Molecular evidence that follicle development is accelerated in vitro compared to in vivo.

In this study, we systematically compared the morphological, functional and molecular characteristics of granulosa cells and oocytes obtained by a three-dimensional in vitro model of ovine ovarian follicular growth with those of follicles recovered in vivo Preantral follicles of 200 µm diameter were recovered and cultured up to 950 µm over a 20-day period. Compared with in vivo follicles, the in vitro culture conditions maintained follicle survival, with no difference in the rate of atresia. However, the in vitro conditions induced a slight decrease in oocyte growth rate, delayed antrum formation and increased granulosa cell proliferation rate, accompanied by an increase and decrease in CCND2 and CDKN1A mRNA expression respectively. These changes were associated with advanced granulosa cell differentiation in early antral follicles larger than 400 µm diameter, regardless of the presence or absence of FSH, as indicated by an increase in estradiol secretion, together with decreased AMH secretion and expression, as well as increased expression of GJA1, CYP19A1, ESR1, ESR2, FSHR, INHA, INHBA, INHBB and FST There was a decrease in the expression of oocyte-specific molecular markers GJA4, KIT, ZP3, WEE2 and BMP15 in vitro compared to that in vivo Moreover, a higher percentage of the oocytes recovered from cultured follicles 550 to 950 µm in diameter was able to reach the metaphase II meiosis stage. Overall, this in vitro model of ovarian follicle development is characterized by accelerated follicular maturation, associated with improved developmental competence of the oocyte, compared to follicles recovered in vivo.

The impact of introducing combined first-trimester trisomy 21 screening in the French population.

BACKGROUND: French state health insurance has funded trisomy 21 prenatal screening for all pregnant women since decades. First-trimester combined screening was introduced nationally and funded in 2010. OBJECTIVE: To evaluate the impact of the introduction, of a national policy of prenatal trisomy 21 first-trimester screening on the reduction of invasive prenatal diagnostic procedures. METHODS: The results of all prenatal trisomy 21 screening and invasive diagnostic procedures were collected for the whole country over the period 2009-12. The screen-positive rates (risk cut-off 1 : 250, including isolated nuchal translucency ≥ 3.5 mm), positive predictive values and percentage of cases diagnosed prenatally were calculated. RESULTS: Over the study period the number of women undergoing serum screening (including first- and second-trimester screening tests) increased from 678 803 to 689 651 (83 to 85% of deliveries, P < 0.0001). By 2012, first-trimester combined screening accounted for 70% of all trisomy 21 screening. The screen-positive rate decreased from 9.5 to 4.8% (P < 0.001) resulting in a 37 478 (47%) drop (P < 0.001) in the number of invasive diagnostic procedures. The positive predictive value of screening increased from 2.6 to 6.1% from 2009 to 2012 (P < 0.001), due to the higher positive predictive value of first-trimester over second-trimester screening (9.1 vs. 1.8% over the period 2010-12, P < 0.001). The percentage of prenatally diagnosed cases remained high at around 80% between 2010 and 2012. CONCLUSIONS: The policy shift from second-trimester to first-trimester trisomy 21 screening allowed to reduce the number of invasive tests. The number of antenatal trisomy 21 diagnoses increased (+2.7%) over the study period.

Expression and regulation of INTELECTIN1 in human granulosa-lutein cells: role in IGF-1-induced steroidogenesis through NAMPT.

INTELECTIN (ITLN) is an adipokine involved in the regulation of insulin sensitivity and inflammatory and immunity responses. Serum ITLN levels are lower in obese, diabetic, and polycystic ovary syndrome (PCOS) women than in control subjects. ITLN has never been studied in ovarian cells. Here, we identified ITLN1 in human ovarian follicles and investigated the molecular mechanisms involved in the regulation of its expression in response to the insulin sensitizers metformin and rosiglitazone, in human granulosa-lutein cells (hGLCs) and in a human ovarian granulosa-like tumor cell line (KGN). We also studied the effects of human recombinant ITLN1 (hRom1) on steroid production and on the activation of various signaling pathways. Using RT-PCR, immunoblotting, and immunohistochemistry, we found that INTL1 is present in human follicular cells. Using ELISA, we showed that INTL levels are similar in plasma and follicular fluid (FF) in control patients, whereas they are higher in FF than in plasma in PCOS patients. In KGN cells and hGLCs, insulin (10(-8) M), insulin-like growth factor-1 (IGF-1; 10(-8) M), and metformin (10(-2) M or 10(-3) M) increased INTL1 expression (mRNA and protein) after 12 and 24 h of stimulation. For metformin, this effect was mediated by adenosine monophosphate-activated kinase (PRKA). Furthermore, hRom1 increased nicotinamide phosphoribosyltransferase (NAMPT) expression in KGN and hGLCs. We also showed that hRom1 increased IGF-1-induced progesterone and estradiol secretion and this was associated with an increase in the STAR and CYP19A1 protein levels and an increase in IGF-1R signaling. Furthermore, all these data were abolished when NAMPT was knocked down in KGN cells, suggesting that INTL1 improves IGF-1-induced steroidogenesis through induction of NAMPT in hGLCs.

