Catherine Patrat

Eutherian mammals use diverse strategies to initiate X-chromosome inactivation during development

X-chromosome inactivation (XCI) in female mammals allows dosage compensation for X-linked gene products between the sexes. The developmental regulation of this process has been extensively investigated in mice, where the X chromosome of paternal origin (Xp) is silenced during early embryogenesis owing to imprinted expression of the regulatory RNA, Xist (X-inactive specific transcript). Paternal XCI is reversed in the inner cell mass of the blastocyst and random XCI subsequently occurs in epiblast cells. Here we show that other eutherian mammals have very different strategies for initiating XCI. In rabbits and humans, the Xist homologue is not subject to imprinting and XCI begins later than in mice. Furthermore, Xist is upregulated on both X chromosomes in a high proportion of rabbit and human embryo cells, even in the inner cell mass. In rabbits, this triggers XCI on both X chromosomes in some cells. In humans, chromosome-wide XCI has not initiated even by the blastocyst stage, despite the upregulation of XIST. The choice of which X chromosome will finally become inactive thus occurs downstream of Xist upregulation in both rabbits and humans, unlike in mice. Our study demonstrates the remarkable diversity in XCI regulation and highlights differences between mammals in their requirement for dosage compensation during early embryogenesis.

Dynamic changes in paternal X-chromosome activity during imprinted X-chromosome inactivation in mice

In mammals, X-chromosome dosage compensation is achieved by inactivating one of the two X chromosomes in females. In mice, X inactivation is initially imprinted, with inactivation of the paternal X (Xp) chromosome occurring during preimplantation development. One theory is that the Xp is preinactivated in female embryos, because of its previous silence during meiosis in the male germ line. The extent to which the Xp is active after fertilization and the exact time of onset of X-linked gene silencing have been the subject of debate. We performed a systematic, single-cell transcriptional analysis to examine the activity of the Xp chromosome for a panel of X-linked genes throughout early preimplantation development in the mouse. Rather than being preinactivated, we found the Xp to be fully active at the time of zygotic gene activation, with silencing beginning from the 4-cell stage onward. X-inactivation patterns were, however, surprisingly diverse between genes. Some loci showed early onset (4–8-cell stage) of X inactivation, and some showed extremely late onset (postblastocyst stage), whereas others were never fully inactivated. Thus, we show that silencing of some X-chromosomal regions occurs outside of the usual time window and that escape from X inactivation can be highly lineage specific. These results reveal that imprinted X inactivation in mice is far less concerted than previously thought and highlight the epigenetic diversity underlying the dosage compensation process during early mammalian development.

XACT Noncoding RNA Competes with XIST in the Control of X Chromosome Activity during Human Early Development

Summary Sex chromosome dosage compensation is essential in most metazoans, but the developmental timing and underlying mechanisms vary significantly, even among placental mammals. Here we identify human-specific mechanisms regulating X chromosome activity in early embryonic development. Single-cell RNA sequencing and imaging revealed co-activation and accumulation of the long noncoding RNAs (lncRNAs) XACT and XIST on active X chromosomes in both early human pre-implantation embryos and naive human embryonic stem cells. In these contexts, the XIST RNA adopts an unusual, highly dispersed organization, which may explain why it does not trigger X chromosome inactivation at this stage. Functional studies in transgenic mouse cells show that XACT influences XIST accumulation in cis. Our findings therefore suggest a mechanism involving antagonistic activity of XIST and XACT in controlling X chromosome activity in early human embryos, and they highlight the contribution of rapidly evolving lncRNAs to species-specific developmental mechanisms.

Timing of intermittent seminal HIV-1 RNA shedding in patients with undetectable plasma viral load under combination antiretroviral therapy.

It was demonstrated that combination antiretroviral therapy (cART) reduces the HIV-1 viral load (VL) in the blood and the seminal compartment. Some studies have reported that the seminal HIV-1 VL is undetectable in individuals with an undetectable blood plasma viral load (bpVL) under cART. However, some recent studies have demonstrated that seminal HIV-1 RNA may still be detected, and potentially infectious, even in the case of an undetectable bpVL. The aim of this retrospective study was to determine the detection rate of a seminal VL and whether shedding could be intermittent over a very short time. From January 2006 to December 2011, 88 HIV-1 infected men, enrolled in an Assisted Reproduction program, provided 306 semen samples, corresponding to 177 frozen sperm samples (two samples delivered at a one-hour interval (n = 129) or one sample (n = 48)). All enrolled men were under cART, with an undetectable bpVL for more than 6 months. HIV-1 RNA was quantified in seminal plasma using a Roche COBAS Ampliprep COBAS TaqMan HIV-1 test. Seminal HIV-1 RNA was detected in 23 samples (7.5%) from 17 patients (19.3%). This detection rate was stable over years. With regards to the freezing of two samples delivered at a one-hour interval, the proportion of discordance between the first and second samples was 9.3% (12/129). Our results confirm the intermittent shedding of HIV-1 in semen. While this finding has been shown by studies examining longer time intervals, to our knowledge, this has never been demonstrated over such a short time interval.

Markers of hepatitis delta virus infection can be detected in follicular fluid and semen.

