Leukaemia inhibitory factor modulates the differentiation of granulosa cells during sheep in vitro preantral to antral follicle development and improves oocyte meiotic competence.

In vitro follicle development from cryopreserved ovarian tissue could become an invaluable assisted reproduction technology for women with early ovarian failure. The challenge lies in producing, from small follicles present in the ovarian cortex, high-quality mature oocytes able to sustain embryo development. In vivo, an optimal combination of hormones and other factors coordinates the development of follicles and their enclosed oocyte. We have investigated the effect of the leukaemia inhibitory factor (LIF) cytokine, alone or in combination with FSH, on sheep in vitro follicle development from the preantral stage onwards. LIF did not alter follicle growth or antrum formation, but it modulated the differentiation of granulosa cells, as revealed by decreased production of anti-Müllerian hormone and abolished FSH-induced stimulation of oestradiol secretion. This modulatory role was also reflected in the abundance of mRNA from 35 genes, analysed by reverse-transcription coupled to microfluidic quantitative PCR. LIF stimulated or at least maintained the expression of genes involved in the dialogue between the oocyte and granulosa cells, through gap junctions (GJA4 encoding connexin 37) or paracrine signalling (Bone morphogenetic protein 15, KIT ligand and their receptors). Finally, the presence of both LIF and FSH during follicle growth strongly improved oocyte meiotic competence: most oocytes (56%) underwent subsequent nuclear maturation, a significant increase compared with their counterparts from follicles of similar size (550-900 µm) cultured with FSH only (28%) or developed in vivo (9%). Their ability to sustain embryo development remains to be evaluated. Combined supplementation with FSH and LIF certainly merits investigation with human follicles.

Breast-cancer anti-estrogen resistance 4 (BCAR4) encodes a novel maternal-effect protein in bovine and is expressed in the oocyte of humans and other non-rodent mammals.

STUDY QUESTION: Does BCAR4 have a role in mammalian embryo development? SUMMARY ANSWER: Expression, localization and functional data support that BCAR4 is a maternal-effect protein in non-rodent mammals. WHAT IS KNOWN ALREADY: BCAR4 was previously identified as an oocyte-specific gene in cattle, and as a marker of certain breast tumors in humans. STUDY DESIGN, SIZE, DURATION: Human oocytes were obtained from patients undergoing IVF, but had failed to mature after ovarian stimulation. Dog oocytes were obtained from ovariectomized bitches. Pig, horse and bovine ovaries were obtained from commercial slaughterhouses for extraction of immature oocyte-cumulus complexes. In vivo matured bovine matured oocytes were obtained after ovulation induction and ovulation inducing treatment of Montbeliard heifers. MATERIALS, SETTING AND METHODS: Expression at the RNA level was analyzed by reverse transcription coupled to polymerase chain reaction. Western blot and immunolabeling coupled to confocal or electronic microscopy were used to analyze bovine protein expression and intracellular localization. For the functional approach, short-interfering RNA were microinjected into mature bovine oocytes, followed by IVF; cleavage and embryo development were recorded. MAIN RESULTS AND THE ROLE OF CHANCE: The BCAR4 gene is conserved in mammalian species from various orders and has been lost in rodents after divergence with lagomorphs. The transcript is expressed in the oocytes of humans and domestic species. We bring the first experimental evidence of the BCAR4 protein in mammals. In cattle, the protein is not detected in immature oocytes but starts to be synthesized during maturation, increases in the zygote and persists until the morula stage. The protein is detected throughout the cytoplasm in mature oocytes, concentrates in and around the pronuclei in the zygote, and appears to shuttle in and out of the nuclei starting in the 2-cell embryo; BCAR4 is also present at the junctions between blastomeres from 2-cell to morula. In our functional approach, targeting the BCAR4 transcript by small-interfering RNA significantly compromised development to the morula or/and blastocyst stages (P < 0.05, logistic regression). LIMITATIONS, REASONS FOR CAUTION: As indicated above, protein expression and function were investigated in cattle and mostly in vitro matured oocytes were used. WIDER IMPLICATIONS OF THE FINDINGS: This study provides a novel candidate gene whose mutation or deregulation may underlie certain cases of unexplained female infertility.

Treating women under 36 years old without top-quality embryos on day 2: a prospective study comparing double embryo transfer with single blastocyst transfer.