Semen quality trends in French regions are consistent with a global change in environmental exposure.

A retrospective study carried out recently in a large sample of men, close to the general population, has reported a significant and strong decline in sperm concentration and morphology in the whole of France between 1989 and 2005. We studied these trends within each region of France. Data were obtained from the Fivnat database. The study sample comprised male partners of sterile women in whom both tubes were absent or blocked. They were located at the assisted reproductive technology center. A Bayesian spatio-temporal model with parametric time trends, adjusted for age, was used to model overall time trends for each region. The results show that sperm concentration decreased in almost all regions of France. Among them, Aquitaine showed the highest decrease and Midi-Pyrénées had the lowest average for the whole period. Regarding total motility, most regions showed a slight increase while Bourgogne showed a steep and significant decrease. While considering sperm morphology, there was a decrease in most of the regions. The decrease in Aquitaine and Midi-Pyrénées was stronger when compared with the overall trend. In conclusion, a decrease in sperm concentration and morphology, already shown at the French metropolitan territory level, was observed in most regions of France. This is consistent with a global change in environmental exposure, according to the endocrine disruptor hypothesis especially. Indeed, ubiquitary exposure to chemicals has been growing in the general population of France since the 1950s, and the results do not appear to support the lifestyle hypothesis. The highest decreases and lowest values are consistently observed in two proximate regions that are both highly agricultural and densely populated.

Semi-quantitative measurement of specific proteins in human cumulus cells using reverse phase protein array.

BACKGROUND: The ability to predict the developmental and implantation ability of embryos remains a major goal in human assisted-reproductive technology (ART) and most ART laboratories use morphological criteria to evaluate the oocyte competence despite the poor predictive value of this analysis. Transcriptomic and proteomic approaches on somatic cells surrounding the oocyte (granulosa cells, cumulus cells [CCs]) have been proposed for the identification of biomarkers of oocyte competence. We propose to use a Reverse Phase Protein Array (RPPA) approach to investigate new potential biomarkers of oocyte competence in human CCs at the protein level, an approach that is already used in cancer research to identify biomarkers in clinical diagnostics. METHODS: Antibodies targeting proteins of interest were validated for their utilisation in RPPA by measuring siRNA-mediated knockdown efficiency in HEK293 cells in parallel with Western blotting (WB) and RPPA from the same lysates. The proteins of interests were measured by RPPA across 13 individual human CCs from four patients undergoing intracytoplasmic sperm injection procedure. RESULTS: The knockdown efficiency of VCL, RGS2 and SRC were measured in HEK293 cells by WB and by RPPA and were acceptable for VCL and SRC proteins. The antibodies targeting these proteins were used for their detection in human CCs by RPPA. The detection of protein VCL, SRC and ERK2 (by using an antibody already validated for RPPA) was then carried out on individual CCs and signals were detected for each individual sample. After normalisation by VCL, we showed that the level of expression of ERK2 was almost the same across the 13 individual CCs while the level of expression of SRC was different between the 13 individual CCs of the four patients and between the CCs from one individual patient. CONCLUSIONS: The exquisite sensitivity of RPPA allowed detection of specific proteins in individual CCs. Although the validation of antibodies for RPPA is labour intensive, RRPA is a sensitive and quantitative technique allowing the detection of specific proteins from very small quantities of biological samples. RPPA may be of great interest in clinical diagnostics to predict the oocyte competence prior to transfer of the embryo using robust protein biomarkers expressed by CCs.

