Surgical management of endometriotic women with pregnancy intention in France: A national snapshot of centers performing a high volume of endometriosis procedures.

OBJECTIVE: To provide a snapshot of the surgical management of endometriosis in French high-volume activity centers. METHODS: Analysis of prospectively collected data between November 2015 and May 2017 in 21 centers with a high volume of endometriosis surgery in France. Each facility could include up to 40 patients undergoing laparoscopy for endometriosis. Data were collected before and two months after surgery. RESULTS: 361 patients were enrolled in the study. Twenty-seven patients (7.48%) were lost to follow-up at the month 2 visit. Endometriosis stage was I-II in 33.70% of patients and III-IV in 66.30%. Uterosacral ligament resection was the most frequently performed procedure (50.97%) followed by rectal surgery (31.58%), ovarian procedures for endometrioma, procedures for ureters (21.33%) and the bladder (11.91%). Antiadhesion agents were employed in 215/361 (59.56%) patients. The median length of hospital stay after surgery was 2 (IQR 1 - 4) days. Post-operative complications were recorded in 9.34% of patients. Rectovaginal fistulae occurred in 8 patients (2.41%), pelvic abscess in 4 (1.20%) and bladder atony in 3 (0.90%). 17 patients (5.14%) required a second surgical procedure after a median time of 31 days (IQR 9 - 81). Two months after surgery, 95.09% of patients reported being satisfied or very satisfied with the surgery. CONCLUSION: Our study shows that surgical management of endometriosis in centers with a high volume of endometriosis surgery, mainly concerns women presenting with severe disease and deep localizations, with an overall risk of major complications inferior to 10% and a high rate of patient satisfaction.

Endometrial receptivity profile in patients with premature progesterone elevation on the day of HCG administration.

The impact of a premature elevation of serum progesterone level, the day of hCG administration in patients under controlled ovarian stimulation during IVF procedure, on human endometrial receptivity is still debated. In the present study, we investigated the endometrial gene expression profile shifts during the prereceptive and receptive secretory stage in patients with normal and elevated serum progesterone level on the day of hCG administration in fifteen patients under stimulated cycles. Then, specific biomarkers of endometrial receptivity in these two groups of patients were tested. Endometrial biopsies were performed on oocyte retrieval day and on day 3 of embryo transfer, respectively, for each patient. Samples were analysed using DNA microarrays and qRT-PCR. The endometrial gene expression shift from the prereceptive to the receptive stage was altered in patients with high serum progesterone level (>1.5 ng/mL) on hCG day, suggesting accelerated endometrial maturation during the periovulation period. This was confirmed by the functional annotation of the differentially expressed genes as it showed downregulation of cell cycle-related genes. Conversely, the profile of endometrial receptivity was comparable in both groups. Premature progesterone rise alters the endometrial gene expression shift between the prereceptive and the receptive stage but does not affect endometrial receptivity.

Comparative gene expression profiling in human cumulus cells according to ovarian gonadotropin treatments.

In in vitro fertilization cycles, both HP-hMG and rFSH gonadotropin treatments are widely used to control human follicle development. The objectives of this study are (i) to characterize and compare gene expression profiles in cumulus cells (CCs) of periovulatory follicles obtained from patients stimulated with HP-hMG or rFSH in a GnRH antagonist cycle and (ii) to examine their relationship with in vitro embryo development, using Human Genome U133 Plus 2.0 microarrays. Genes that were upregulated in HP-hMG-treated CCs are involved in lipid metabolism (GM2A) and cell-to-cell interactions (GJA5). Conversely, genes upregulated in rFSH-treated CCs are implicated in cell assembly and organization (COL1A1 and COL3A1). Interestingly, some genes specific to each gonadotropin treatment (NPY1R and GM2A for HP-hMG; GREM1 and OSBPL6 for rFSH) were associated with day 3 embryo quality and blastocyst grade at day 5, while others (STC2 and PTX3) were related to in vitro embryo quality in both gonadotropin treatments. These genes may prove valuable as biomarkers of in vitro embryo quality.

Transcriptome analysis during human trophectoderm specification suggests new roles of metabolic and epigenetic genes.

