Sophie Debrock

Chromosome instability is common in human cleavage-stage embryos

Chromosome instability is a hallmark of tumorigenesis. This study establishes that chromosome instability is also common during early human embryogenesis. A new array-based method allowed screening of genome-wide copy number and loss of heterozygosity in single cells. This revealed not only mosaicism for whole-chromosome aneuploidies and uniparental disomies in most cleavage-stage embryos but also frequent segmental deletions, duplications and amplifications that were reciprocal in sister blastomeres, implying the occurrence of breakage-fusion-bridge cycles. This explains the low human fecundity and identifies post-zygotic chromosome instability as a leading cause of constitutional chromosomal disorders.

Single-cell chromosomal imbalances detection by array CGH

Genomic imbalances are a major cause of constitutional and acquired disorders. Therefore, aneuploidy screening has become the cornerstone of preimplantation, prenatal and postnatal genetic diagnosis, as well as a routine aspect of the diagnostic workup of many acquired disorders. Recently, array comparative genomic hybridization (array CGH) has been introduced as a rapid and high-resolution method for the detection of both benign and disease-causing genomic copy-number variations. Until now, array CGH has been performed using a significant quantity of DNA derived from a pool of cells. Here, we present an array CGH method that accurately detects chromosomal imbalances from a single lymphoblast, fibroblast and blastomere within a single day. Trisomy 13, 18, 21 and monosomy X, as well as normal ploidy levels of all other chromosomes, were accurately determined from single fibroblasts. Moreover, we showed that a segmental deletion as small as 34 Mb could be detected. Finally, we demonstrated the possibility to detect aneuploidies in single blastomeres derived from preimplantation embryos. This technique offers new possibilities for genetic analysis of single cells in general and opens the route towards aneuploidy screening and detection of unbalanced translocations in preimplantation embryos in particular.

POTENTIAL INVOLVEMENT OF THE IMMUNE SYSTEM IN THE DEVELOPMENT OF ENDOMETRIOSIS

This article presents an overview of immunological factors and their role in the development of endometriosis, with emphasis on inflammatory cytokines, growth and adhesion factors. Although retrograde menstruation is a common phenomenon among women of reproductive age, not all women who have retrograde menstruation develop endometriosis. The development of endometriosis is hypothesised to be a complex process, which may be facilitated by several factors, including the quantity and quality of endometrial cells in peritoneal fluid (PF), increased inflammatory activity in PF, increased endometrial-peritoneal adhesion and angiogenesis, reduced immune surveillance and clearance of endometrial cells, and increased production of autoantibodies against endometrial cells. Potential biomarkers like cytokines and autoantibodies upregulated during development of endometriosis may be useful in the development of a non-surgical diagnostic tool. Although endometriosis can be treated using hormonal suppression, there is need for non-hormonal drugs, which can inhibit the development of endometriosis and alleviate pain or infertility without inhibition of ovulation. New molecules that modulate immune function in endometriosis should be the targets for future research.

Recombinant Human TNFRSF1A (r-hTBP1) Inhibits the Development of Endometriosis in Baboons: A Prospective, Randomized, Placebo- and Drug-Controlled Study

Abstract Endometriosis is associated with chronic inflammation, including an increased macrophage activity with increased secretion of cytokines, such as tumor necrosis factor (TNF) or TNF superfamily member 2, previously known as TNFα. In the present study, we tested the hypothesis that recombinant human TNFRSF1A (r-hTBP1) can inhibit the development of endometriotic lesions in the baboon, an established model for the study of endometriosis. Endometriosis was induced using intrapelvic injection of menstrual endometrium in 20 baboons with a normal pelvis. In the first part of the study, 14 baboons were randomly assigned to subcutaneous treatment with r-hTBP1, placebo, or GnRH antagonist (positive control). In the second part of the study, menstrual endometrium from 6 baboons was randomly incubated with either PBS or r-hTBP1 before intrapelvic seeding. Video laparoscopy was performed 25 days later to document the number, surface area, and estimated volume of endometriotic lesions and adhesions; to calculate the revised American Fertility Society (rAFS) score and stage; and to confirm the histological presence of endometriosis. In the first part, baboons treated with r-hTBP1 or with Antide (Bachem) had a lower endometriosis rAFS score, a lower surface area and estimated volume of peritoneal endometriotic lesions, and a lower histological confirmation rate compared with controls. Because of less adnexal and cul-de-sac adhesions, the number of baboons with endometriosis of stage II, III, or IV was lower among baboons treated with r-hTBP1 or Antide than among controls. In the second part, the surface area of endometriotic lesions was lower, and less severe endometriosis was observed in r-hTBP1-treated baboons. No hypoestrogenic effects were observed in baboons treated with r-hTBP1. In conclusion, r-hTBP1 can effectively inhibit the development of endometriosis without hypoestrogenic effects in baboons.

