Age, lifestyle, and disease affect ciliary transport in the human fallopian tube.

There was no significant difference in the CBF of ampulla (Fig.  1 A, circles) and isthmus (Fig.  1 B, circles) when comparing all patients ( n  = 27, n  = 34 tubes) (Fig.  1 C, generalised linear model for repeated measurements, p  = 0.772). CBF was not significantly different in right and left fallopian tubes (Fig.  1 C, generalised linear model for repeated measurements, p  = 0.160). Therefore, ampulla and isthmus and right and left oviduct were taken together in the subsequent analyses (Table  1 ). There was no significant difference in the CBF of control patients (risk reduction) in the secretory phase ( n  = 2, 19.3 ± 7.96 Hz) and proliferative phase ( n  = 2, 15.7 ± 5 Hz) of their menstrual cycle. There was no significant difference in the CBF of cycling ( n  = 4) and menopausal patients ( n  = 3, 17.3 ± 1.9 Hz) (Fig.  1 D, post hoc Bonferroni test, p  = 1.000). When analysing the effect of age on CBF in control patients ( n  = 7), a significant relationship was identified (Fig.  1 E). In patients under 45 years, increasing age was inversely proportional to CBF (Spearman’s rho, rho=−0.708, p  < 0.001). In patients between 45 and 50 years, CBF increased, subsequently plateaued and then decreased in the patients aged 50–55-years (Spearman’s rho, rho=−0.752, p   24.9 (Movie 2) examining control (risk reduction) patients ( n  = 5) and patients with benign pathologies ( n  = 9). In control patients (risk reduction), and patients with benign uterine myoma and ovarian cysts, the mean CBF decreased with increasing BMI (Fig.  2 A). CBF was significantly correlated with BMI (Fig.  2 A, Spearman’s rho: −0.543, generalised linear model, p  = 0.024). Control patients (risk reduction) and patients with benign pathologies who smoked ( n  = 5) revealed a significantly higher mean CBF (18.0 ± 1.8 Hz) than non-smokers ( n  = 9, 14.9 ± 4.6 Hz) (Fig.  2 B, generalised linear model, p  = 0.025). CBF was not significantly different in patients (risk reduction, benign diseases) who consumed alcohol on a regular basis ( n  = 13) compared to patients who abstained from alcohol ( n  = 3) (Fig.  2 C, generalised linear model, p  = 0.815). When comparing the CBF in patients with uterine benign myomas ( n  = 3) and ovarian cysts ( n  = 6) to the CBF of cycling and postmenopausal control patients (risk reduction), CBF was significantly decreased in benign uterine myoma (Fig.  3 A, generalised linear model for repeated measurements, p  = 0.027). CBF was significantly increased in menopausal ( n  = 4) and perimenopausal ( n  = 1) ovarian cyst patients compared to a cycling ovarian cyst patient ( n  = 1) in the proliferative phase (Fig.  3 B, post hoc with Bonferroni correction, p  < 0.001). When comparing menopausal patients, CBF was significantly reduced in patients with ovarian cysts (Fig.  3 C, generalised linear model for repeated measurements, p  = 0.038). When compared to patients in the proliferative phase, CBF was significantly reduced in the patient with the ovarian cyst (Fig.  3 D, generalised linear model for repeated measurements, p  = 0.004). To evaluate the effects of malignant gynaecological cancer on CBF, ciliated cells in cycling control patients (Fig.  4 A, circles, see Movie 3) and menopausal control patients (Fig.  4 B, circles) were compared to similarly cycling cervical cancer patients or menopausal endometrial cancer patients. In ampullae of patients with cervical cancer, selected ciliated cells were seen to beat more slowly compared to adjacent cells (Fig.  4 B, circle, see Movie 4). The mean CBF of cervical cancer patients ( n  = 6) was 11.9 ± 2.5 Hz, which was a significant reduction when compared to controls who averaged 17.5 ± 5.8 Hz (Fig.  4 C, generalised linear model for repeated measurements, p  = 0.002). Similarly, mean CBF of menopausal endometrial cancer patients ( n  = 5, see Movie 5) (14.4 ± 2.8 Hz) was significantly lower than the CBF of menopausal controls ( n  = 3) (17.3 ± 1.9 Hz) (Fig.  4 D, generalised linear model for repeated measurements, p  = 0.003). When the effects of cancer subtype and prognosis on CBF were assessed, CBF was significantly increased (17.1 Hz) in high-grade serous endometrial carcinoma (HGSEC) ( n  = 1) when compared to low-grade stage I disease ( n  = 2) (13.4 ± 4.2 Hz) (Fig.  4 E, generalised linear model for repeated measurements, p  = 0.018). This result was further refined by grouping FIGO stage I and II endometrial cancer as prognostically favourable ( n  = 3) and compared CBF to FIGO stage III endometrioid adenocarcinoma and HGSEC (prognostically poor, n  = 2) (Fig.  4 F). Endometrial cancer patients with a clinically favourable prognosis revealed a significantly lower CBF (13.2 ± 2.9 Hz) than those with poor prognosis (16.2 ± 1.4 Hz) (Fig.  4 F, generalised linear model for repeated measurements, p  = 0.021).

