Increased pregnancy rate using standardized coculture on autologous endometrial cells and single blastocyst transfer : a multicentre randomized controlled trial.

Despite excellent published results, the lack of well-designed, multicentre, randomized clinical trials results in an absence of general consensus on the efficacy of autologous endometrial cells coculture (AECC) in Assisted Reproductive Technology (ART). An open, multicentre, prospective, randomized controlled trial was designed to compare the pregnancy rate (PR) after the transfer of one blastocyst on day 5 after AECC to the transfer of one embryo on day 3 (control group). Patients were women aged 18 to 36, undergoing an ART cycle with no more than 1 embryo transfer failure. Sample size was calculated at 720 for a superiority trial involving an intermediate analysis at 300 patients. We present the results of the intermediate analysis that resulted in the study ending considering the observed difference. Three hundred thirty nine patients were randomized: 170 in the AECC group and 169 in the control group. The clinical PR per transfer was 53.4% with AECC and 37.3% in the control group (p=0.025). The quality of embryos was improved with AECC. These results suggest that implementation of the AECC technique to a large number of In-Vitro Fertilization (IVF) centres could lead to a substantial improvement in the proportion of successful assisted reproduction. The study was supported by the Laboratoires Genévrier, France.

[Importance of the determination of MTHFR SNPs (Methylene Tetrahydrofolate Reductase Single Nucleotide Polymorphisms) in couple infertility]. / Intérêt de la mise en évidence des isoformes de la MTHFR (Méthylènetétrahydrofolate réductase) en infertilité du couple.

OBJECTIVE: MTHFR SNPs (Methylene Tetrahydrofolate reductase Single Nucleotide polymorphisms) are biochemical modifications decreasing the capacity to form 5 MTHF 5 methyltetrahydrofolate (5MTHF). Their presence reduces the capacity of the One Carbon cycle, and so the regeneration of Homocysteine (Hcy) and in fine strongly perturbs all the methylation processes. As methylation processes are major regulators in gametogenesis and embryogenesis. We have determined the prevalence of the 2 most important SNPs A1298C and C677T in our population of patients consulting for infertility. METHODS: Determination of the MTHFR SNPs A1298C and C677T, by hybridization using the LAMP Human MTHFR mutation KITs. RESULTS: Only 15.8% of our patients (861) do not carry any SNP (WT wid type). Close to 20% of the patients are homozygotes for one mutation or the other. A total of 19.7% are composite heterozygous. A total of 43% of our population is considered "at risk", based on observations collected for the repeat miscarriages. CONCLUSIONS: Determination of the 2 major MTHFR SNPs is not a "first row" choice, but it must not be neglected and should be carried out in case of repeat ART failures and repeat miscarriages. Some simple therapeutic options can be proposed: they are based on the use of 5MTHF (5MethyleneTetraHydroFolate) the compound downstream the MTHFR.

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.

Natural cycle IVF and oocyte in-vitro maturation in polycystic ovary syndrome: a collaborative prospective study.

In-vitro maturation (IVM) was performed in 350 cycles for 262 unstimulated patients diagnosed with polycystic ovary syndrome who were primed with human chorionic gonadotrophin (HCG) before oocyte retrieval. In order to improve nuclear and cytoplasmic maturation, growth hormone was added to the maturation medium. Oocytes were recovered in 94.8% of the cycles, with a mean number of nine cumulus-oocyte complexes retrieved. Within 28 h, 62% of the oocytes reached the metaphase II (MII) stage, and 17.6% were MII after a further 20 h in culture. An ongoing pregnancy rate of 15.2% was obtained, but with a high miscarriage rate, 28% of the total with a positive betaHCG test assessed after embryo transfer. Cytogenetic and DNA fragmentation analysis of the embryos was not fundamentally different from what is classically observed in routine IVF. This observation implies that the results are not necessarily due to compromised oocyte quality after IVM, and that endometrial receptivity should also be considered, especially in IVM cycles where the follicular phase is dramatically shortened.

[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.

Metabolic enhancers supporting 1-carbon cycle affect sperm functionality: an in vitro comparative study.