In humans, successful pregnancy depends on a cascade of dynamic events during early embryonic development. Unfortunately, molecular data on these critical events is scarce. To improve our understanding of the molecular mechanisms that govern the specification/development of the trophoblast cell lineage, the transcriptome of human trophectoderm (TE) cells from day 5 blastocysts was compared to that of single day 3 embryos from our in vitro fertilization program by using Human Genome U133 Plus 2.0 microarrays. Some of the microarray data were validated by quantitative RT-PCR. The TE molecular signature included 2,196 transcripts, among which were genes already known to be TE-specific (GATA2, GATA3 and GCM1) but also genes involved in trophoblast invasion (MUC15), chromatin remodeling (specifically the DNA methyltransferase DNMT3L) and steroid metabolism (HSD3B1, HSD17B1 and FDX1). In day 3 human embryos 1,714 transcripts were specifically up-regulated. Besides stemness genes such as NANOG and DPPA2, this signature included genes belonging to the NLR family (NALP4, 5, 9, 11 and 13), Ret finger protein-like family (RFPL1, 2 and 3), Melanoma Antigen family (MAGEA1, 2, 3, 5, 6 and 12) and previously unreported transcripts, such as MBD3L2 and ZSCAN4. This study provides a comprehensive outlook of the genes that are expressed during the initial embryo-trophectoderm transition in humans. Further understanding of the biological functions of the key genes involved in steroidogenesis and epigenetic regulation of transcription that are up-regulated in TE cells may clarify their contribution to TE specification and might also provide new biomarkers for the selection of viable and competent blastocysts.

Analysis of cytokines in the peritoneal fluid of endometriosis patients as a function of the menstrual cycle stage using the Bio-Plex® platform.

OBJECTIVE: Endometriosis is a painful disease affecting 10-15% of reproductive-age women. Concentrations of several cytokines and angiogenic factors in peritoneal fluid (PF) have been found to correlate with the severity of the disease. However, levels of some analytes vary across the menstrual cycle, and an ideal biomarker of endometriosis has not yet been identified. We have compared the PF concentrations of different cytokines in proliferative and secretory phases in women with and without the disease using the Bio-Plex platform. METHODS: PF was aspirated during laparoscopy (N = 133) and the PF concentrations of 18 cytokines from Bio-Plex panels I and II determined with the serum protocol. RESULTS: Increased PF concentrations of IL-6, IL-18, eotaxin, and MCP-1 were found in endometriosis with no changes with menstrual cycle. Levels of IL-12(p70), ICAM-1, and GRO-α were higher in the secretory phase, while eotaxin concentrations were lower. CONCLUSION: Of the 18 cytokines tested, IL-6, IL-18 and MCP-1 were the best PF markers of endometriosis.

Differences in transcriptomic profiles of human cumulus cells isolated from oocytes at GV, MI and MII stages after in vivo and in vitro oocyte maturation.

BACKGROUND: Oocyte maturation and competence to development depends on its close relationship with cumulus cells (CCs). However, the maturation conditions of human cumulus-oocyte complexes (COCs) might affect gene expression in both oocyte and CCs. We thus compared the transcriptome profiles of CCs isolated from in vivo and in vitro matured COCs at different nuclear maturation stages. METHODS: Three groups of CCs from patients who underwent ICSI were included: CCs of patients with polycystic ovary syndrome (PCOS) referred for in vitro maturation (IVM), CCs from patients with PCOS for in vivo maturation (used as controls) and CCs from normal responders referred for in vivo maturation. CCs were isolated from COCs at the germinal vesicle, metaphase I and metaphase II stages. Microarray technology was used to analyse the global gene expression and significance analysis of microarray to compare the expression profiles of CCs from COCs at different nuclear maturation stages following IVM or in vivo maturation. Selected genes were validated by RT-qPCR. RESULTS: In CCs isolated after IVM, genes related to cumulus expansion and oocyte maturation, such as EREG, AREG and PTX3, were down-regulated, while cell cycle-related genes were up-regulated in comparison with CCs from in vivo matured COCs from PCOS and normal responder patients. Moreover, irrespective of the stage of oocyte maturation, genes involved in DNA replication, recombination and repair were up-regulated in CCs after IVM. CONCLUSIONS: The CC transcriptomic signature varies according to both the oocyte maturation stage and the maturation conditions. Our findings suggest a delay in the acquisition of the mature CC phenotype following IVM, opening a new perspective for the improvement in IVM conditions.

Insights into human endometrial receptivity from transcriptomic and proteomic data.

The appreciation of endometrial receptivity is a crucial step in assisted reproductive technology as implantation failures are thought to result, in large part, from abnormal endometrial receptivity. Using emerging omics technologies, investigators have begun to define both molecular signatures and specific biomarkers of receptive endometrium. The aim of this review was to analyse the new perspectives brought to the appreciation of endometrial receptivity by transcriptomic and proteomic technologies, involving the analysis of gene- or protein-expression-profile shifts between the pre-receptive and receptive secretory stages and how they might lead to new strategies for endometrial receptivity assessments. The use of omics as molecular tools to determine the effects of stimulation protocols on endometrial gene expression and clinical outcomes has also been investigated.