Full ethical approval for this study was granted from The Mater Misericordiae University Hospital (1/378/2140 & 1/378/1645) Dublin, Ireland), the Rotunda Hospital (REC-2014-004, Dublin, Ireland) and University College Dublin (LS-E-21–116, Dublin, Ireland). All experimental procedures were performed under the guidelines and regulations of the Mater Misericordiae University Hospital, the Rotunda Hospital and University College Dublin Institutional Review Boards in accordance with General Data Protection Regulation (GDPR) and the Declaration of Helsinki. Informed and written consent was obtained from all patients prior to surgery. By filling questionnaires all patient information regarding lifestyle, reproductive history, menstrual cycle stage, medical history, cancer diagnosis (if any) and familial history of cancer was obtained. 0.5–1 cm sections of human fallopian tube tissue (ampulla and isthmus) from patients ( n  = 43) were collected from the hospitals immediately after routine hysterectomy. In total, samples of 43 women were collected. 11 patients receiving hormonal therapy within the preceding 2 months prior to surgery had to be excluded from the study due to potential confounding effects on CBF. Samples of 5 patients, which did not provide precise data during the live cell imaging analysis due to degradation or viscous secretions covering the cilia, were also excluded from the analysis. Control patients ( n  = 7) were free of gynaecological disease and underwent risk reduction surgery due to family history. Further to that, patients diagnosed with cervical cancer ( n  = 6) or endometrial cancer ( n  = 5), as well as with ovarian cysts ( n  = 6), and with benign uterine fibroids (myomas) or polyps ( n  = 3) were examined. In a number of patients both samples from ampulla and isthmus and right and left oviduct were obtained which were included in the study after we had shown that CBF is independent of the anatomical location. Samples were obtained immediately after the removal of the genital tract and after securing the samples for routine pathological diagnosis. They were kept in cold phosphate buffered saline (PBS, Thermo Scientific, Dublin, Ireland) solution at 4 °C until use. Ciliary beat frequencies in the fallopian tubes of patients with cervical cancer ( n  = 6), endometrial cancer ( n  = 5), ovarian cysts ( n  = 6), benign uterine fibroids/polyps ( n  = 3) and control patients (risk reduction) ( n  = 7) were compared. CBF was evaluated from 5 randomly selected regions of interest (ROIs) containing clear motile cilia. Tubal specimens were imaged with a 40x water immersion lens (UMPLFL 40 × W/0.8, Olympus, Hamburg, Germany). Video recordings (100 frames/s (FPS) were carried out using StreamPix ® 7.0 software (NorPix, Canada) connected to a SUMMIX Mx7 camera (Sumix, CA, USA) mounted to a BX51WI fixed-stage upright microscope (Olympus, Hamburg, Germany). Fast Fourier Transformation (FFT) with AutoSignal ® (Systat Software, GmbH) and Image-Pro Plus ® version 7.1 ( https://my.mediacy.com/support/updates?swp=11 ) software (Media Cybernetics, Inc.) was used to convert the changes in the shades of grey caused by beating cilia into a frequency (Hz). Statistical analysis was performed by using SPSS 24.0 (IBM, New York. US). Normality of distribution was determined by Shapiro-Wilk test. The generalised linear model for repeated measurements was used to test for significant differences between ampulla and isthmus as well as contralateral and ipsilateral tubes from all patients. This statistical test was also used to investigate significant changes in the CBF of control patients according to cycle stage (with Bonferroni correction), and to clarify whether patients with benign disease could be included in our control cohort. Additional assessment of the effects of cycle stage within the ovarian cyst patient group was performed using the generalised linear model for repeated measurements with post hoc pairwise comparison according to Bonferroni. To investigate the relationship between patient age and BMI on CBF, the correlation coefficient Spearman’s rho was determined. When comparing the effects of cervical cancer and endometrial