BACKGROUND Embryologists currently face a challenge when counselling patients regarding the stage and the number of embryos to transfer when no top-quality embryos (TQE) are available. METHODS The aim of this study was to evaluate the efficacy of single blastocyst transfer (SBT) in comparison with the transfer of two cleavage-stage embryos in women under 36 years old. A total of 450 women under 36 years undergoing their first or second IVF treatment who had no TQE on Day 2 were included in this prospective study. Couples were assigned to either a SBT or a double cleavage-stage embryo transfer (DET). The clinical end-points monitored were rates of implantation, delivery and multiple deliveries. RESULTS The rate of transfer was significantly lower for couples assigned to the SBT group compared with the DET group (88 versus 100%, respectively, P < 0.001) while the delivery rate per oocyte retrieval was similar in both groups (26.7%). By contrast, the rate of multiple deliveries was significantly lower in the SBT group compared with the DET group (3.3 versus 23.3%, respectively, P < 0.01). Blastocyst cryopreservation was twice as high in the SBT group compared with the DET group (39 versus 18%, respectively, P < 0.001). CONCLUSIONS These findings show the value of extended embryo culture for couples without TQE. In such situations, delaying embryo transfer in order to select a single blastocyst with the highest potential for implantation can reduce the number of multiple pregnancies. Furthermore, our results demonstrate that extended culture allows blastocyst cryopreservation from embryos not available for Day 2 cryopreservation.

Does early morphology provide additional selection power to blastocyst selection for transfer?

Routine early developmental parameters are widely used in IVF centres to evaluate embryo development and fresh single-blastocyst transfer currently seems superior to single-embryo transfer. Would early morphological parameters help to choose the single blastocyst to be transferred, thereby improving the chances of implantation and live birth rate? This prospective observational study analysed the individual outcomes of 2617 embryos from 511 IVF couples scheduled for a single-blastocyst transfer. Embryo and blastocyst scores were constructed. There was a clear relationship between the kinetics and morphology of blastocysts and further implantation and live birth rate. There was a limited predictive value of embryo score with regard to blastocyst development and growth kinetics. Implanted and non-implanted blastocysts showed similar embryo scores. Thus usual morphological parameters on days 1 and 2 seem to have no additional value in indicating the right blastocyst to transfer. Non-invasive approaches might be helpful to increase the chances of implantation in the future.

[Non invasive assessment of embryo quality: proteomics, metabolomics and oocyte-cumulus dialogue]. / Approches non invasives de l'embryon : protéomique, métabolomique, dialogue ovocyte-cumulus.

Preimplantation embryo development is one of the key features with implantation itself to achieve a pregnancy. Assisted Reproductive Technologies both in human and animal have improved our knowledge on these events, although it remains elusive to predict embryo potential to give a baby. Among various ways to define embryo viability, noninvasive approaches get a serious advantage linked to the final transfer of the embryo. Techniques devoted to characterize the embryo secretome using proteomic or metabolomic approaches may be non invasive. Based on a direct identification of products of the embryo metabolism or an assessment of profile(s) related with embryo viability, they have greatly improved their sensitivity to allow their use in clinical embryology, once validated. Oocyte-cumulus dialogue, as a key factor for oocyte competence to meiosis and embryo development, was particularly concerned with both genomic and proteomic assessment of cumulus cells. While it is not possible to designate at the time being which among these approaches will be robust and cost-efficient enough to help routinely the clinical embryologist in assisted reproductive techniques (ART), one can predict that our ability to select the "right" embryo will combine morphological criteria already available with validated biomarkers.

Single Day 2 embryo versus blastocyst-stage transfer: a prospective study integrating fresh and frozen embryo transfers.

BACKGROUND: Whether extended culture allowing selection of embryos with high development potential has any advantage over cleavage-stage embryo transfer remains a matter of debate. Among the currently unsolved questions, the cumulative delivery rate resulting from fresh and frozen embryo transfers needs to be taken into account in both strategies. The aim of our study was, therefore, to compare the efficacy of single embryo transfer either on Day 2 or on Day 5/6 combining fresh and frozen embryo transfers. METHODS: A prospective study including 478 couples assigned on a voluntary basis to undergo elective single embryo transfer (eSET, n = 243) on Day 2 or single blastocyst transfer (SBT, n = 235) on Day 5/6 was performed. The primary outcome measurement was the cumulative delivery rate including fresh and frozen-thawed cycles in both groups. RESULTS: The delivery rate per cycle following fresh embryo transfer was significantly higher in the SBT group compared with the eSET group (P < 0.01). Conversely, frozen embryo and/or blastocyst transfers tended to result in a higher number of deliveries in the eSET compared with the SBT group. Altogether, the cumulative delivery rate per couple, including fresh and frozen embryo transfers, was similar between the two groups (37.9% versus 34.2% in the SBT and eSET groups, respectively). CONCLUSIONS: The observed cumulative delivery rates in this study do not allow us to take a position in favor of SBT or eSET. An improvement in blastocyst cryopreservation may change this attitude.