Resistin decreases insulin-like growth factor I-induced steroid production and insulin-like growth factor I receptor signaling in human granulosa cells.

OBJECTIVE: To identify resistin in human ovarian follicles and investigate the effect and the molecular mechanisms associated with resistin on steroidogenesis in human granulosa cells (GCs). DESIGN: The effects of recombinant human resistin on the secretion of progesterone (P) and estradiol (E2) by cultured human GCs were investigated. SETTING: Academic institutions. PATIENT(S): Twenty infertile and healthy women undergoing IVF. INTERVENTION(S): Primary human GC cultures stimulated with recombinant human resistin (10 ng/mL). MAIN OUTCOME MEASURE(S): Determination of messenger RNA (mRNA) and protein expression of resistin in fresh human GCs by reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblot and immunohistochemistry, respectively; measurement of P and E2 levels in the conditioned media by radioimmunoassay; determination of cell proliferation by tritiated thymidine incorporation; and analysis of signaling pathways activation by immunoblot analysis. RESULT(S): Human GCs and theca cells express resistin. In primary human GCs, resistin decreases P and E2 secretion in response to insulin-like growth factor I (IGF-I). This was associated with a reduction in the P450 aromatase and P450scc (cholesterol side-chain cleavage cytochromes P450) (P450scc) protein levels but not those of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) or steroidogenic acute regulatory protein (StAR) and with a decrease in IGF-I-induced IGF-I receptor and mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Resistin treatment does not affect IGF-I-induced cell proliferation and basal steroidogenesis (there is no IGF-I or follicle-stimulating hormone stimulation). In the basal state, resistin rapidly stimulates Akt and MAPK ERK1/2 and p38 phosphorylation in primary human GCs. CONCLUSION(S): Resistin is present in human GCs and theca cells. It decreases P and E2 secretion, P450scc and P450 aromatase protein levels, and IGF-IR signaling in response to IGF-I in primary human GCs.

Visfatin is expressed in human granulosa cells: regulation by metformin through AMPK/SIRT1 pathways and its role in steroidogenesis.

Visfatin is a cytokine hormone and an enzyme involved in metabolic (obesity, type II diabetes) and immune disorders. Some data suggest a role of visfatin in ovarian function. Here, we identified visfatin in human follicles and investigated the molecular mechanisms involved in the regulation of its expression in response to insulin sensitizers, metformin (MetF) and rosiglitazone, in primary human granulosa cells (hGCs) and in a human ovarian granulosa-like tumour cell line (KGN). We also studied the effects of human recombinant visfatin (RhVisf) on steroid production and on the activation of various signalling pathways. By RT-PCR, immunoblotting and immunohistochemistry, we showed that visfatin is expressed not only in hGCs and KGN cells, but also in human cumulus cells and oocytes. In hGCs and KGN cells, MetF increased visfatin mRNA in a dose-dependent manner (0.1, 1 and 10 mM), and rosiglitazone increased visfatin mRNA expression (only at 10 µM) after treatments for 24 h, whereas both reduced it after 48 h of incubation. This regulation was confirmed at the protein level for the MetF treatment only. Using the compound C and Aicar, inhibitor and activator of AMP-activated protein kinase (AMPK), respectively, and Sirtinol, an inhibitor of sirtuin-1 (SIRT1), we observed that these MetF effects on visfatin expression were mediated through the AMPK/SIRT1 signalling pathways. RhVisf (10 ng/ml) significantly increased insulin-like growth factor-1 (IGF-1) (10 nM)- but not FSH (10 nM)-induced secretion of progesterone and estradiol as determined by radioimmunoassay and IGF-1-induced thymidine incorporation in hGCs and KGN cells. Finally, rhVisf rapidly activates the mitogen-activated protein kinase pathway via ERK1/2, P38 and Akt phosphorylation under basal conditions in primary hGC cells. In conclusion, visfatin is present in ovarian human follicles, and in hGCs and KGN cells, visfatin increases IGF-1-induced steroidogenesis and cell proliferation and MetF regulates visfatin expression through the AMPK/SIRT1 signalling pathway.

Is NMR metabolic profiling of spent embryo culture media useful to assist in vitro human embryo selection?