cancer on CBF to controls, the generalised linear model for repeated measurements was applied. An additional post hoc pairwise comparative test with Bonferroni correction was applied to evaluate any differences in the CBFs of the two cancer groups relating to different cycle stages, histological subtype and prognosis. P  ≤ 0.05 was deemed significant and p  ≤ 0.001 was considered as highly significant. Fig. 1 Effects of anatomical site, cycle stage, BMI, and age on ciliary beat frequency (CBF) in the fallopian tube of control patients (risk reduction). A , B : Live cell image (LCI) of ciliated cells (circles) in the ampulla ( A ) and isthmus ( B ). C : CBF is not significantly different in ampulla and isthmus (generalised linear model for repeated measurements, p  = 0.772). There is no significant difference in right and left oviducts (generalised linear model for repeated measurements, p  = 0.160). D : CBF is similar in cycling and menopausal patients (post hoc Bonferroni test, p  = 1.000). E : There is a significant relationship between patient age and CBF (Spearman’s rho, age < 45 years rho=−0.708 p   45 years rho=−0.752 p  < 0.001). Scale bars: A , B : 20 μm. Effects of anatomical site, cycle stage, BMI, and age on ciliary beat frequency (CBF) in the fallopian tube of control patients (risk reduction). A , B : Live cell image (LCI) of ciliated cells (circles) in the ampulla ( A ) and isthmus ( B ). C : CBF is not significantly different in ampulla and isthmus (generalised linear model for repeated measurements, p  = 0.772). There is no significant difference in right and left oviducts (generalised linear model for repeated measurements, p  = 0.160). D : CBF is similar in cycling and menopausal patients (post hoc Bonferroni test, p  = 1.000). E : There is a significant relationship between patient age and CBF (Spearman’s rho, age < 45 years rho=−0.708 p   45 years rho=−0.752 p  < 0.001). Scale bars: A , B : 20 μm. Fig. 2 Effect of lifestyle factors (BMI, smoking and alcohol consumption) on tubal CBF. A : In controls, benign uterine myoma and ovarian cysts, CBF is significantly reduced as patient BMI is increased (Spearman’s rho, −0.543, p  = 0.024, see movies 1 and 2). B : Control patients (risk reduction), benign uterine myoma patients, ovarian cyst patients who smoke reveal significantly higher mean CBF than non-smokers (generalised linear model, p  = 0.025). C : CBF is similar in patients who consume alcohol when compared to those who abstain (generalised linear model, p  = 0.815). Effect of lifestyle factors (BMI, smoking and alcohol consumption) on tubal CBF. A : In controls, benign uterine myoma and ovarian cysts, CBF is significantly reduced as patient BMI is increased (Spearman’s rho, −0.543, p  = 0.024, see movies 1 and 2). B : Control patients (risk reduction), benign uterine myoma patients, ovarian cyst patients who smoke reveal significantly higher mean CBF than non-smokers (generalised linear model, p  = 0.025). C : CBF is similar in patients who consume alcohol when compared to those who abstain (generalised linear model, p  = 0.815). Fig. 3 Effect of benign disease on tubal CBF. A : CBF is significantly decreased in patients with uterine myomas or polyps as compared to controls (generalised linear model for repeated measurements, p  = 0.027). B : CBF in ovarian cyst patients is significantly increased in the postmenopausal and perimenopausal patients compared to a patient in the proliferative phase (post hoc Bonferroni test, p  < 0.001). C : CBF is significantly reduced in menopausal ovarian cyst patients (generalised linear model for repeated measurements, p  = 0.038). D : CBF in control patients (proliferative phase) is significantly higher than in ovarian cyst (proliferative phase) patients (generalised linear model for repeated measurements, p  = 0.004). Effect of benign disease on tubal CBF. A : CBF is significantly decreased in patients with uterine myomas or polyps as compared to controls (generalised linear model for repeated measurements, p  = 0.027). B : CBF in ovarian cyst patients is significantly increased in the postmenopausal and perimenopausal patients compared to a patient in the proliferative