The sperm plasma membrane is a sensitive target to oxidative stress. The most representative reactive oxygen species (ROS) scavengers in the genital tract, hypotaurine and glutathione, require, for their synthesis, cysteine whose availability is associated with the 1-carbon cycle (1-CC). Human, bovine and ascidian spermatozoa were incubated with compounds supporting the 1-CC (Vitamin B6, Methylcobalamin, 5 Methyl Tetrahydrofolate, Zinc Bisglycinate and N-acetyl-cysteine) (TRT) and compared to the effects induced solely by N-acetyl-cysteine (NAC). In control groups (CNTRL), spermatozoa were incubated with medium alone. After 90 and 180 minutes of incubation, the mitochondrial membrane potential (ΔΨM) in TRT and NAC was significantly (P < 0.01) higher than in CNTRL. At H2DCFDA evaluation, ROS production differed between species whereas, at 2-OH Ethidium, it significantly decreased in bovine TRT group. Intracellular pH (pHi) did not significantly vary in relation to treatment. In ascidian spermatozoa, the NAC supplementation decreased external pH, which in turn brought to a pHi lowering. Buffering seawater with NaHCO3 reversed the beneficial effects of N-acetyl-cysteine supplementation. In conclusion, both fully supporting the 1-CC and treatment with N-acetyl-cysteine alone improved kinetics, ΔΨM and ROS production in mammalian sperm demonstrating for the first time the direct in vitro effects of these compounds on sperm functionality.

[Oocyte capacity to repair DNA damage induced in sperm]. / L'ovocyte réparateur de l'ADN du spermatozoïde.

Germ cells perform a unique and critical biological function: they propagate the DNA that will be used to direct development of the next generation. Genetic integrity of germ cell DNA is essential for producing healthy and reproductively fit offspring. There is accumulating evidence that genetic damage, in the form of unrepaired DNA lesions or de novo mutations, may be transmitted via sperm to offspring. Such genetic damage may arise from environmental exposure or via endogenously formed reactive species, in stem cells or during spermatogenesis. Based on results from our own and other laboratories, we discuss the current knowledge of different pathways of DNA repair, especially in human oocyte.

[Impact of sperm DNA fragmentation on ART outcome]. / La fragmentation de l'ADN du spermatozoïde: impact en Assistance médicale à la procréation.

We have used the sperm chromatin structure assay (SCSA) test in order to determine if correlations can be found between sperm DNA fragmentation and spermogram parameters. Only necrospermia and DNA fragmentation index are strongly correlated (P<0.0001). Neither fertilization rates for ICSI and IVF, nor blastocyst formation rates are impaired by a high DFI. However when the critical DFI>30% is reached, the chances of having ongoing pregnancies after blastocyst transfer are reduced by three. Treatments with antioxidants are of limited efficacy even though we obtained 2 deliveries after DFI treatments with such treatments. New strategies in order to improve the pregnancy rates for these peculiar cases of reduced fertility are discussed.

Cholinesterases isoenzymes: a comparative study in the skin and plasma.

Acetylcholinesterase (Ache) and pseudocholinesterase (BUche) activities were studied quantitatively in healthy skin by spectrophotometric methods and qualitatively by polyacrylamide gel electrophoresis. The results were compared to those obtained in plasma. The substrates used to reveal enzyme activities were acetylthiocholine (ATC) iodide and butyrylthiocholine (BTC) iodide, respectively. A linear relationship exists between the values of BUche and Ache activities in plasma and those in skin. Six isoenzymes of different electrophoretic mobility were observed in the skin. One of them, which is never found in plasma extracts, appears to be specific to the skin. On gradient gel electrophoresis, with both substrates (ATC and BTC), a single band of enzyme activity, corresponding to a molecular weight of 600,000 was observed. These results suggest that in the skin there is only one enzyme, most probably butyryl cholinesterase, which cleaves BTC somewhat faster than ATC. This methodology, when applied to the study of dermatoses in which abnormalities of cutaneous nerve terminals are suspected, should furnish precise functional pathophysiological details.

Purification and characterization of ubiquitin from mammalian testis.

Ubiquitin was extracted from testis of 4 mammals and purified to homogeneity by gel filtration chromatography. Amino acid compositions and NH2-terminal sequences were found to be identical in the 4 species and with calf thymus ubiquitin. Ubiquitin conformation was shown to be very sensitive to oxidation. Improved methods for radioimmunoassay of ubiquitin in tissue extracts are also discussed.

Co-culture of the early human embryo: factors affecting human blastocyst formation in vitro.