OBJECT: The prediction of embryo viability by usual morphological analysis is currently unsatisfactory. New non-invasive techniques such as high-resolution nuclear magnetic resonance ((1)H-NMR) spectroscopy that allows assessment of metabolic profiling in spent culture media might help embryologists to predict embryo development. MATERIALS AND METHODS: Individual microdrops of culture media were analysed after 24 h of embryo culture (from day 3 to day 4) by spectroscopy using a 1 mm microliter probe allowing analysis without sample dilution. Embryos were divided into two groups on day 5: non-arrested embryos (n = 19) and arrested embryos unable to reach the blastocyst stage (n = 20). Multivariate analysis techniques such as Principal Component Analysis (PCA) and Orthogonal Partial Least Square Discriminant Analysis (OPLS-DA) were performed to compare extracellular metabolite balance. RESULTS: (1)H-NMR used in combination with a 1 mm probe suggested that in vitro cultured human embryos that have a high developmental potential modify their environment slightly compared to embryos that cease to develop. However, differences between the two groups did not reach statistical significance and multivariate statistical analysis did not allow clustering of the two groups. CONCLUSION: This study indicated that this technique would not be sufficiently powerful alone to provide information that might help to assess the developmental potential of individual embryos for in vitro fertilisation (IVF).

Genomic assessment of human cumulus cell marker genes as predictors of oocyte developmental competence: impact of various experimental factors.

BACKGROUND: Single embryo transfer (SET) is the most successful way to reduce the frequency of multiple pregnancies following in vitro fertilisation. However, selecting the embryo for SET with the highest chances of pregnancy remains a difficult challenge since morphological and kinetics criteria provide poor prediction of both developmental and implantation ability. Partly through the expression of specific genes, the oocyte-cumulus interaction helps the oocyte to acquire its developmental competence. Our aim was therefore to identify at the level of cumulus cells (CCs) genes related to oocyte developmental competence. METHODOLOGY/PRINCIPAL FINDINGS: 197 individual CCs were collected from 106 patients undergoing an intra-cytoplasmic sperm injection procedure. Gene expression of CCs was studied using microarray according to the nuclear maturity of the oocyte (immature vs. mature oocyte) and to the developmental competence of the oocyte (ability to reach the blastocyst stage after fertilisation). Microarray study was followed by a meta-analysis of the behaviour of these genes in other datasets available in Gene Expression Omnibus which showed the consistency of this list of genes. Finally, 8 genes were selected according to oocyte developmental competence from the 308 differentially expressed genes (p<0.0001) for further validation by quantitative PCR (qPCR). Three of these 8 selected genes were validated as potential biomarkers (PLIN2, RGS2 and ANG). Experimental factors such as inter-patient and qPCR series variability were then assessed using the Generalised Linear Mixed Model procedure, and only the expression level of RGS2 was confirmed to be related to oocyte developmental competence. The link between biomarkers and pregnancy was finally evaluated and level of RGS2 expression was also correlated with clinical pregnancy. CONCLUSION/SIGNIFICANCE: RGS2, known as a regulator of G protein signalling, was the only gene among our 8 selected candidates biomarkers of oocyte competence to cover many factors of variability, including inter-patient factors and experimental conditions.

Chemerin inhibits IGF-1-induced progesterone and estradiol secretion in human granulosa cells.