phase (post hoc Bonferroni test, p  < 0.001). C : CBF is significantly reduced in menopausal ovarian cyst patients (generalised linear model for repeated measurements, p  = 0.038). D : CBF in control patients (proliferative phase) is significantly higher than in ovarian cyst (proliferative phase) patients (generalised linear model for repeated measurements, p  = 0.004). Fig. 4 Effects of cervical and endometrial cancer on tubal CBF. A : CBF (circle) of control cycling patients in the secretory phase (see movie 3). B : CBF (circle) in cervical cancer patients (see movie 4). C : CBF is significantly decreased in cervical cancer (generalised linear model for repeated measurements, p  = 0.002). D : CBF is significantly reduced in postmenopausal endometrial cancer patients (generalised linear model for repeated measurements, p  = 0.003). E : CBF is significantly increased in patients with high-grade serous endometrial carcinoma (generalised linear model for repeated measurements, p  = 0.018). F : Patients with endometrial cancers associated with favourable prognoses (e.g., endometrial adenocarcinoma FIGO stage I-II) reveal significantly lower CBF than those with poor prognosis (generalised linear model for repeated measurements, p  = 0.021). Scale bars: A , B : 40 μm. Effects of cervical and endometrial cancer on tubal CBF. A : CBF (circle) of control cycling patients in the secretory phase (see movie 3). B : CBF (circle) in cervical cancer patients (see movie 4). C : CBF is significantly decreased in cervical cancer (generalised linear model for repeated measurements, p  = 0.002). D : CBF is significantly reduced in postmenopausal endometrial cancer patients (generalised linear model for repeated measurements, p  = 0.003). E : CBF is significantly increased in patients with high-grade serous endometrial carcinoma (generalised linear model for repeated measurements, p  = 0.018). F : Patients with endometrial cancers associated with favourable prognoses (e.g., endometrial adenocarcinoma FIGO stage I-II) reveal significantly lower CBF than those with poor prognosis (generalised linear model for repeated measurements, p  = 0.021). Scale bars: A , B : 40 μm. Table 1 Mean ciliary beat frequencies (CBF) of the patient subgroups. Patients were grouped according to cycle stage, pathological diagnosis and lifestyle factors. The effects of smoking, alcohol intake and BMI on CBF was evaluated in patients without gynaecological malignancy. Abbreviations: RA: right ampulla, LA: left ampulla, RI: right isthmus, LI: left isthmus, SCC: squamous cell carcinoma, ECA: endocervical adenocarcinoma, EAC: endometrial adenocarcinoma, HGSEC: high-grade serous endometrial cancer, BMI: body mass index. Patient group Number of patients Number of tubal samples Mean CBF (Hz) Anatomical location Details Control cycling 4 6 17.5 ± 5.8 RA & LA Proliferative phase: n  = 2, secretory phase: n  = 2 Control menopausal 3 3 17.3 ± 1.9 LA & LI 12 months without menstruation Ovarian cysts 6 8 14.9 ± 3.1 RA & LA & RI Unilateral or bilateral ovarian cysts Benign uterine pathologies 3 3 12.8 ± 4.5 RA & LA Benign uterine fibroid and endometrial polyp Cervical cancer 6 9 11.9 ± 2.5 RA & LA & LI SCC/ECA Endometrial cancer 5 5 14.4 ± 2.8 RA & LA & RI EAC/HGSEC Smokers 5 6 18.0 ± 1.8 RA & LA Duration of nicotine exposure unknown Non-smokers 9 14 14.9 ± 4.6 RA & LA & RI & LI Never smoked Drinkers 13 17 15.6 ± 4.0 RA & LA & RI & LI Moderate to heavy alcohol consumption (up to 20 units/week) Abstinent 3 3 13.2 ± 4.9 RA & LA Do not drink alcohol/drink less than one unit/week High BMI 10 13 14.1 ± 3.3 RA & LA & RI BMI > 24.9 Normal BMI 4 5 19.9 ± 3.1 RA & LA BMI = 18.5–24.9 Mean ciliary beat frequencies (CBF) of the patient subgroups. Patients were grouped according to cycle stage, pathological diagnosis and lifestyle factors. The effects of smoking, alcohol intake and BMI on CBF was evaluated in patients without gynaecological malignancy. Abbreviations: RA: right ampulla, LA: left ampulla, RI: right isthmus, LI: left isthmus, SCC: squamous cell carcinoma, ECA: endocervical adenocarcinoma, EAC: endometrial adenocarcinoma, HGSEC: high-grade serous endometrial cancer, BMI: body mass index.