Preimplantation genetic screening for aneuploidy of embryos after in vitro fertilization in women aged at least 35 years: a prospective randomized trial

OBJECTIVE To test the hypothesis that patients with advanced maternal age (AMA) have a higher implantation rate (IR) after embryo transfer of embryos with a normal chromosomal pattern for the chromosomes studied with preimplantation genetic screening (PGS) compared with patients who had an embryo transfer without PGS. DESIGN Prospective randomized controlled trial (RCT). SETTING Academic tertiary setting. PATIENT(S) Patients with AMA (> or =35 years). INTERVENTION(S) In an RCT, the clinical IR per embryo transferred was compared after embryo transfer on day 5 or 6 between the PGS group (analysis of chromosomes 13, 16, 18, 21, 22, X, and Y) and the Control group without PGS. MAIN OUTCOME MEASURE(S) No differences were observed between the PGS group and the Control group for the clinical IR (15.1%; 14.9%; rate ratio 1.01; exact confidence interval [CI], 0.25-5.27), the ongoing IR (at 12 weeks) (9.4%; 14.9%), and the live born rate per embryo transferred (9.4%; 14.9%; rate ratio 0.63; exact CI, 0.08-3.37). Fewer embryos were transferred in the PGS group (1.6 +/- 0.6) than in the Control group (2.0 +/- 0.6). A normal diploid status was observed in 30.3% of the embryos screened by PGS. CONCLUSION(S) In this RCT, the results did not confirm the hypothesis that PGS results in improved reproductive outcome in patients with AMA.

Neutralization of plasminogen activator inhibitor-1 inhibitory properties: identification of two different mechanisms

Plasminogen activator inhibitor-1 (PAI-1), a unique member of the serpin superfamily, plays an important role in fibrinolysis and is an established risk factor for cardiovascular diseases. PAI-1 can occur in three interconvertible conformations: an active, a latent and a substrate form. To study conformational and functional relationships in PAI-1, a wide variety of monoclonal antibodies were evaluated for their influence on PAI-1 activity. Out of 77 monoclonal antibodies, directed against human PAI-1, six were selected for their strong inhibitory effect towards PAI-1 activity, i.e., 80 to 100% inhibition in the presence of a 1- to 16-fold molar excess of monoclonal antibody. Detailed analysis of the reaction products formed during the interaction between PAI-1 and its target proteinases tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA), in the presence of these monoclonal antibodies, revealed two distinct mechanisms of PAI-1 inactivation. Incubation of PAI-1 with one series of monoclonal antibodies resulted in the absence of any reaction indicative for direct interaction with the reactive-site loop or a facilitated conversion to the latent conformation. The loss of PAI-1 activity in the presence of the other group of monoclonal antibodies was associated with the concomitant formation of a 41 kDa cleavage product after interaction with the target proteinase. The latter observation demonstrates that binding of these antibodies induced a conformational change thereby converting the inhibitory, active conformation to the non-inhibitory substrate conformation. No conformational changes could be observed in latent PAI-1 under these conditions. Analysis of cross-reactivity revealed that some of these functionally important epitopes were conserved throughout PAI-1 obtained from various species including rabbit mouse and/or pig, resulting in similar functional and conformational effects induced by these antibodies. Thus, we have demonstrated the occurrence of two distinct mechanisms by which the inhibitory activity of PAI-1 can be neutralized. This may have implications for the design of therapeutic or preventive strategies to interfere with PAI-1 activity. Cross-reactivity of these inhibitory antibodies with PAI-1 from various species may also allow their application in experimental animal models studying the in vivo role of PAI-1 in various diseases (e.g. atherosclerosis, thrombosis, angiogenesis,...).

