Milena Myllykangas

Reproduction is one of the most fundamental biological processes in the human body, yet the molecules governing it remain incompletely understood. Here, we have characterized the role of PKHD1L1 and its globally relatively common splice donor variant rs17368310 in female fertility. We demonstrate estrogen-responsive expression of PKHD1L1 in the human endometrial and Fallopian tube epithelium, identify the change in the rs17368310 mRNA sequence in endometrial tissue, and assess the possible effects of the variant on the PKHD1L1 protein through structural modeling. We reveal that women homozygous for rs17368310 have a persistently lower child count compared to other genotypes not only among all women but also among women who have undergone medical treatments for infertility in the Finnish population. We further show that rs17368310 associates with female infertility-related traits also in the Mexican population. These findings elucidate the effects of rs17368310 on fertility in millions of reproductive-age women across different populations.

STUDY QUESTION: Does the endometrium of oligo-ovulatory women with polyendocrine metabolic ovarian syndrome (PMOS) exhibit menstrual cycle phase-specific transcriptomic alterations compared to women without PMOS? SUMMARY ANSWER: Compared to healthy controls, women with PMOS exhibit endometrial transcriptomic dysregulations which are menstrual cycle phase-specific and, most notably, related to mitochondrial function. WHAT IS KNOWN ALREADY: Infertility and gestational dysfunction in women with PMOS are increasingly attributed to endometrial abnormalities, yet the underlying mechanisms behind PMOS-related endometrial dysfunction remain unclear. Previous studies have focused on anovulatory phenotypes, resulting in a poor understanding of cycle phase-specific defects in the endometria of women with PMOS. STUDY DESIGN, SIZE, DURATION: Endometrial biopsies were obtained from oligo-ovulatory women with PMOS and healthy controls at four distinct menstrual cycle phases (proliferative, early-, mid-, and late-secretory phases) and subjected to RNA sequencing. Parallel in vitro experiments were conducted using cultured endometrial stromal cells (ESCs) and epithelial organoids (EEOs) derived from an additional endometrial sample cohort. PARTICIPANTS/MATERIALS, SETTING, METHODS: RNA sequencing was performed on biopsies from 78 (oligo-)ovulatory women, age- and BMI-matched between PMOS (n = 39) and control (n = 39) groups. Phase-specific transcriptomic profiles and pathway enrichment analyses were performed. For functional assays, endometrial cells were isolated from four PMOS and four control donors per BMI group (lean: < 25 kg/m2; obese: ≥30 kg/m2). Mitochondrial function, including respiration, ATP and reactive oxygen species production, and membrane potential, was assessed in both cell types before and after hormonal exposure. MAIN RESULTS AND THE ROLE OF CHANCE: Endometrial transcriptomes clustered primarily by cycle phase; however, PMOS-specific dysregulation was observed related to extracellular matrix organization, estrogen receptor (ESR) signaling, ion transport, and mitochondrial respiratory function. Gene trajectory analyses revealed phase-specific alterations in progesterone-, insulin signaling-, and mitochondria-related gene expression during the early-secretory phase, and epithelial function during the mid-secretory phase. Functionally, decidualized ESCs from obese PMOS women displayed reduced mitochondrial respiration, whereas EEOs from lean PMOS patients exhibited increased mitochondrial activity. LARGE SCALE DATA: Sequencing data are available from the corresponding author upon request. LIMITATIONS, REASONS FOR CAUTION: Women with PMOS were oligo-ovulatory while controls exhibited regular cycles. The sample size for in vitro studies was modest, warranting validation in larger cohorts. Additionally, the use of bulk RNA sequencing may obscure cell-type-specific transcriptomic signatures, emphasizing the need for future single-cell approaches to dissect cellular heterogeneity in PMOS endometrium. WIDER IMPLICATIONS OF THE FINDINGS: This study uncovers dynamic, cycle phase-dependent molecular and bioenergetic alterations in the endometrium of oligo-ovulatory women with PMOS. Mitochondrial dysfunction, particularly during the implantation window, emerges as a key pathological feature. The observed BMI-dependent divergence in mitochondrial function suggests that personalized therapeutic strategies may be necessary to improve reproductive outcomes in women with PMOS. FUNDING: Funded by the Jusélius Foundation, Novo Nordisk Foundation, Research Council of Finland, and the Horizon 2020 Marie-Curie MATER Innovative Training Network (all to T.T.P.), with additional support from the University of Oulu Scholarship Foundation (L.L.), the Estonian Research Council (E.V.M. and I.R.), and the Estonian Research Council grant no. PRG1076, Swedish Research Council grant no. 2024-02530, Novo Nordisk Foundation grant no. NNF24OC0092384 and Horizon Europe grant NESTOR, grant no. 101120075 (A.S.). DISCLOSURES: The authors declare no competing interests.