Co-culture systems have been designed to overcome the embryonic developmental arrest observed in vitro in conventional culture media. Oviduct and uterine epithelial cells can sustain embryonic development, as can trophoblastic tissue and transport epithelia of non-genital origin. Its benefits involve neither hormone dependency nor histo-specificity. Fibroblasts do not overcome the developmental arrest in most mammalian species, but whether they do in humans is still unsure. In all systems, the quality of the feeder cells and the co-culture medium are very important. Using the Vero cell line, 60% of human IVF embryos reach the blastocyst stage. The quality of the sperm seems to affect results. We have observed: For 10% of the patients with unexplained fertility, blastocyst stage is not attained; this probably involves a maternal (ovarian) problem. When at least one blastocyst is transferred, the pregnancy rate per transfer is 31%. The implantation rate in pregnant women is higher than after transfer at day 2. After repeated failures of transfer at early stages (2-6 cells), transfer at the blastocyst stage gives high pregnancy rates (40%). This indicates an in vitro selection. There is a strong paternal effect on blastocyst formation: poor quality sperm give lower rates of blastocyst. Co-culture helps to understand treatment failures related to male factors. Around 60% of the patients having spare embryos have had blastocysts frozen. Transfers of frozen-thawed blastocysts give a 20% pregnancy rate and an implantation rate per embryo of 11%. Co-culture is a new tool which has to be carefully evaluated in human IVF programs. It does not impair "a minima" embryo viability and it allows in vitro selection.(ABSTRACT TRUNCATED AT 250 WORDS)

Enkephalin production by the corpus luteum.

In search of Early pregnancy factors, on the base of Kentsin, the sole molecule embryo-specific, we detected the presence of Enkephalin in the Mouse ovary, in Bovine and Human corpus luteum. In the Mouse the Met-Enkephalin release by the ovary seems to be stimulated by the oviduct in presence of the embryo. In vitro Met enkephalin release by Bovine Corpus luteum is about 0.5 to 1. pMole/mg of fresh tissue/24 Hours. This release is not increased in presence of trophoblastic tissue. The content of the fresh tissue is between 0.7 and 1.9 picomoles par gram of Human tissue, and 0.9 picomoles for Bovine tissue. We determine the presence of Leu-Enkephalin and Met-Enkephalin Arg-Gly-Leu. The ratios observed confirm a ProEnkephalin A expression in the Ovary. The roles of these opioid peptides is discussed in term of ovum transport, Granulosa cell physiology and Early pregnancy factors.

Central opioid-like influence of a tetrapeptide from hamster embryo (kentsin) on gastrointestinal motility in dogs.

The effects of intracerebroventricular (i.c.v.) vs. intravenous (i.v.) administration of kentsin (H-Thr-Pro-Arg-Lys-OH) on gastrointestinal motility were investigated in fasted dogs. Administered i.c.v. at doses of 20 and 100 ng/kg, this peptide inhibited by 51.2 and 76.1% the antral motility index and disrupted the jejunal migrating motor complex for 2 and 4 h respectively. Similar effects were only obtained after a 25 fold higher dose administered i.v. and these effects were abolished after a previous i.c.v. administration of naloxone (50 micrograms/kg) suggesting that they are mediated through opiate receptors.

Serum is not necessary in human in vitro fertilization, early embryo culture, and transfer.

Human in vitro fertilization (IVF), embryo culture (EC), and embryo transfer (ET) are commonly performed in various media supplemented with blood serum. To determine whether serum is really necessary, the results of IVF, EC, and ET were compared by using two types of media: B2 medium supplemented with human cord serum and B3 medium without any serum. B3 medium is similar to B2, except that it contains 1% pure controlled human serum albumin in place of 1% bovine serum albumin. We did not observe any difference between the results obtained by using B2 or B3 at any phase of IVF, EC, and ET processes. Both media give an overall evolutive pregnancy rate of about 16%. B3 medium with serum seems to increase slightly the cleavage speed. Our results indicate that there are no positive effects when serum is used for human IVF-ET. To avoid serum supplementation is of evident interest for the homogeneity of the results. This will also lead to a better understanding of human early development and control of the egg quality by metabolic analysis of the media following in vitro EC.

Freezing cocultured human blastocysts.

