Hormonal stimulation reduces numbers and impairs function of human uterine natural killer cells during implantation.

STUDY QUESTION: How does an altered maternal hormonal environment, such as that seen during superovulation with gonadotropins in ART, impact human uterine immune cell distribution and function during the window of implantation? SUMMARY ANSWER: Hormonal stimulation with gonadotropins alters abundance of maternal immune cells including uterine natural killer (uNK) cells and reduces uNK cell ability to promote extravillous trophoblast (EVT) invasion. WHAT IS KNOWN ALREADY: An altered maternal hormonal environment, seen following ART, can lead to increased risk for adverse perinatal outcomes associated with disordered placentation. Maternal immune cells play an essential role in invasion of EVTs, a process required for proper establishment of the placenta, and adverse perinatal outcomes have been associated with altered immune cell populations. How ART impacts maternal immune cells and whether this can in turn affect implantation and placentation in humans remain unknown. STUDY DESIGN, SIZE, DURATION: A prospective cohort study was carried out between 2018 and 2021 on 51 subjects: 20 from natural cycles 8 days after LH surge; and 31 from stimulated IVF cycles 7 days after egg retrieval. PARTICIPANTS/MATERIALS, SETTING, METHODS: Endometrial biopsies and peripheral blood samples were collected during the window of implantation in subjects with regular menstrual cycles or undergoing superovulation. Serum estradiol and progesterone levels were measured by chemiluminescent competitive immunoassay. Immune cell populations in blood and endometrium were analyzed using flow cytometry. uNK cells were purified using fluorescence-activated cell sorting and were subjected to RNA sequencing (RNA-seq). Functional changes in uNK cells due to hormonal stimulation were evaluated using the implantation-on-a-chip (IOC) device, a novel bioengineered platform using human primary cells that mimics early processes that occur during pregnancy in a physiologically relevant manner. Unpaired t-tests, one-way ANOVA, and pairwise multiple comparison tests were used to statistically evaluate differences. MAIN RESULTS AND THE ROLE OF CHANCE: Baseline characteristics were comparable for both groups. As expected, serum estradiol levels on the day of biopsy were significantly higher in stimulated (superovulated) patients (P = 0.0005). In the setting of superovulation, we found an endometrium-specific reduction in the density of bulk CD56+ uNK cells (P < 0.05), as well as in the uNK3 subpopulation (P = 0.025) specifically (CD103+ NK cells). In stimulated samples, we also found that the proportion of endometrial B cells was increased (P < 0.0001). Our findings were specific to the endometrium and not seen in peripheral blood. On the IOC device, uNK cells from naturally cycling secretory endometrium promote EVT invasion (P = 0.03). However, uNK cells from hormonally stimulated endometrium were unable to significantly promote EVT invasion, as measured by area of invasion, depth of invasion, and number of invaded EVTs by area. Bulk RNA-seq of sorted uNK cells from stimulated and unstimulated endometrium revealed changes in signaling pathways associated with immune cell trafficking/movement and inflammation. LIMITATIONS, REASONS FOR CAUTION: Patient numbers utilized for the study were low but were enough to identify significant overall population differences in select immune cell types. With additional power and deeper immune phenotyping, we may detect additional differences in immune cell composition of blood and endometrium in the setting of hormonal stimulation. Flow cytometry was performed on targeted immune cell populations that have shown involvement in early pregnancy. A more unbiased approach might identify changes in novel maternal immune cells not investigated in this study. We performed RNA-seq only on uNK cells, which demonstrated differences in gene expression. Ovarian stimulation may also impact gene expression and function of other subsets of immune cells, as well as other cell types within the endometrium. Finally, the IOC device, while a major improvement over existing in vitro methods to study early pregnancy, does not include all possible maternal cells present during early pregnancy, which could impact functional effects seen. Immune cells other than uNK cells may impact invasion of EVTs in vitro and in vivo, though these remain to be tested. WIDER IMPLICATIONS OF THE FINDINGS: These findings demonstrate that hormonal stimulation affects the distribution of uNK cells during the implantation window and reduces the proinvasive effects of uNK cells during early pregnancy. Our results provide a potential mechanism by which fresh IVF cycles may increase risk of disorders of placentation, previously linked to adverse perinatal outcomes. STUDY FUNDING/COMPETING INTEREST(S): Research reported in this publication was supported by the University of Pennsylvania University Research Funding (to M.M.), the Eunice Kennedy Shriver National Institute of Child Health and Human Development (P50HD068157 to M.M., S.S., and S.M.), National Center for Advancing Translational Sciences of the National Institutes of Health (TL1TR001880 to J.K.), the Institute for Translational Medicine and Therapeutics of the Perelman School of Medicine at the University of Pennsylvania, the Children's Hospital of Philadelphia Research Institute (to S.M.G.), and the National Institute of Allergy and Infectious Diseases (K08AI151265 to S.M.G.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. All authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Diagnostic accuracy of transvaginal ultrasound examination for assigning a specific diagnosis to adnexal masses.