BACKGROUND: Chemerin is a novel adipokine involved in the regulation of adipocyte development, inflammation and metabolic functions. To date, no role of this adipokine in reproductive functions has been described. In the present study, we identified chemerin and its receptor, CMKLR1 (chemokine-like receptor 1), in primary human granulosa cells (hGCs) and in a human ovarian granulosa-like tumour cell line (KGN). We also investigated the effects of recombinant human chemerin (rhChem) on steroid production and on various signalling pathways. METHODS AND RESULTS: By RT-PCR immunoblotting and immunohistochemistry, we showed that chemerin and CMKLR1 are expressed in hGCs and KGN cells. By ELISA, we also found chemerin in human follicular fluid and we observed that in 8 of 10 women the chemerin level was at least 2-fold higher in follicular fluid than in plasma. rhChem (10 or 100 ng/ml) significantly decreased insulin-like growth factor-1 (IGF-1) (10(-8) M)-induced secretion of progesterone and estradiol (as determined by radioimmunoassay) but did not affect basal-or FSH (10(-8) M)-induced steroid secretion in hGCs and KGN cells. In parallel, it also decreased IGF-1-induced p450 aromatase protein levels without affecting the protein levels of other factors involved in steroidogenesis (steroidogenic acute regulatory protein, 3-beta-hydroxysteroid dehydrogenase and p450 side-chain cleavage enzyme) in hGCs cells. All these changes were associated with a decrease in the IGF-1-induced tyrosine phosphorylation of IGF-1 receptor beta subunit and phosphorylation of mitogen-activated protein kinase extracellular signal-regulated kinases 1/2 (MAPK ERK1/2) and Akt. In hGCs and KGN cells, rhChem also decreased IGF-1-induced thymidine incorporation. Finally, we showed that rhChem rapidly activates MAPK ERK1/2, MAPK P38 and Akt phosphorylation and more slowly AMP-activated protein kinase phosphorylation under basal conditions (no IGF-1 or FSH) in primary hGC cells. CONCLUSIONS: Taken together, chemerin and its receptor (CMKLR1) are present and active in hGCs. Chemerin reduces IGF-1-induced steroidogenesis and cell proliferation through a decrease in the activation of IGF-1R signalling pathways in primary hGCs.

Preferential ß-arrestin signalling at low receptor density revealed by functional characterization of the human FSH receptor A189 V mutation.

The A189 V inactivating mutation of the human FSH receptor (FSHR) leads to subfertility in men and primary ovarian failure in women. This mutation has previously been associated with intracellular retention of the FSHR and impaired cAMP production. Here, we show that the A189 V FSHR stably expressed in HEK293N cells provoked ERK MAP kinases phosphorylation through ß-arrestins, independently of the canonical cAMP/PKA pathway. Interesting, both the A189 V and wild-type (Wt) FSHRs selectively activated cAMP-independent ERK phosphorylation when expressed at low plasma membrane densities. These data indicate that the selective intracellular signalling triggered by the A189 V FSHR resulted from reduced membrane expression rather than by switching receptor coupling. Hence, receptor density at the plasma membrane might control the balance between distinct signal transduction mechanisms. Furthermore, our results help to clarify why mutations of FSHß are more deleterious to human fertility than the FSHR A189 V mutation which preserves parts of receptor signalling repertoire.

Conjoined twins after intracytoplasmic sperm injection and transfer of a single day 2 embryo: case report.

OBJECTIVE: To describe a case of conjoined twins after intracytoplasmic sperm injection (ICSI) and transfer of a single day 2 embryo. DESIGN: Case report. SETTING: University hospital. PATIENT(S): A 30-year-old woman underwent ICSI with a transfer of a single day 2 embryo. INTERVENTION(S): Vaginal ultrasound examination was performed during early pregnancy. RESULT(S): The diagnosis of conjoined twins was made by vaginal ultrasonography at 9 weeks' gestation. The conjoined fetuses were diagnosed as thoracopagus twins with a single beating heart shared between the thoraxes. Extensive ultrasound examination suggested an extremely poor prognosis, Therapeutic termination was performed at 11 weeks. CONCLUSION(S): Given that single embryo transfers are able to shed light on the real incidence of conjoined twins developed on day 2/3 or on day 5/6, this is of interest mechanistically. Thus, it should be taken into account when trying to determine the true incidence of conjoined twins and ultimately trying to understand why these anomalies occur. The importance of early expert vaginal ultrasonography in pregnancies resulting from assisted reproductive technologies is emphasized.

Top quality embryos at day 2: a prerequisite for single blastocyst transfer? An observational cohort study in women under 36.

PURPOSE: While extended culture has been considerably improved, some questions remain regarding the application of Single Blastocyst Transfer (SBT). METHODS: An observational cohort study was undertaken with 456 women under 36 years old and assigned to SBT on a voluntary basis. The main outcome was the cumulative delivery rate per couple according to the number of Top Quality Embryos (TQE) on day 2 (Group 1= > or =2 TQE, Group 2= 1 TQE and Group 3= 0 TQE). RESULTS: Rate of transfer and mean number of frozen blastocyts were higher in Group 1 compared to Group 3. As a consequence, the cumulative delivery rate per couple was higher in Group 1 (47.9%) compared to Group 3 (34.9%). CONCLUSIONS: Single blastocyst transfer combining fresh and frozen cycles, might be a worthwhile strategy irrespective of embryo quality on day 2 providing good delivery rates while keeping the rate of multiple deliveries low.