Human cumulus cells molecular signature in relation to oocyte nuclear maturity stage.

The bi-directional communication between the oocyte and the surrounding cumulus cells (CCs) is crucial for the acquisition of oocyte competence. We investigated the transcriptomic profile of human CCs isolated from mature and immature oocytes under stimulated cycle. We used human Genome U133 Plus 2.0 microarrays to perform an extensive analysis of the genes expressed in human CCs obtained from patients undergoing intra-cytoplasmic sperm injection. CC samples were isolated from oocyte at germinal vesicle, stage metaphase I and stage metaphase II. For microarray analysis, we used eight chips for each CC category. Significance analysis of microarray multiclass was used to analyze the microarray data. Validation was performed by RT-qPCR using an independent cohort of CC samples. We identified differentially over-expressed genes between the three CC categories. This study revealed a specific signature of gene expression in CCs issued from MII oocyte compared with germinal vesicle and metaphase I. The CC gene expression profile, which is specific of MII mature oocyte, can be useful as predictors of oocyte quality.

Endothelin-dependent vasoconstriction in human uterine artery: application to preeclampsia.

BACKGROUND: Reduced uteroplacental perfusion, the initiating event in preeclampsia, is associated with enhanced endothelin-1 (ET-1) production which feeds the vasoconstriction of uterine artery. Whether the treatments of preeclampsia were effective on ET-1 induced contraction and could reverse placental ischemia is the question addressed in this study. We investigated the effect of antihypertensive drugs used in preeclampsia and of ET receptor antagonists on the contractile response to ET-1 on human uterine arteries. METHODOLOGY/PRINCIPAL FINDINGS: Experiments were performed, ex vivo, on human uterine artery samples obtained after hysterectomy. We studied variations in isometric tension of arterial rings in response to the vasoconstrictor ET-1 and evaluated the effects of various vasodilators and ET-receptor antagonists on this response. Among antihypertensive drugs, only dihydropyridines were effective in blocking and reversing the ET-1 contractile response. Their efficiency, independent of the concentration of ET-1, was only partial. Hydralazine, alpha-methyldopa and labetalol had no effect on ET-1 induced contraction which is mediated by both ET(A) and ET(B) receptors in uterine artery. ET receptors antagonists, BQ-123 and BQ-788, slightly reduced the amplitude of the response to ET-1. Combination of both antagonists was more efficient, but it was not possible to reverse the maximal ET-1-induced contraction with antagonists used alone or in combination. CONCLUSION: Pharmacological drugs currently used in the context of preeclampsia, do not reverse ET-1 induced contraction. Only dihydropyridines, which partially relax uterine artery previously contracted with ET-1, might offer interesting perspectives to improve placental perfusion.

Involvement of BCL2 family members in the regulation of human oocyte and early embryo survival and death: gene expression and beyond.

In women, up to 99.9% of the oocyte stockpile formed during fetal life is decimated by apoptosis. Apoptotic features are also detected in human preimplantation embryos both in vivo and in vitro. Despite the important consequences of cell death processes to oocyte competence and early embryonic development, little is known about its genetic and molecular control. B cell lymphoma-2 (BCL2) family proteins are major regulators of cell death and survival. Here, we present a literature review on BCL2 family expression and protein distribution in human and animal oocytes and early embryos. Most of the studies focused on the expression of two antagonistic members: the founding and survival family member BCL2 and its proapoptotic homolog BAX. However, recent transcriptomic analyses have identified novel candidate genes related to oocyte and/or early embryonic viability (such as BCL2L10) or commitment to apoptosis (e.g. BIK). Interestingly, some BCL2 proteins appear to be differentially distributed at the subcellular level during oocyte maturation and early embryonic development, a process probably linked to the functional compartmentalization of the ooplasm and blastomere. Assessment of BCL2 family involvement in regulating the survival of human oocytes and embryos may be of particular value for diagnosis and assisted reproductive technology. We suggest that implications of not only aberrant gene expression but also abnormal subcellular protein redistribution should be established in pathological conditions resulting in infertility.

Dynamic changes in gene expression during human early embryo development: from fundamental aspects to clinical applications.