This study is the first of its kind to explore the effects of age, lifestyle and disease on human tubal transport function under near in vivo conditions using a state-of-the-art live cell imaging technology under near in vivo conditions. In doing so, this study has established that age, smoking, obesity, uterine myoma, and cervical and endometrial cancer exert profound effects on tubal transport. According to our live cell imaging studies, the mean CBF in the human fallopian tube ranges between 16 and 19 Hz in healthy patients independently of cycle stage (secretory or proliferative) or menopause. These results coincides with findings by Lyons et al. 10 who also found no significant difference in ampullar CBF of cycling secretory and proliferative control patients, albeit the baseline frequencies recorded in that study were lower than our findings. This is likely due to the variety of technologies applied to calculate ciliary activity as such discrepancies can be seen across a plethora of mammalian species 56 – 58 . CBF is similar in the right and left tube and – when looking at one oviduct – does not differ in the various anatomical sites such as ampulla and isthmus. This finding is supported by Scully et al. 19 when examining the bovine fallopian tube. Whereas anatomical location and cycle stage do not affect ciliary function, the patient`s age has significant effects on CBF. Thus, there is a gradual decline in CBF in patients under 45 years of age, which is followed by an increase in ciliary beating in 45–50-year-old patients, and then a reduction of CBF when patients begin to reach 55 years. This gradual decline in CBF might be attributed to the decreasing levels of oestradiol in the years leading up to menopause, in line with ovarian aging 59 . The sudden rise in CBF in a patient just over 45 years might be due to rapid changes in the ratio of progesterone and oestrogen in the perimenopause 60 . This is supported by a study of Gordon et al. 61 who recorded increases in oestradiol levels in perimenopausal patients. Besides age, the patient`s body mass index (BMI) significantly affects CBF in patients with benign conditions and control patients (risk reduction). To date, the relationship between body mass index and ciliary activity has mainly been studied in the respiratory system of animal models. Thus, in obese mice, regular exercise improves the response to influenza infection by activation of bronchoalveolar-lavage cell infiltration and by increasing tracheal ciliary beat frequency 62 . In rat oviducts, the presence of high levels of leptin, which is a common feature of obesity in humans 63 , has been shown to be associated with reduced CBF and impaired oocyte transport 64 . Consequently, high levels of leptin might account for the decreased CBF of obese patients in our studies. Smoking is another lifestyle factor that significantly affects tubal function. Patients who smoke reveal a significantly higher CBF as compared to non-smokers. To date, there are no studies that have investigated the effect of cigarette smoke on ciliary beating in the human fallopian tube under near in vivo conditions. In regard to embryo transport, which is the result of ciliary beating, smooth muscle contraction and fluid flow, Horne et al. and Guo et al. were able to show that smoking leads to impaired transport of the early embryo in the fallopian tubes. This might be associated with higher ectopic pregnancy rates in smoking women 65 , 66 . Whereas there are only few studies which elucidate the effect of nicotine in the genital tract, there has been significant inquiry into the effect of cigarette smoking on the function of ciliated epithelium in the nasal cavity, trachea and lung in animal models under in vitro conditions 67 – 69 . A study using tracheal epithelium from ferrets found that CBF was increased after exposure to nicotine 70 . Similarly, Perniss et al. 71 reported that nicotine stimulates ciliary activity in the trachea of mice. In regard to human ciliary activity in the respiratory tract, several studies reported in the early stages of the COVID-19 pandemic that smokers appeared to fare better than non-smokers when infected with the Sars-Cov-2 virus 72 . This phenomenon was explained by the fact that smokers have higher CBFs in their nasal tracts which might improve protection against Sars-Cov-2 infection by increased mucociliary clearance 72 . In animals, there is one study which reports a reduction in CBF following exposure to cigarette smoke under in vitro conditions in the hamster oviduct 36 . These results might be due to the duration and dosage of exposure. As shown