. On the other hand, some of the haploid cells of round spermatid size did not possess proacrosin immunoreactivity. The structural pattern of proacrosin immunoreactivity was highly variable both in spermatids and in younger spermatogenic cells. These data show that cell size is the main criterion to be used for the identification of ejaculated round spermatids, whereas the presence of the developing acrosome represents only an auxiliary criterion. The scoring of acrosomal development in ejaculated spermatids may be useful as part of pre-treatment diagnosis before the inclusion of infertile couples in a spermatid conception programme.

[Microinjection in France: report of the BLEFCO multicentric study]. / La micro-injection en France: rapport de l'enquête multicentrique des BLEFCO.

In order to evaluate the assisted fertilizations in France, BLEFCO association has analysed 436 and 1,781 attempts for SUZI and ICSI respectively. For SUZI, the transfer rate was 62% and the pregnancy per attempt rate was 10%. For ICSI, whatever indications, the transfer rate was 86% and the pregnancy per attempt rate was 21%. ICSI in cases of altered sperm without previous IVF represented 46% of ICSI attempts. No relationship between the transfer rate and the indication of ICSI has been observed. However, in case of unexplained fertilization failures, the pregnancy per attempt rate is significantly lower when compared to the other indications.