Adiponectin increases insulin-like growth factor I-induced progesterone and estradiol secretion in human granulosa cells.

OBJECTIVE: To identify adiponectin and its receptors (AdipoR1 and AdipoR2) in human granulosa cells (GC) and to study the effects of recombinant human adiponectin on P and E(2) secretion from these cells. DESIGN: The effects of recombinant human adiponectin on the secretion of P and E(2) by cultured human GCs were investigated. SETTING: Academic institutions. PATIENT(S): Seventeen infertile and healthy women undergoing IVF. INTERVENTION(S): Primary human GC cultures stimulated with human recombinant adiponectin (5 microg/mL). MAIN OUTCOME MEASURE(S): Determination of messenger RNA (mRNA) and protein expression of adiponectin and its receptors AdipoR1 and AdipoR2 in fresh human GCs by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblot, respectively. Measurement of P and E(2) levels in the conditioned media by RIA and determination of cell proliferation by tritied thymidine incorporation. RESULT(S): Human GCs express adiponectin receptors AdipoR1 and AdipoR2 but not adiponectin. In primary human GCs, adiponectin increases P and E(2) secretion in response to insulin-like growth factor I (IGF-I). This was associated with an increase in the p450 aromatase protein level but not those of p450scc, 3 beta HSD, or StAR. Adiponectin treatment does not affect IGF-1-induced cell proliferation and basal steroidogenesis (no IGF-1 or FSH stimulation). Adiponectin rapidly stimulates MAPK ERK1/2 and p38 phosphorylation in primary human GCs. CONCLUSION(S): Adiponectin receptors AdipoR1 and AdipoR2, but not adiponectin, are present in human GCs. Adiponectin increases IGF-1-induced P and E(2) secretion in primary human GCs.

Revised guidelines for good practice in IVF laboratories.

The 'ESHRE Guidelines for Good Practice in IVF Laboratories' were drawn up by the Special Interest Group (SIG) in Embryology and published in the year 2000, and since then they constitute the minimal requirements for any laboratory offering assisted reproduction techniques (ART). In the understanding that the embryologist has a responsibility for the correct and justified application of ART in the laboratory, the implementation of these guidelines requires a quality management programme to be in place that encompasses and integrates the operative units, the processes and procedures that represent the core of ART clinics. In March 2004, the European Parliament issued the Directive 2004/23/EC 'On setting standards of quality and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells'. The Directive applies to human tissues and cells, including fresh or frozen reproductive cells for application to the human body, and is mainly concerned with increasing quality and safety through the implementation of a quality management system. Therefore, the European Society of Human Reproduction and Embryology (ESHRE) undertook a series of initiatives aiming to promote assurance of good laboratory practice and to define the concept of qualified embryologists. One ESHRE initiative was to revise the guidelines for good practice in IVF laboratories, not only in response to the need of embryologists for support and guidance in their duties, but also as a complement to the requirements issued by the Tissue and Cell Directive. The SIG in Embryology hopes that this document may assist the laboratory staff to operate according to the requirements of harmonization, implementation, inspection and certification that are now common to all European member states.

Zygote arrest 1 gene in pig, cattle and human: evidence of different transcript variants in male and female germ cells.