by Owhor et al. 7 tubal inflammation first leads to increased CBF in an attempt to optimize oviductal clearance and then decreases with the progression of the disease and the accumulation of toxins 7 . Thus, the relationship between smoking and ciliary transport is complex and warrants careful consideration of both the duration and timing of exposure. This study does not differentiate between individuals with chronic smoking habits and those with acute or recent exposure to cigarette smoke. Further studies elucidating the different effects of acute and chronic nicotine exposure will have to be performed to precisely determine whether chronic exposure is associated with cumulative harm. In contrast to smoking regular alcohol consumption does not impact ciliary beating. To the best of our knowledge, the effect of alcohol intake on human tubal ciliary beat frequency has not been investigated to date. In animal studies, in vivo mouse experiments have shown that acute ethanol intake during early pregnancy is associated with reduced embryo transport, delayed embryo development, loss of ciliated cells and disrupted ciliary structural integrity 73 . It is important to note that our study fully relied on self-reported data of the patients (units of alcohol consumed weekly) and therefore might not fully reflect the actual intake. Further to that, experiments using direct exposure of tubal explants to ethanol in animal models are likely to reveal effects which are not seen during oral intake of a variety of alcoholic beverages in humans. Besides lifestyle, diseases of the female genital tract affect tubal ciliary function. In patients with uterine myomas (fibroids) and polyps, there is a significant drop in CBF compared to control patients. As progesterone synthesis is increased by uterine fibroid smooth muscle cells 74 and both maintains and enhances fibroid growth in an in vivo xenograft model 75 , reduced CBF might be due to increased progesterone levels. Further to that, the glandular epithelium in endometrial polyps reveal increased expression of oestrogen and progesterone receptors compared to healthy endometrial epithelia 76 , thus further increasing the effect of progesterone on tubal CBF. When looking at patients with ovarian cysts, tubal CBF was increased in menopausal patients and decreased in patients during the proliferative phase. This finding highlights the continuous interaction between ovarian and tubal cells and points to the fact that diseases of the ovary always affect tubal function and that alterations in the tube impact ovarian function. This is supported by the increasing evidence that many high-grade serous carcinomas of the ovary originate from the epithelium of the distal fallopian tube 77 – 79 . The fact that there is controverse action of the ovarian cysts on CBF in cycling and menopausal patients might be due to the fluctuating oestrogen and progesterone levels in the cycling patients and the decreased oestrogen levels in menopausal patients, which are associated with the degradation and loss of cilia 80 . Our finding that myomas and ovarian cysts impair tubal function are highly consequential for the analysis and publication of patient data. The majority of clinical research in regard to the fallopian tube use the patients with myomas and ovarian cysts as control group, which - according to the results of our studies – should be avoided as these diseases of ovary and uterus do affect tubal function. In regard to malignant gynaecological cancer, CBF is significantly decreased in patients with cervical cancer and in menopausal patients with endometrial cancer. This reduction in CBF may point towards a cancer-induced pro-inflammatory response within the female genital tract, which has had an upstream effect on the function of the fallopian tube. Thus, increased levels of cytokines, such as interleukin-6 (IL-6), have been shown to decrease CBF in the human fallopian tube 27 . High levels of serum IL-6 have also been found in patients with cervical cancers and is associated with adverse prognosis 81 . In endometrial cancer, increased production of IL-6 has been linked to cancer growth 82 and high serum levels of the cytokine have been detected in patients with advanced cancer 83 . Interestingly, CBF in patients with high-grade endometrial cancer had higher CBF when compared to patients with early-stage disease. High-grade serous endometrial tumours tend to manifest more aggressively and are more frequently subject to chemotherapeutic resistance 84 . They are often oestrogen or progesterone receptor negative 85 , which not only mitigates the use of targeted