Gene expression in human cumulus cells: one approach to oocyte competence.

BACKGROUND: Dialogue between the oocyte and cumulus cells is essential for oocyte maturation. A prospective laboratory research project was designed to evaluate transcription of specific genes in cumulus cells harvested before intracytoplasmic sperm injection from pre-ovulatory follicles, according to individual oocyte nuclear maturity and developmental competence. Genes were chosen because their expression was induced by the LH peak [Steroidogenic Acute Regulatory protein (STAR), Cyclooxygenase 2 (COX2 or PTGS2), Amphiregulin (AREG)] or because they were involved in oocyte lipidic metabolism [Stearoyl-Coenzyme A Desaturase 1 and 5 (SCD1 and SCD5)] or in gap-junctions [Connexin 43 (CX43 or GJA1)]. METHODS: mRNA levels in cumulus cells were assessed by real-time PCR. RESULTS: Expression levels of all genes investigated, except Cx43, were increased after resumption of meiosis. Nuclear maturation was thus associated with increased expression of STAR, COX2, AREG, SCD1 and SCD5 by cumulus cells. When considering only cumulus associated with metaphase II oocytes, gene expression was independent of morphological status at Day 2. In contrast, transcript levels were lower and distributed over a narrower range in cumulus enclosing oocytes achieving blastocyst development at Day 5/6 than in cumulus enclosing oocytes unable to develop beyond the embryo stage. CONCLUSION: Further developmental potential from embryo to blastocyst stage was associated with lower expression in a narrow range for these genes.

[Sequential assessment of individually cultured embryos in IVF]. / Suivi individuel des embryons en FIV.

OBJECTIVE: It is a challenge for IVF centers to propose a method to select the most viable embryo to transfer, thereby minimizing the risk of multiple births. In this study, a prospective investigation was made to determine if non-invasive developmental markers on day 1 combined to conventional evaluation on day 2 can predict in vitro blastocyst development. PATIENTS AND METHODS: A total of 4190 individually cultured embryos from patients undergoing IVF/ICSI treatment at the Tours University Hospital Center from January 2002 to December 2004 were included. Individual embryos were cultured in sequential media in microdrops under mineral oil from j1 to j5/j6 allowing to record their sequential growth until the blastocyst stage. RESULTS: The results showed a significant positive relationship between pattern 0 zygote, early cleavage, 4 cells embryos with < 20% fragmentation on day 2 and the rate of blastocyst development on day 5 (P < 0.05). In our hands, zygote pattern does not bring additional benefit to better select embryo. DISCUSSION AND CONCLUSION: Zygote and early cleavage assessments on day 1, morphological appearance on day 2 are some other parameters related individually to blastocyst development on days 5 and 6. These parameters can be used collectively to establish a predictive in vitro sequential embryo assessment model for routine use in IVF clinics.

[Oocyte-cumulus dialog]. / Le dialogue ovocyte-cumulus.

The dialog between oocyte and cumulus cells brings a major contribution for oocyte meiotic and developmental competence. On the one hand, the oocyte will modulate follicle growth through specific gene expression (Figalpha, GDF-9, BMP15) as well as its meiosis (GPR3 et PDE3A). Beyond its action on proliferation, oocyte will control in part the differentiation of cumulus cells with a particular involvement of GDF-9, BMP15 in this late maturation process. On the other hand, somatic cells are the main targets of gonadotropins and will modulate both oocyte growth and maturation. Gap-junctions between oocyte and cumulus cells have a major role in this interaction, since they allow the action of some oocyte specific genes (GDF9) but also the control of its own metabolism and calcium movements. While ovulation will involve gonadotropins action on somatic cells, EGF-like factors recruited at the cumulus level will participate in this process. Finally we may suspect that improving the knowledge on oocyte-cumulus dialog will contribute to better define oocyte competence, while bringing some clues for in vitro maturation.

Characterization of the fertility of Kit haplodeficient male mice.

The role of the proto-oncogene Kit expression during gonadal development, then in differentiated spermatogonia has been thoroughly established. The present study was designed to investigate the consequences of a partial defect in Kit gene expression on sperm fertilizing ability, using Kit haplodeficient mice (kitW-lacZ/+). Same inbred mice (kit+/+) were used as controls. Epididymal sperm characteristics and in vivo fertility were assessed, then in vitro-fertilization experiments were carried out for mice of both genotypes. Epididymal sperm count was drastically reduced, and sperm motility was also decreased in kitW-lacZ/+ compared with kit+/+ males. Both in vivo or in vitro fertility were greatly reduced in kitW-lacZ/+ compared with kit+/+ males. By contrast, the fertility of kitW-lacZ/+ females was apparently unaffected. Additionally, a higher number of spermatozoa with undetected acrosomal contents was revealed by fluorescein isothiocyanate-labelled Pisum sativum agglutinin acrosomal staining after epididymal sperm retrieval in kitW-lacZ/+ mice, whereas no difference was observed after induction of acrosomal reaction in mice of either genotype. Ultra-structural data confirmed the higher frequency of abnormal acrosome in spermatozoa of kitW-lacZ/+ mice. Thus, sperm production is impaired in Kit haplodeficient mice both on a quantitative and a qualitative basis. Finally, we show that one single copy of Kit gene is not sufficient to maintain genuine fertility in male mice.