OBJECTIVES: To determine the sensitivity and specificity of subjective evaluation of gray-scale and Doppler ultrasound findings (here called pattern recognition) when used by experienced ultrasound examiners with regard to making a specific diagnosis of adnexal masses. METHODS: Within the framework of a European multicenter study, the International Ovarian Tumor Analysis study, comprising nine ultrasound centers, women with at least one adnexal mass were examined with gray-scale and color Doppler ultrasonography by experienced ultrasound examiners. A standardized examination technique, and standardized terms and definitions were used. Using pattern recognition the examiners classified each mass as benign or malignant and suggested a specific diagnosis (e.g. dermoid cyst or endometrioma). The reference standard was the histology of the surgically removed adnexal tumors. RESULTS: A total of 1066 women were included, of whom 800 had a benign mass and 266 a malignant mass. A specific diagnosis based on ultrasound findings was suggested in 899 (84%) tumors. The specificity was high for all diagnoses (range, 94-100%). The sensitivity was highest for benign teratoma/dermoid cysts (86%, 100/116), hydrosalpinges (86%, 18/21), peritoneal pseudocysts (80%, 4/5) and endometriomas (77%, 153/199), and lowest for functional cysts (17%, 4/24), paraovarian/parasalpingeal cysts (14%, 3/21), benign rare tumors (11%, 1/9), adenofibromas (8%, 3/39), simple cysts (6%, 1/18) and struma ovarii (0%, 0/5). The positive and negative likelihood ratios of pattern recognition with regard to dermoid cysts, hydrosalpinges and endometriomas were 68.2 and 0.14, 38.9 and 0.15, and 33.3 and 0.24, respectively. Dermoid cysts, hydrosalpinges, functional cysts, paraovarian cysts, peritoneal pseudocysts, fibromas/fibrothecomas and simple cysts were never misdiagnosed as malignancies by the ultrasound examiner, whereas more than 10% of inflammatory processes, adenofibromas and rare benign tumors including struma ovarii were misdiagnosed as malignancies. CONCLUSIONS: Using subjective evaluation of gray-scale and Doppler ultrasound findings it is possible to make an almost conclusive diagnosis of a dermoid cyst, endometrioma and hydrosalpinx. Many other adnexal pathologies can be recognized but not confidently confirmed or excluded.

Changes in ultrasound morphology of the uterus and ovaries during the menopausal transition and early postmenopause: a 4-year longitudinal study.

OBJECTIVES: To describe changes in uterine and ovarian size and morphology as determined by ultrasonography from 2 years before to 2 years after menopause. METHODS: Twenty 50-year-old women with fairly regular vaginal bleeding at the start of the study underwent transvaginal ultrasound examination every 3 months until 12 months postmenopause, then every 6 months until 24 months postmenopause. The results are presented from 2 years before to 2 years after the menopause. RESULTS: In the 2 years preceding menopause all the women were in menopausal transition. From 2 years before to 2 years after menopause uterine anteroposterior diameter decreased by 22% (mean) and left and right ovarian volumes by 45 and 20% (median), respectively. At 2 years before the menopause the total number of intraovarian follicle-like cystic structures varied from 0 to 5, at the menopause from 0 to 7, and at 1 and 2 years after the menopause from 0 to 4 and from 0 to 2, respectively. Premenopause, the most common finding was that of ovaries containing either no follicles or a few follicles with at least one measuring >/= 11 mm and simultaneously a hyperechogenic endometrium of varying thickness and not manifesting any midline echo or triple-layer appearance. Images compatible with the late follicular phase were found in 6% (9/150) of examinations ('cycle day' 8-196) and images compatible with the luteal phase in 7% (10/150) ('cycle day' 11-56). Intraovarian cystic structures (3-25 mm) were seen in 14 women after the menopause. CONCLUSION: We have described sonographic changes in the uterus and ovaries occurring during the transition from premenopause to postmenopause.