What next for preimplantation genetic screening? High mitotic chromosome instability rate provides the biological basis for the low success rate

Preimplantation genetic screening is being scrutinized, as recent randomized clinical trials failed to observe the expected significant increase in live birth rates following fluorescence in situ hybridization (FISH)-based screening. Although these randomized clinical trials are criticized on their design, skills or premature stop, it is generally believed that well-designed and well-executed randomized clinical trials would resolve the debate about the potential benefit of preimplantation genetic screening. Since FISH can analyze only a limited number of chromosomal loci, some of the embryos transferred might be diagnosed as ‘normal’ but in fact be aneuploid for one or more chromosomes not tested. Hence, genome-wide array comparative genome hybridization screening enabling aneuploidy detection of all chromosomes was thought to be a first step toward a better design. We recently showed array screening indeed enables accurate determination of the copy number state of all chromosomes in a single cell. Surprisingly, however, this genome-wide array screening revealed a much higher frequency and complexity of chromosomal aberrations in early embryos than anticipated, with imbalances in a staggering 90% of all embryos. The mitotic error rate in cleavage stage embryos was proven to be higher than the meiotic aneuploidy rate and as a consequence, the genome of a single blastomere is not representative for the genome of the other cells of the embryo. Hence, potentially viable embryos will be discarded upon screening a single blastomere. This observation provides a biological basis for the failure of the randomized clinical trials to increase baby-take-home rates using FISH on cleavage stage embroys.

Accelerated Conversion of Human Plasminogen Activator Inhibitor-1 to Its Latent Form by Antibody Binding

The serpin plasminogen activator inhibitor-1 (PAI-1) slowly converts to an inactive latent form by inserting a major part of its reactive center loop (RCL) into its β-sheet A. A murine monoclonal antibody (MA-33B8), raised against the human plasminogen activator (tPA)·PAI-1 complex, rapidly inactivates PAI-1. Results presented here indicate that MA-33B8 induces acceleration of the active-to-latent conversion. The antibody-induced inactivation of PAI-1 labeled with the fluorescent probeN,N′-dimethyl-N-(acetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene diamine (NBD) at P9 in the RCL caused a fluorescence enhancement and shift identical to those accompanying the spontaneous conversion of the P9·NBD PAI-1 to the latent form. Like latent PAI-1, antibody-inactivated PAI-1 was protected from cleavage by elastase. The rate constants for MA-33B8 binding, measured by NBD fluorescence or inactivation, were similar (1.3–1.8 × 104 m −1 s−1), resulting in a 4000-fold faster inactivation at 4.2 μm antibody binding sites. The apparent antibody binding rate constant, at least 1000 times slower than one limited by diffusion, indicates that exposure of its epitope depends on an unfavorable equilibrium of PAI-1. Our observations are consistent with this idea and suggest that the equilibrium involves partial insertion of the RCL into sheet A: latent, RCL-cleaved, and tPA-complexed PAI-1, which are inactive loop-inserted forms, bound much faster than active PAI-1 to MA-33B8, whereas two loop-extracted forms of PAI-1, modified to prevent loop insertion, did not bind or bound much more weakly to the antibody.

Future directions in endometriosis research

Endometriosis is an important gynecological disease, pathologically defined by the ectopic presence of both endometrial glands and stroma, and clinically associated with pelvic pain and infertility. Our current knowledge of the pathogenesis, pathophysiology of related infertility, and spontaneous evolution is still limited, although endometriosis has been described for many years. Future research in endometriosis needs to focus on pathogenesis studies in the baboon model and on the early interactions between endometrial and peritoneal cells in the pelvic cavity at the time of menstruation. Proteomic and genomic approaches are needed to detect potential differences between eutopic endometrium and myometrium in women with and without endometriosis. Immunomodulatory drugs inhibiting endometriosis-associated pelvic inflammation may offer new medical treatment for endometriosis in the future.

Breakage–fusion–bridge cycles leading to inv dup del occur in human cleavage stage embryos

Recently, a high incidence of chromosome instability (CIN) was reported in human cleavage stage embryos. Based on the copy number changes that were observed in the blastomeres it was hypothesized that chromosome breakages and fusions occur frequently in cleavage stage human embryos and instigate subsequent breakage‐fusion‐bridge cycles. In addition, it was hypothesized that the DNA breaks present in spermatozoa could trigger this CIN. To test these hypotheses, we genotyped both parents as well as 93 blastomeres from 24 IVF embryos and developed a novel single nucleotide polymorphism (SNP) array‐based algorithm to determine the parental origin of (aberrant) loci in single cells. Paternal as well as maternal alleles were commonly rearranged in the blastomeres indicating that sperm‐specific DNA breaks do not explain the majority of these structural variants. The parent‐of‐origin analyses together with microarray‐guided FISH analyses demonstrate the presence of inv dup del chromosomes as well as more complex rearrangements. These data provide unequivocal evidence for breakage–fusion–bridge cycles in those embryos and suggest that the human cleavage stage embryo is a major source of chromosomal disorders. Hum Mutat 32:783–793, 2011.