[Molecular mechanisms of stimulation and desensitization of Sertoli cells by follicle-stimulating hormone]. / Mécanismes moléculaires de stimulation et désensibilisation de la cellule de Sertoli par l'hormone folliculo-stimulante.

Many Sertoli functions are regulated by the receptor-mediated action of follicle-stimulating hormone (FSH). The interaction of FSH with its specific cell surface receptors leads to stimulation of a number of intracellular events, including the activation of guanine nucleotide binding protein (G protein), adenylate cyclase and the cAMP-dependent protein kinase (PKA) pathway. In addition to positive regulation of cell functions, a phenomenon of refractoriness occurs after primary exposure of target cells to the hormone. Different sites of lesion have been suggested including down-regulation of FSH receptor, uncoupling of the receptor and the G protein/adenylate cyclase complex, and stimulation of nucleotide phosphodiesterases or inhibition of PKA activity. Alterations of cell responsiveness are mediated by a combination of these different mechanisms occurring over different time-scales and hormonal concentrations.

Protein kinases and protein synthesis are involved in desensitization of the plasminogen activator response of rat Sertoli cells by follicle-stimulating hormone.

Tissue-type plasminogen activator (tPA) secretion is a specific response of Sertoli cells to follicle-stimulating hormone (FSH), which is lower after preincubation of the cells with low FSH concentrations because of FSH receptor/Gs protein uncoupling. In this report, we present evidence that this desensitization induced by the lowest FSH concentrations is suppressed by specific peptidic inhibitors of endogenous PKA and PKC in permeabilized Sertoli cells. In contrast, desensitization promoted by slightly higher FSH concentrations is not mediated through PKA or PKC activation but is dependent on protein neosynthesis.

Structure-function relationships and mechanism of action of pituitary and placental gonadotrophins.

Data from the author's laboratory on relationships between structure and function of equine luteinizing hormone, follicle-stimulating hormone and choriogonadotrophin as well as their mechanisms of action are reviewed and compared with their human counterparts. Polymorphism of these hormones and problems associated with their purification are discussed as well as the association and dissociation of their alpha- and beta-subunits. The affinity of receptor binding, the superactivity of membrane transduction and homologous desensitization of target cells by non-stimulatory doses of the gonadotrophins are also reviewed.

Involvement of cyclic adenosine monophosphate-dependent protein kinase isozymes in tissue plasminogen activator secretion by rat Sertoli cells stimulated with follicle-stimulating hormone in vitro.

This study was undertaken to investigate, in freshly isolated rat Sertoli cells, the physiological function of the type I and type II cyclic adenosine monophosphate (cAMP)-dependent protein kinase isozymes in tissue-type plasminogen activator secretion and the regulation of this cAMP process by follicle-stimulating hormone (FSH). Follicle-stimulating hormone-induced tissue-type plasminogen activator secretion depends upon intracellular cAMP levels. The changes in cAMP amounts required to activate maximally the tissue-type plasminogen activator secretion are extremely small, a cAMP threshold having to be reached for triggering the tissue-type plasminogen activator output. Intact Sertoli cells were incubated with combinations of cAMP analogs specific for each cAMP-dependent protein kinase type and complementary in their cAMP binding site on the cAMP-dependent protein kinase regulatory subunits: 8-aminohexylamino-cAMP = type 1, site 1; 8-thiomethyl-cAMP = type II, site 1 and N6-benzoyl-cAMP = types I/II, site 2. This allowed us to activate selectively each cAMP-dependent protein kinase type in a synergistic manner and then to evaluate their respective influence in the specific tissue-type plasminogen activator response. We establish that both of the cAMP-dependent protein kinase types are present and functional; the activity of the type I isozyme is preponderant (60%) in the cAMP-dependent tissue-type plasminogen activator secretion. Likewise, when these cAMP analogs were coupled with endogenously generated cAMP by FSH or forskolin, both of the cAMP-dependent protein kinase types were involved in the tissue-type plasminogen activator production.(ABSTRACT TRUNCATED AT 250 WORDS)