hormonal intervention 86 , 87 , but also might cause altered hormonal action on ciliary beating. In addition, high-grade endometrial carcinomas are associated with chronic inflammation 88 and increased prostaglandin E2 secretion 89 – 92 which has been shown to increase CBF in vitro 93 . In contrast, lower-grade endometrioid adenocarcinomas are commonly associated with excessive oestrogen production 94 , 95 which is able to increase ciliary beat frequency in several mammalian species 21 , 96 . However, Mahmood et al. 9 showed that oestradiol alone did not impact CBF in the human fallopian tube but rather negated a reduction in CBF when tissues were co-incubated with progesterone 9 . It is important to highlight that the study has limitations concerning sample size and data collection. Although samples of 43 patients were collected, every single patient was different, revealing an individual combination of lifestyle and medical traits. To provide the best possible precision every single aspect was considered in the multifactorial analyses. This leads to smaller patient groups which is the nature of human studies, because each patient is individual and different. As it has become a routine procedure in human pathology to check the entire fallopian tube for serous tubal intraepithelial carcinoma after removal, tissue availability for experimental procedures is further reduced. Further research is warranted to explore the mechanisms involved and to test the potential for clinical interventions aimed at improving CBF in populations at risk for reproductive challenges. Overall, this study is the first to highlight the effects of cycle stage, age, lifestyle, benign disease, and malignant cancer on tubal transport function in the ex vivo organ culture using state-of-the-art live cell imaging technology. Our findings point to substantial functional alterations in the fallopian tubes of patients who are obese and who smoke. This highlights the importance of individual counselling in patients, emphasizing how changes in lifestyle may benefit fertility and pregnancy outcomes. Of importance, benign diseases of ovary and uterus impact ciliary beating in the oviduct. The fact that myomas and ovarian cysts impair tubal function imply that only patients undergoing hysterectomy with salpingoopherectomy for risk reduction are to be considered as control group in statistical analyses of research involving the oviduct. The fact that CBF is reduced in patients with cervical and endometrial cancer may be of particular significance to pre-menopausal cancer patients wishing to opt for fertility preserving surgery and may explain why some women struggle to conceive following their treatment. The oviduct does not only provide transport, but also ensures nutrition of the early embryo, which reaches the uterus through fluid movement in the genital tract due to ciliary beating and smooth muscle contraction. Thus, optimizing ciliary transport might offer a potential unexplored pathway towards improving fertility outcomes both in infertile and cancer patients.

Ciliary beating in the fallopian tube is vital for the successful capture of the cumulus-oocyte-complex by the infundibulum after ovulation as well as for the timely transport of the early embryo in direction to the uterus 1 – 3 . It also contributes to the circulation of the oviductal fluid containing nutrients and factors that are essential for sperm capacitation, fertilization and early embryonic development 4 – 6 . Further to that, ciliary beating helps to clear dead cells and debris and maintains a healthy luminal environment in the fallopian tube. As shown by Owhor et al. 7 inflammation in the oviduct initially induces a rise in ciliary beat frequency (CBF) which might be associated with increased oviductal clearance. CBF is species-specific. In humans, the CBF ranges between 2 and 23 Hz 8 – 13 . In mice, Shi et al. 14 reported 11 Hz and 9 Hz for dioestrus and oestrus mice, whereas Bylander et al. 15 and Noreikat et al. 16 reported baseline CBFs of 23 and 21 Hz, respectively. Similarly, in the bovine, CBF has been reported to range between 8 and 23 Hz 17 – 19 . Due to different measurements technologies CBF numbers may vary widely 20 – 22 . Ciliary beating is driven by a primary motor function that is calcium dependent and is powered by the hydrolysis of ATP 23 . The motor protein composition of human tubal cilia is identical to that of respiratory cilia 24 . Numerous neuronal factors as well as hormones have been shown to affect CBF 11 , 25 , 26 . Thus, progesterone (P4) and oestradiol (E 2 ) as well as cytokines such as interleukin-6 (Il-6), prostaglandins and angiotensin-II 10 , 20 , 27 , 28 have been reported to modulate human CBF. In oviductal explants progesterone reduces CBF by 40–50% after 24 hs of treatment 9 , whilst oestradiol enhances ciliary beat frequency (CBF) by 25% in ex vivo studies 29 . Ovariectomy in rabbits results in the loss of tubal ciliated cells and a reduction of cilia length in the oviduct highlighting that ovarian hormones play a vital role for ciliary formation and function 30 . Tubal explants exposed to follicular fluid reveal increased CBF compared to controls 11 . CBF is also increased in oviductal explants treated with peritoneal fluid from patients in the secretory phase when compared to explants treated with peritoneal fluid obtained during the proliferative phase 11 . Incubation with testosterone has been shown to decrease ciliary beating in human tubal explants in vitro 31 . The protein composition of oviductal fluid varies in line with the reproductive cycle stage, and is low at ovulation and high at menstruation 32 . This variation alters the viscosity of tubal fluid, which consequentially alters the flow rate. Changes in protein concentration are detected by tubal cells via transient receptor potential vanilloid 4 (TRPV4) which then upregulates CBF to mitigate the decreased viscous fluid flow 33 . To date, the effects of age, lifestyle, and disease on human CBF have not fully been elucidated. As increasing age has a significant effect on steroid hormone expression in the tube 34 and is associated with deciliation 35 . Thus,  changes in CBF might occur as age increases. The influence of lifestyle factors on reproductive success such as cigarette smoking have been extensively studied in animal models. In vitro exposure to cigarette smoke has been shown to significantly reduce CBF 36 , inhibit oocyte pick up 37 and reduce smooth muscle contraction 38 in the hamster oviduct. In addition, mice exposed to cigarette smoke reveal significantly decreased numbers of cilia 39 . In regard to obesity, it is known that the disruption of endocrine signalling results in reduced reproductive success 40 . Thus, the infertility risk is three times higher in obese women than in non-obese women and patients who are overweight or obese are more likely to experience negative outcomes when undergoing IVF treatment 40 – 42 . With respect to benign diseases, pelvic inflammatory diseases such as salpingitis and cystic ovary disease (COD) have been associated with reduced ciliary beating and smooth muscle contraction in the bovine fallopian tube 7 , 19 . In humans, exposure to elevated testosterone, which is a common feature of polycystic ovarian syndrome, results in reduced CBF 31 . In addition, patients with tubal endometriosis have significantly decreased CBF and smooth muscle contractions when compared to controls 12 . In regard to gynaecological malignancies their effects on tubal ciliary activity in humans have not been investigated yet. To date, merely adverse effects of oncogenic signalling pathways on ciliary function and formation have been reported 43 . Thus, the activation of serine threonine kinase Aurora-A (AURKA), which acts as oncogene in several tumor types, causes ciliary degradation in humans in both malignant and non-malignant cells 44 , 45 . Additionally, the oncogene Kirsten rat sarcoma viral oncogene homologue (KRAS) has been shown to suppress ciliary formation 46 . Cervical cancer predominantly affects pre-menopausal patients who are likely to opt for fertility-sparing surgery after the diagnosis 47 – 49 . Recent studies have highlighted that successful pregnancy outcomes are reduced in this group 50 , 51 highlighting the necessity to elucidate the effects of gynaecological cancer on tubal function. Although endometrial cancer is predominantly diagnosed in menopausal patients, increased numbers in the 30–39-year age group have been reported recently 52 – 55 . This implies that precise knowledge on oviductal function in cancer patients may prove highly valuable for those patients wishing to conceive after their diagnosis. Although it has been known for a long time that fertility is affected by age, lifestyle, and disease, it is still unknown to date how these factors precisely affect tubal function. Therefore, we set out to investigate the effect of anatomical site and cycle stage, patient age, smoking, alcohol, and obesity as well as the impact of benign diseases and gynaecological cancer on ciliary function of the fallopian tube using novel live-cell imaging technologies in real-time and under near in vivo conditions.

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