X-chromosome inactivation patterns depend on age and tissue but not conception method in humans

Chromosome Res (2023) 31:4 https://doi.org/10.1007/s10577-023-09717-9 RESEARCH X‑chromosome inactivation patterns depend on age and tissue but not conception method in humans Patrycja Juchniewicz · Anna Kloska · Karolina Portalska · Joanna Jakóbkiewicz‑Banecka · Grzegorz Węgrzyn · Joanna Liss · Piotr Głodek · Stefan Tukaj · Ewa Piotrowska Received: 16 September 2022 / Revised: 27 November 2022 / Accepted: 6 December 2022 © The Author(s) 2023 Abstract Female somatic X-chromosome inactivation (XCI) balances the X-linked transcriptional dosages between the sexes, randomly silencing the maternal or paternal X chromosome in each cell of 46,XX females. Skewed XCI toward one parental X has been observed in association with ageing and in some female carriers of X-linked diseases. To address the problem of non-random XCI, we quantified the XCI skew in different biological samples of naturally conceived females of different age groups and girls conceived after in vitro fertilization (IVF). Generally, XCI skew differed between saliva, blood, Responsible Editor: Beth Sullivan Supplementary Information The online version contains supplementary material available at https://doi. org/10.1007/s10577-023-09717-9. P. Juchniewicz · A. Kloska (*) · J. Jakóbkiewicz‑Banecka · J. Liss Department of Medical Biology and Genetics, Faculty of Biology, University of Gdańsk, Wita Stwosza 59, 80‑308 Gdańsk, Poland e-mail: K. Portalska · G. Węgrzyn · S. Tukaj · E. Piotrowska (*) Department of Molecular Biology, Faculty of Biology, University of Gdańsk, Wita Stwosza 59, 80‑308 Gdańsk, Poland e-mail: and buccal swabs, while saliva and blood had the most similar XCI patterns in individual females. XCI skew increased with age in saliva, but not in other tissues. We showed no significant differences in the XCI patterns in tissues of naturally conceived and IVF females. The gene expression profile of the placenta and umbilical cord blood was determined depending on the XCI pattern. The increased XCI skewing in the placental tissue was associated with the differential expression of several genes out of 40 considered herein. Notably, skewed XCI patterns (> 80:20) were identified with significantly increased expression levels of four genes: CD44, KDM6A, PHLDA2, and ZRSR2. The differences in gene expression patterns between samples with random and non-random XCI may shed new light on factors contributing to the XCI pattern outcome and indicate new paths in future research on the phenomenon of XCI skewing. Keywords X-chromosome inactivation · XCI · Dosage compensation · Human development · Placenta · Methods of conception Abbreviations IVF In vitro fertilization XCI X-chromosome inactivation J. Liss · P. Głodek Research and Development Center, INVICTA, Sopot, Poland Vol.: (0123456789) 13 4 Page 2 of 19 Introduction X-chromosome inactivation (XCI) is an epigenetically regulated mechanism of dosage compensation balancing expression levels of X-linked genes between females (XX) and males (XY). In this process, either maternally or paternally inherited X chromosome present in individual cells of the developing female embryo is marked for inactivation. It means that several hundred physically linked loci become concomitantly and stably silenced (Patrat et al. 2020); this silencing is inherited through subsequent somatic cell divisions. Still, up to one-third of human X-chromosomal genes escape this process and are expressed from both the active and inactive X chromosomes in female cells (Tukiainen et al. 2017). The degree of XCI incompleteness varies between genes, tissues, and individuals (Shvetsova et al. 2019), and remains poorly characterized. Sixty years after Mary Lyon put forth the hypothesis of random X-chromosome inactivation (Lyon 1961), skewed XCI has been reported to associate with many rare and common disorders (Plenge et al. 2002; Migeon 2020; Juchniewicz et al. 2021). As for the general female population, the literature is much scarcer. The most comprehensive population studies of healthy women and girls showed XCI skewing ratio > 70:30 in 25–27% of the analysed females, and a ratio > 80:20 in 8–10%, while the ratio > 90:10 was identified only in 1.8% (Amos-Landgraf et al. 2006; Shvetsova et al. 2019). However, when the threshold for a skewed XCI ratio was defined as ≥ 65:35, nearly 50% of the population demonstrated imbalanced inactivation of the two X chromosomes (Shvetsova et al. 2019). Besides the subjectively defined threshold for preferential inactivation of either parental chromosome, the pattern of XCI (random or skewed) depends on the age of a woman and the type of examined tissues (Sharp et al. 2000; Amos-Landgraf et al. 2006; Ørstavik 2009). The randomness of XCI in different tissues may range from 50:50 to 80:20 in the same individual which can be attributed to the fact that cells with high mitotic activity have a more skewed pattern than cells with lower mitotic activity (Sharp et al. 2000; Knudsen et al. 2007; Bolduc et al. 2008). Still, most studies on XCI have been performed in DNA from peripheral blood with intensely dividing cells (Ørstavik 2009). When XCI patterns were determined in Vol:. (1234567890) 13 Chromosome Res (2023) 31:4 easily accessible tissues (buccal epithelium, blood, and hair follicle) and compared with XCI patterns in several inaccessible tissues (heart, thyroid, kidney, liver, muscle, and ovary), it appeared that buccal epithelium was preferable over peripheral blood cells for predicting XCI pattern in inaccessible tissues. The ovary was the only inaccessible tissue showing a poor correlation to the buccal epithelium and blood cells but had a good correlation to hair follicles instead (de Hoon et al. 2015). The frequency of XCI skewing was suggested to increase with age, especially over 55 years (Kristiansen et al. 2005). Longitudinal studies of XCI performed for the same females at an average interval of two decades confirmed a significant difference in the degree of XCI skewing with time in females who were 60 years or older at the time of the first sampling (Sandovici et al. 2004). On the other hand, Shvetsova et al. (2019) did not observe an increase in skewing with age while studying the population with the age range of 20–64 years. If the age-related skewing of XCI is more prevalent in the general population, its biological significance remains unclear, but one of its possible consequences might be the manifestation of X-linked disorders in elderly females (Cazzola et al. 2000; Au et al. 2004). XCI occurs in early embryonic life, but the exact timing in humans is still elusive (Goto and Monk 1998; Patrat et al. 2020). The current knowledge of human XCI is mainly based on the studies performed on embryos obtained by in vitro fertilization (IVF) or pluripotent stem cells. Epidemiological data indicate that children conceived in vitro have a greater relative risk of low birth weight, major and minor birth defects, and rare disorders involvin (...truncated)