BACKGROUND: The first week of human embryonic development comprises a series of events that change highly specialized germ cells into undifferentiated human embryonic stem cells (hESCs) that display an extraordinarily broad developmental potential. The understanding of these events is crucial to the improvement of the success rate of in vitro fertilization. With the emergence of new technologies such as Omics, the gene expression profiling of human oocytes, embryos and hESCs has been performed and generated a flood of data related to the molecular signature of early embryo development. METHODS: In order to understand the complex genetic network that controls the first week of embryo development, we performed a systematic review and study of this issue. We performed a literature search using PubMed and EMBASE to identify all relevant studies published as original articles in English up to March 2010 (n = 165). We also analyzed the transcriptome of human oocytes, embryos and hESCs. RESULTS: Distinct sets of genes were revealed by comparing the expression profiles of oocytes, embryos on Day 3 and hESCs, which are associated with totipotency, pluripotency and reprogramming properties, respectively. Known components of two signaling pathways (WNT and transforming growth factor-ß) were linked to oocyte maturation and early embryonic development. CONCLUSIONS: Omics analysis provides tools for understanding the molecular mechanisms and signaling pathways controlling early embryonic development. Furthermore, we discuss the clinical relevance of using a non-invasive molecular approach to embryo selection for the single-embryo transfer program.

Controlled ovarian hyperstimulation for in vitro fertilization alters endometrial receptivity in humans: protocol effects.

The impact of gonadotropin-releasing hormone (GnRH) agonist long compared with GnRH antagonist protocols, under in vitro fertilization conditions on endometrial receptivity, is still debated. Therefore, we compared the effect of both GnRH antagonist and agonist long protocols on the endometrial receptivity by analyzing, to our knowledge for the first time, the global gene expression profile shift during the prereceptive and receptive stages of stimulated cycles under the two GnRH analogue protocols compared with natural cycles in the same patients. For the same normal-responder patients, endometrial biopsies were collected on the day of oocyte retrieval and on the day of embryo transfer after human chorionic gonadotropin administration of a stimulated cycle with either GnRH agonist long or GnRH antagonist protocols and compared with the prereceptive and receptive stages of a natural cycle. Samples were analyzed using DNA microarrays. Gene expression profiles and biological pathways involved during the prereceptive stage to the receptive endometrial transition of stimulated and natural cycles were analyzed and compared for each patient. Both protocols affect endometrial receptivity in comparison with their natural cycle in the same patients. Major differences in endometrial chemokines and growth factors under stimulated cycles in comparison with natural cycles were observed. Such an effect has been associated with gene expression alterations of endometrial receptivity. However, the endometrial receptivity under the GnRH antagonist protocol was more similar to the natural cycle receptivity than that under the GnRH agonist protocol.

Phylogeny, diversity and host specialization in the phylum Synergistetes with emphasis on strains and clones of human origin.

Members of the phylum Synergistetes have been demonstrated in several environmental ecosystems and mammalian microflorae by culture-independent methods. In the past few years, the clinical relevance of some uncultivated phylotypes has been demonstrated in endodontic infections, and uncultured Synergistetes have been demonstrated in human mouth, gut and skin microbiota. However, Synergistetes are rarely cultured from human samples, and only 17 isolates are currently reported. Twelve members of Synergistetes isolated in the course of various infectious processes, including 3 Jonquetella anthropi, 2 Cloacibacillus evryensis, 2 Pyramidobacter piscolens and 5 unidentified strains, as well as 56 clones obtained by specific PCR from the normal vaginal microflora, were studied. 16S rRNA gene-based phylogeny showed that the clones were grouped into 3 clusters, corresponding to the genus Jonquetella, P. piscolens and one novel Synergistetes taxon. The presence and diversity of Synergistetes were reported for the first time in the vaginal microflora. Synergistetes were found in healthy patients, suggesting that they could play a functional role in human microflorae, but may also act as opportunistic pathogens. Studying the phylogenetic relationships between environmental and mammalian strains and clones revealed clearly delineated independent lineages according to the origin of the sequences.

Endometriosis and in vitro fertilisation: a review.

This review aims to evaluate whether severe endometriosis has an impact on the outcome of in vitro fertilisation (IVF), whether IVF is associated with specific complications in this context, whether a specific ovarian stimulation protocol is most appropriate, whether the endometrial condition progresses following ovarian stimulation, and whether endometrial cysts pose a specific problem for IVF. In patients with severe endometriosis, IVF represents an effective treatment option for infertility, as a complement to surgery. The prognostic parameters of IVF are identical to those of other patients. However, the risks related to the severity of endometriosis, particularly the risk of ovarian deficiency, need to be considered. Because of this issue, to which endometriosis-related pain often adds, IVF treatment should be initiated as early as possible, using appropriate protocols and after having fully informed the patient about the specific oocytes retrieval-related risks.

Total fertilization failure and molecular abnormalities in metaphase II oocytes.