OBJECTIVE: To determine the possibility of obtaining good pregnancy rates (PRs) after freezing and thawing cocultured blastocysts. DESIGN: Human blastocysts were frozen first according to a protocol available from literature. Two other protocols including the addition of glycerol were designed to improve the results. SETTING: All the patients were under clinical management at the Institut Rhonalpin pour l'Etude de la Reproduction Humaine in Lyons, France. PATIENTS: Patients involved in the in vitro fertilization program have had their supernumerary embryos frozen according to the three protocols presented here. MAIN OUTCOME MEASURES: Embryo recovery after freezing and thawing. Clinical and ongoing pregnancies after embryo transfer (ET). RESULTS: A protocol including sucrose addition and reduction of steps in the preparation of the blastocysts for freezing gave us a 21% PR per transfer (15 ongoing) of 101 transfers (106 thawings). CONCLUSIONS: Freezing of cocultured human blastocysts allow good PRs. This can represent an alternative for repeated failures of ETs at early stages.

Birth weight and sex ratio after transfer at the blastocyst stage in humans.

OBJECTIVE: To analyze the birth weights and sex ratio of infants born after blastocyst transfer. DESIGN: Retrospective analysis. SETTING: Three infertility clinics. PATIENT(S): Patients admitted for IVF with blastocyst transfer. INTERVENTION: None. MAIN OUTCOME MEASURE(S): Birth weights and sex ratio of infants born after blastocyst transfer. RESULT(S): Statistically significantly more male infants were born after transfer of fresh blastocysts, either cocultured or cultured in sequential media. No specific differences in birth weight were observed between infants born after blastocyst transfer and those born after spontaneous conception. CONCLUSION(S): More male infants than female infants were born after blastocyst transfer when transfers were performed as soon as the blastocyst stage was reached and male embryos had a faster cleavage rate.

Cocultured blastocyst cryopreservation: experience of more than 500 transfer cycles.

OBJECTIVE: To present our experience using cocultured cryopreserved and transferred blastocysts. DESIGN: Retrospective study of patients undergoing transfer of cryopreserved blastocysts. SETTING: Three different IVF centers. PATIENTS: Four hundred sixty-seven thawed cycles from January 1991 to June 1994. MAIN OUTCOME MEASURE: Pregnancy rate per cycle after transfer of pre-embryos developed from thawed blastocysts. RESULTS: One thousand two hundred thirty-nine blastocysts were thawed. Of these, 1,033 (83%) survived thawing and were transferred. Five hundred sixty-three thawed cycles resulted in 516 (92%) receiving intrauterine transfer. One hundred twelve clinical pregnancies were established, resulting in a 21.7% pregnancy per transfer with a 19% ongoing rate. The implantation rate of 13.4% results from 138 implanted pre-embryos. There was a higher PR in the programmed cycle (79/302; 26.2%) compared with the natural cycle (6/47;13%). CONCLUSIONS: Freezing at the blastocyst stage is a proven and reliable method in IVF technology. Although there may be fewer pre-embryos, their ability to implant appears to approach the potential of a fresh transfer.

Enzymes in the seminal plasma from azoospermic men: correlation with the origin of their azoospermia.

Semi-quantitative estimations of the activities of 65 enzymes were studied for cases of azoospermia in an attempt to correlate the seminal enzyme profile with the origin of the azoospermia. One single enzyme--alpha-glucosidase--showed variation. All other specificities tested gave identical results for all the azoospermic samples and for normal controls. The alpha-glucosidase activity that is present in normal semen was absent from samples from patients with azoospermia associated with a complete obstruction of the genital tract, and also from all semen samples from vasectomized subjects. This enzyme appears to be secreted by the epididymis, and the activity present in azoospermic semen can provide information concerning the origin of the azoospermia and the functional state of the epididymis. Positive but reduced alpha-glucosidase activity appears to be frequently associated with inflammatory disease. The consequences of these findings are discussed.

[Insulin-like growth factors I and II in follicular and oocyte maturation]. / Insulin-like growth factors I et II dans la maturation folliculaire et ovocytaire.

Insulin-like growth factors I and II are synthesized from animal and human follicles. Their reproduction is FSH-, LH- and oestrogen-dependent. Growth hormone is also involved. IGF-I receptor numbers increase in the presence of FSH, while estradiol has a synergic effect. IGF-I and IGF-II stimulate growth of different follicular cell elements. TGF-alpha has the same activity but seems to be mediated by an increase in IGFs. Conversely, TGF beta and activin inhibit follicular growth accompanied by a reduction in IGFs. In contrast to TGF alpha, IGFs do not cause acquisition of meiotic competence. Injection of IGF-I into cultures of prepuberal splenic primary follicles reduced IGF-II production.