STUDY QUESTION: Are we able to establish endometrium epithelial organoids (EEOs) from endometrial samples obtained from women with PCOS, and do they differ from non-PCOS EEOs? SUMMARY ANSWER: We were able to establish, for the first time, PCOS EEOs which capture endometrial abnormalities present in women with PCOS, including increased inflammation and decreased receptivity-related gene expression. WHAT IS KNOWN ALREADY: Patient-derived EEOs could serve as a tool to study endometrial dysfunction, as diseased tissue-derived organoid models typically retain the disease-related traits. In PCOS, endometrial dysfunction likely contributes to subfertility and pregnancy complications, yet previous research on the endometrial epithelial compartment has been scarce and, so far, no PCOS-derived EEOs have been established. STUDY DESIGN, SIZE, DURATION: EEOs were established from endometrial biopsies from two cohorts of women with PCOS—including overweight/obese (O-PCOS, n = 4) and lean (L-PCOS, n = 4)—along with BMI-matched controls (overweight/obese control (O-Ctrl), n = 4; lean control (L-Ctrl), n = 4). EEOs were exposed to combinations of steroid hormones (β-estradiol (E2), progesterone, cAMP, and the Wnt/β-catenin signaling (WNT) inhibitor XAV-939) for 6 days to simulate the proliferative or secretory phases of the menstrual cycle, with or without simultaneous androgen exposure with dihydrotestosterone (DHT). PARTICIPANTS/MATERIALS, SETTING, METHODS: Bulk RNA-sequencing was conducted to identify variations in gene expression between PCOS and Ctrl EEOs, while reverse-transcription quantitative PCR RT-qPCR was employed to validate these results. Morphological assessment of EEOs was performed using hematoxylin and eosin staining and immunostaining. The size of EEOs was evaluated after 6 days of hormonal exposure. MAIN RESULTS AND THE ROLE OF CHANCE: PCOS EEOs from both BMI groups demonstrated increased inflammation-related gene expression (including increased expression of Oncostatin M Receptor (OSMR) and Intercellular Adhesion Molecule 1 (ICAM1)) and showed a reduced diameter compared to their respective control EEOs. The O-PCOS EEOs displayed an aberrant response to steroid exposure with E2 and progesterone (including reduced expression of receptivity-related genes progestagen-associated endometrial protein and leukemia inhibitory factor) as compared to control EEOs. Addition of DHT to the culture media did not affect EEO transcriptome, aligning with the minimal androgen receptor (AR) expression in the EEOs. LARGE SCALE DATA: Sequencing data are available from the corresponding author upon request. LIMITATIONS, REASONS FOR CAUTION: The study should be replicated with a larger number of samples and with other PCOS phenotypes apart from different weight categories. Furthermore, as this work is the first one to establish PCOS EEOs, future studies should focus on incorporating other endometrial cell types, including immune cells, in a co-culture system. WIDER IMPLICATIONS OF THE FINDINGS: This novel in vitro organoid model for PCOS captures the endometrial abnormalities present in the two weight categories of women with PCOS, thereby providing a valuable tool to gain insights into PCOS-related endometrial dysfunction. Our findings propose potential links to the increased risk of pregnancy complications in women with PCOS, such as the role of altered receptivity and implantation environment including increased inflammation, which may contribute to aberrant placentation and subsequent placental dysfunction. STUDY FUNDING/COMPETING INTEREST(S): Jusélius Foundation, Novo Nordisk Foundation, Research Council of Finland, Horizon 2020 Marie-Curie MATER Innovative Training Network (all to T.T.P.), Fund for Scientific Research Flanders-Belgium (FWO, G0A6719N to J.V. and GO99023N to H.V.); KU Leuven Research Fund (C14/21/116 to H.V. and C14/24/152 to J.V.), University of Oulu Scholarship Foundation Grant (to L.L.), and PhD grant of China Scholarship Council (CSC, to M.W.). The authors have no conflicts of interest to declare.

Polycystic ovary syndrome (PCOS) is a common hyperandrogenic and metabolic condition in women. The syndrome is linked to subfertility and pregnancy complications, yet the independent effects of exposure to hyperandrogenism and obesity on endometrial function remain unclear. Here, PCOS-like mice were generated using prenatal androgenization (PNA) with dihydrotestosterone, followed by a prepubertal high-fat (HF) or standard diet. In ovariectomized mice, PNA impaired uterine closure during the implantation window, disrupted decidualization, and altered extracellular matrix– and inflammation-related gene expression. The effects were aggravated by the HF diet. In naturally mated, ovary-intact mice, PNA and HF diet affected decidual and placental gene expression, suggestive of placental dysfunction and inflammation, and induced fetal growth restriction. This study underlines the role of the uterus in adverse pregnancy outcomes in PCOS and identifies possible underlying mechanisms for future studies. Prepregnancy interventions targeting metabolic health and hyperandrogenism should be the next steps to optimize PCOS pregnancy outcomes.