BACKGROUND: Zygote arrest 1 (ZAR1) is one of the few known oocyte-specific maternal-effect genes essential for the beginning of embryo development discovered in mice. This gene is evolutionary conserved in vertebrates and ZAR1 protein is characterized by the presence of atypical plant homeobox zing finger domain, suggesting its role in transcription regulation. This work was aimed at the study of this gene, which could be one of the key regulators of successful preimplantation development of domestic animals, in pig and cattle, as compared with human. METHODS: Screenings of somatic cell hybrid panels and in silico research were performed to characterize ZAR1 chromosome localization and sequences. Rapid amplification of cDNA ends was used to obtain full-length cDNAs. Spatio-temporal mRNA expression patterns were studied using Northern blot, reverse transcription coupled to polymerase chain reaction and in situ hybridization. RESULTS: We demonstrated that ZAR1 is a single copy gene, positioned on chromosome 8 in pig and 6 in cattle, and several variants of correspondent cDNA were cloned from oocytes. Sequence analysis of ZAR1 cDNAs evidenced numerous short inverted repeats within the coding sequences and putative Pumilio-binding and embryo-deadenylation elements within the 3'-untranslated regions, indicating the potential regulation ways. We showed that ZAR1 expressed exclusively in oocytes in pig ovary, persisted during first cleavages in embryos developed in vivo and declined sharply in morulae and blastocysts. ZAR1 mRNA was also detected in testis, and, at lower level, in hypothalamus and pituitary in both species. For the first time, ZAR1 was localized in testicular germ cells, notably in round spermatids. In addition, in pig, cattle and human only shorter ZAR1 transcript variants resulting from alternative splicing were found in testis as compared to oocyte. CONCLUSION: Our data suggest that in addition to its role in early embryo development highlighted by expression pattern of full-length transcript in oocytes and early embryos, ZAR1 could also be implicated in the regulation of meiosis and post meiotic differentiation of male and female germ cells through expression of shorter splicing variants. Species conservation of ZAR1 expression and regulation underlines the central role of this gene in early reproductive processes.

A mechanistic overview on male infertility and germ cell cancers.

The testis is devoted to two important tasks: haploid cell production and sexual steroid synthesis. A number of highly sophisticated and unique strategies operate during spermatogenesis, a process crucial for reproduction, heredity and evolution. It is particularly important to decipher the underlying molecular mechanisms whose function can be perverted in pathological situations, such as infertility and testicular cancers, which represent an increasing biomedical issue today. This review summarises the currently available data concerning some key molecular components that are altered or potentially involved in male infertility and testicular tumors, with the aim of defining some common "hot spots". We particularly focused on genetically engineered in vivo models in which testicular functions are altered and we pinpointed to the potential involvement of the targeted genes in testicular pathologies. Those molecular mechanisms peculiar to the male gonad can be envisioned as a basis for the design of novel drugs potentially dedicated to testicular dysfunction.

Overnight incubation improves selection of frozen-thawed blastocysts for transfer: preliminary study using supernumerary embryos.

A study was undertaken to determine whether the interval between thawing and transfer influences both biological and clinical outcomes of cryopreserved blastocysts, using supernumerary embryos cultured in sequential media. One hundred and seventy-two patients who underwent blastocyst thawing without any exclusion criteria were included in this single center prospective study of blastocyst thawing cycles. Outcome of 338 blastocysts originating from culture of supernumerary embryos in sequential media was analyzed after 4 or 20 h of culture between thawing and transfer. Survival rate, re-expansion and hatching rates for surviving blastocysts, implantation rates (IRs), pregnancy and miscarriage rates were studied. Blastocyst survival was not influenced by the incubation time after thawing; however both re-expansion and hatching rates were increased after 20-h incubation. Moreover, the IR per thawed or transferred blastocyst was increased three-fold after 20-h incubation compared to 4-h incubation. Increasing the interval between thawing and transfer appears to be beneficial in order to better select for transfer frozen-thawed blastocysts.

Cohort follow-up of couples with primary infertility in an ART programme using frozen donor semen.

BACKGROUND: This study was designed to determine the crude cumulative live-birth rates in a cohort initiating frozen donor semen treatment until completion. METHODS: This cohort study included 588 couples with primary infertility in one University Hospital centre. The treatment sequence involved first artificial insemination (AID) followed by IVF if necessary (IVF-D). Live birth, drop-out for personal or medical reasons and recourse to IVF-D were recorded for all patients. Live births and drop-out were expressed both as rates per cycle and crude cumulative rates. RESULTS: At the completion of AID and IVF-D cycles, 406 couples in the cohort (69%) achieved a live-birth and 182 couples (31%) discontinued treatment. In most cases, couples stopped treatment for personal reasons (74%) whereas fewer couples were denied further treatment for medical reasons (26%). CONCLUSIONS: This is the first report on the crude cumulative live-birth rate in a cohort after AID and IVF-D cycles. Although calculation based on crude cumulative live-birth rate shows lower results in comparison with life table analysis, this method allows patients to obtain an insight into their actual chances of achieving a successful pregnancy.