How to view the female reproductive tract through single-cell looking glasses

Single-cell technologies have emerged as an unprecedented tool for biologists and clinicians, allowing them to assess organs and tissues at the level of individual cells. In the field of women's reproductive biology, single-cell studies have provided insights into the cellular and molecular processes that regulate reproductive and obstetrical functions in health and disease. The knowledge that these studies generate is helping clinicians to improve the understanding and diagnosis of infertility related issues or pregnancy complications and to find new avenues for their treatment. However, navigating the expansive landscape of this type of transcriptomic data analysis represents a pivotal challenge in current research. Single cell RNA sequencing involves isolating cells into droplets, reverse transcribing RNA to generate complementary DNA, with each droplet content uniquely labeled by a barcode. Upon sequencing the complementary DNAs, the barcodes enable the reassignment of sequencing reads to individual droplets, facilitating the reconstruction of the cellular landscape of the sample obtained from a tissue or organ and beyond. Researchers, equipped with the metaphorical 'single-cell glasses,' must adequately choose from a plethora of strategies to dissect and interpret cellular information. Sophisticated algorithms and the decision-making process are often underestimated, resulting in artefactual or cumbersome interpreted results. Computational biologists apply and innovate computational tools designed to process, model, and interpret expansive datasets. The ramifications of their work extend far beyond the realm of data processing; they give shape to the outcome of analyses, playing a pivotal role in drawing meaningful conclusions from the wealth of information garnered. In this review, we describe the wide variety of approaches and analytical steps available with enough detail to gain a concise picture of what a complete examination of a single-cell dataset would be. We commence with a discussion on key points in experimental design, highlighting crucial questions one should consider. Following this, we delve into the various preprocessing and quality control steps essential for any single-cell dataset. The subsequent section offers a detailed guide on constructing a single-cell atlas, exploring nuances such as differential characteristics in visualization and clustering techniques, as well as strategies for assigning identity to cell populations through gene marker annotations. Moving beyond the creation of an atlas, we explore methods for investigating pathological conditions. This involves conducting cell population comparison tests between conditions and analyzing specific cell-to-cell communications and cellular differentiation trajectories in both health and disease scenarios. This work aims to furnish a newcomer researcher and/or clinician with essential guidelines to embark on a single-cell adventure without succumbing to common pitfalls. By bridging the gap between theory and practice, it facilitates the translation of single-cell technologies into clinically relevant applications. Throughout the manuscript, practical examples of its usage in women's reproductive health studies are provided. Various sections delve into specific clinical scenarios, demonstrating how these guidelines can be instrumental in unraveling the molecular landscapes of diseases and physiological processes related to women's reproduction.