Total fertilization failures (TFF) are rare events of IVF by intracytoplasmic sperm injection (ICSI). When male factor is excluded, the lack of identifiable aetiological criteria raises the question of the reliable clinical management. The goal of this study was to identify molecular abnormalities in metaphase II (MII) oocytes yielding TFF. The nuclear mature MII oocytes mRNA expression profiles were compared between a 30-year-old patient who had experienced three successive TFF (egg number = 39) and control patients with fertile cohorts diagnosed with tubal or male infertility. The mRNA abundance for the 30,000 genes of the genome was evaluated by microarray and, for selected genes, by quantitative-polymerase chain reaction analysis. Transcriptional analysis of unfertilized MII oocytes revealed an altered gene expression profile associated with TFF. Meiosis, cell growth and apoptosis controlling genes were mis-expressed with important fold changes. The results reveal that, despite passing the pre-IVF morphological examination, high-grade oocytes may carry substantial molecular abnormalities at the gene expression level associated with failure of MII oocyte activation. In the absence of an identifiable defect causing TFF, this microarray approach allows improvement of clinical therapeutic management: informed counselling about alternate therapeutic solutions could be proposed.

Optimal timing of ultrasonographic and Doppler evaluation of uterine receptivity to implantation.

In IVF programmes, transvaginal ultrasonography is used as a non-invasive method to evaluate uterine receptivity. The aim of this study was to determine when to perform this investigation in order to optimize prediction of the likelihood of pregnancy. Over 9 months, 124 patients undergoing IVF or intracytoplasmic sperm injection were studied. The ultrasonographic evaluation included endometrial thickness, endometrial pattern, uterine artery pulsatility index, protodiastolic notch, end-diastolic blood flow, and endometrial-subendometrial blood flow distribution pattern. All patients underwent ultrasonographic investigation on the days of human chorionic gonadotrophin (HCG) administration, oocyte retrieval, and embryo transfer. Statistical analysis was done using recursive-partitioning analysis. The pregnancy and implantation rates per transfer were 33 and 19.8% respectively. In terms of single parameters, women with an end-diastolic blood flow, an endometrial-subendometrial blood flow and a multilayered endometrium were more likely to be pregnant than women without one or more of these signs. The most effective combination for evaluation of uterine receptivity was end-diastolic blood flow, endometrial pattern and endometrial thickness. Sensitivity and specificity of this combination were around 81%, positive predictive value was 68.2%, and negative predictive value 89.7%. The best sensitivity and specificity were obtained on the day of HCG administration: respectively 81.1 and 81.3%.

Identifying new human oocyte marker genes: a microarray approach.

The efficacy of classical IVF techniques is still impaired by poor implantation and pregnancy rates after embryo transfer. This is mainly due to a lack of reliable criteria for the selection of embryos with sufficient development potential. Several studies have provided evidence that some gene expression levels could be used as objective markers of oocyte and embryo competence and capacity to sustain a successful pregnancy. These analyses usually use reverse transcription-polymerase chain reaction to look at small sets of pre-selected genes. However, microarray approaches allow the identification of a wider range of cellular marker genes which could include additional and perhaps more suitable genes that could serve as embryo selection markers. Microarray screenings of around 30,000 genes on U133P Affymetrix gene chips made it possible to establish the expression profile of these genes as well as other related genes in human oocytes and cumulus cells. This study identifies new potential regulators and marker genes such as BARD1, RBL2, RBBP7, BUB3 or BUB1B, which are involved in oocyte maturation.

Comparative protein expression profiling in human cumulus cells in relation to oocyte fertilization and ovarian stimulation protocol.

Comparative profiling was performed on proteins synthesized in human cumulus cells (CC) from individual oocytes recovered after two different ovarian stimulation protocols for classical IVF (cIVF). Using high-resolution two-dimensional protein electrophoresis after metabolic labelling with [35S]-methionine, protein expression was profiled in CC of metaphase II oocytes obtained after two different ovarian stimulation protocols (rFSH versus human menopausal gonadotrophin). Analysis was done on CC from two cIVF cycles in the same patient and then extended to CC from individual oocytes from two groups of patients. CC from single oocytes have robust levels of protein expression into 600-800 protein spots. Comparison of CC protein expression from oocytes obtained from the same patient but after two different stimulation protocols shows that the type of hormonal treatment influences CC protein expression. In contrast, CC from oocytes obtained under the same stimulation protocol but with different fertilization outcome show a high profile similarity with differences in only a few spots. Comparison of two groups of patients indicates that dissimilarities in protein pattern between patients become very high, even when comparing the same stimulation protocol and oocyte fertilization outcome. Thus protein expression profiling of human CC may provide a correlation between the synthesis of specific cumulus proteins and maturity and fecundity.