BACKGROUND Hepatitis delta virus (HDV) is a satellite of HBV and needs this latters envelope for its morphogenesis and propagation. An estimated 5-20% of HBV-infected patients are also infected with HDV. No studies have ever been performed to determine the presence of HDV in follicular fluid (FF) and semen of HDV-infected patients. OBJECTIVES To investigate the presence of HDV markers in the FF or in the semen of two HDV-infected patients. DESIGN Two unrelated HDV-infected patients, a woman and a man pursuing in vitro fertilization (IVF), participated in this study. FF was collected after analysis of oocyte retrieval. The supernatant of seminal plasma (SP) and the final pellet (FP) were fractionated from freshly ejaculated semen. Serological and molecular markers of HDV infection were searched for in these different samples. RESULTS The woman was infected with an HDV-7 genotype strain and her HDV plasma viral load (VL) was 6 log copies/mL. HDV antibodies and RNA were also detected in the FF, however the RNA VL value there was lower by more than 4 log. The man was infected with an HDV-1 strain and his plasma VL was 6.7 log copies/mL. Total anti-HDV antibodies were positive in the serum, in the SP and in the FP, while IgM were detected only in the serum. However, HDV RNA was negative in the SP and in the FP. CONCLUSION HDV markers can be found in the follicular fluid or in the semen of infected patients.

Dextran-Based Hydrogel as a New Tool for BALB/c 3T3 Cell Cryopreservation Without Dimethyl Sulfoxide

INTRODUCTION Cryopreservation provides an efficient way to preserve cells for a broad range of medical applications, including cell therapy. In clinical practice, cells are frozen in solutions containing dimethyl sulfoxide (DMSO) cryoprotectant agents (CPAs) to reduce their damage during the cooling process. In the current cell preservation methods, polysaccharides such as dextran, a nonpenetrating CPA, are used. However, the cell viability decreases when the solution concentration in polysaccharides increases. MATERIALS AND METHODS To overcome this limitation, we have developed a dextran-based hydrogel (PSH) as a new CPA. Three molecular weight PSHs (PSH40, PSH70, and PSH500) were synthesized. The physicochemical characteristics of PSHs were studied. Then, their biocompatibility properties were studied in vitro in BALB/c 3T3 cells according to ISO standard 10993-5/12. Crystallization temperature (Tc), that is, ice-crystal formation, was determined using the thermocouple method. Finally, PSHs were used as CPAs in a slow freezing procedure of BALB/c 3T3 cells with Voluven® (Fresenius Kabi, Sèvres, France), and were compared with the DMSO procedure. RESULTS Our results showed that PSHs were biocompatible and did not modify the osmolality of the Voluven cryopreservation solution. PSHs decreased the Tc when compared with the DMSO procedure. Furthermore, without adding DMSO, PSH500 cryopreserved the viability of BALB/c 3T3 cells, and the result was similar to that of the control conditions. CONCLUSION PSH500 could represent an alternative to DMSO. It could be used as a new medical device while avoiding DMSO side effects on patients.

COL09-01 : Procréation chez les couples VIH séro-différents : TasP, PrEP ou AMP ? (ANRS 12008)

Introduction – objectifs Le but de cette etude etait d’evaluer le risque residuel de transmission sexuelle du VIH, le cout et le rapport cout-efficacite (CE) de 5 strategies de procreation chez les couples sero-differents fertiles dont l’homme VIH+ a une charge virale indetectable sous ARV : 1) TasP = rapports sexuels non proteges, 2) FertilD = TasP seulement en periode ovulatoire (test urinaire), 3) TasP + PrEP continue (tenofovir/emtricitabine), 4) TasP + PrEP en periode ovulatoire, 5) AMP (lavage du sperme et ≤ 6 inseminations intra-uterines). Materiels et methodes Le modele d’aide a la decision utilise incluait les parametres issus de la litterature internationale avec un taux de naissances vivantes de 85 % pour chaque strategie, un risque de transmission sexuelle (β), de transmission mere-enfant, et de malformations liees aux ARV en cas d’infection maternelle a la conception respectivement de 0,0083 %/mois, 1 % et 4,4 %. La diminution du risque β pour les strategies PrEP et FertilD a ete estimee a 80 % et 67 %, respectivement, et a 93,4 % pour la PrEP ovulatoire (1−(1−0,80) × (1−0,67)). La PrEP etait supposee ne pas etre a l’origine des malformations. L’analyse a ete realisee du point de vue societal francais (euros 2013), avec un taux d’actualisation annuel a 4 %. Resultats Le risque β le plus eleve concernait la strategie TasP et le plus bas la AMP, suivie de la PrEP ovulatoire (respectivement 5,4, 0 et 0,3 infections/10 000 couples). La PrEP continue etait moins efficace et plus couteuse que FertilD qui disposait des couts les plus bas et d’un risque β a 0,9/10 000. Le rapport CE PrEP ovulatoire/FertilD etait de 1 128 000 €/annee de vie gagnee (AVG), et celui de AMP/PrEP ovulatoire de 2 419 000 €/AVG. Ces resultats etaient robustes aux analyses de sensibilite multivariees. Conclusion Chez les couples sero-differents fertiles dont l’homme est VIH+, la strategie TasP pendant la periode ovulatoire est a faible risque de transmission sexuelle. La PrEP ovulatoire et la AMP diminuent encore ce risque, mais sont associees a des rapports CE defavorables.