Frailty is a geriatric syndrome characterized by anabolic-catabolic imbalance and multisystem dysregulation resulting in increased adverse health outcomes, and is closely related with dietary habits in the general population. Although chronic inflammatory diseases are thought to accelerate development of frailty, correlations between rheumatoid arthritis (RA), frailty and dietary habits have not been examined. We performed a cross-sectional study using our cohort database (KURAMA cohort), and classified 306 participants into three groups  according to the Study of Osteoporotic Fracture (SOF) criteria. Multivariate logistic analysis revealed that the presence of frailty/prefrailty was significantly correlated with the disease activity score (DAS28-ESR) (OR 1.70 (1.30–2.22), p < 0.0001). Additional analyses of frailty and food intake showed that 5 foods  of 20 groups on the questionnaire were inversely associated with the prevalence of frail/prefrail categories. In multivariate analysis with the five nutrients, fish intake (> two times a week) was an independent covariate negatively correlated with frailty/prefrailty (OR 0.35 (0.19–0.63), p = 0.00060). In conclusion, habitual fish intake may play a key role in nutritional intervention to prevent progression of frailty and RA. RA patients show increased prevalence of comorbidities 3, and declining functional status and body weight loss are reported as risk factors for mortality5. Especially, the elderly with RA often have multimorbidity and drug intolerance, and tend to experience functional decline due to poor treatment response as well as limited therapeutic options7.Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joint destruction; cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) play key roles in systemic inflammation8. Frailty has a complex multifactorial etiology, and chronic inflammation is a crucial factor that increases catabolism and interferes with homeostatic signaling10. Serum levels of inflammatory molecules are elevated in the frail elderly11, and it has been proposed that several inflammatory diseases accelerate anabolic resistance and the development of frailty12. Although RA is a representative of chronic inflammatory disease, little is known of the interaction between frailty and RA-related factors such as current disease activity and therapeutic agents.Frailty, a key concept in geriatric medicine, is a state of compromised physiologic reserve and increased risk for adverse outcomes and mortality13. In the RA population, seafood, fruits and vegetables have beneficial effects on disease activity14, whereas high-fat diets have the opposite effect by leading to a pro-inflammatory response15. Even though several nutrients are expected to have therapeutic effects both on frailty and inflammatory diseases, clinical correlations between dietary habits and frailty with RA have not been examined.Recently, a number of studies have reported the impact of dietary habits on frailty and several other diseases. Nutritional interventions such as protein supplementation were found to improve physical function of the frail elderly in several clinical trialsIn the current study, we conducted a cross-sectional study using the Kyoto University RA Management Alliance cohort (KURAMA) to clarify the relationships among frailty, dietary habits and disease activity of RA patients.16. A total of 388 outpatients with RA who were over 18 years of age and visited the Kyoto University Hospital between 1st May and 31st December 2014 were recruited for the study. After we excluded 82 patients for lack of a complete data set or incomplete answers to the food intake questionnaire, we included the remaining 306 patients in the study. This research complied with the Declaration of Helsinki and was approved by the Medical Ethics Committee of Kyoto University Graduate School and Faculty of Medicine . Informed consent was obtained from all subjects, including the use of human blood samples and data.In the present study, we conducted a cross-sectional study of female outpatients with RA from the KURAMA (Kyoto University Rheumatoid Arthritis Management Alliance) cohort database17. The SOF index comprises the following three components: 1. weight loss (5% or more reduction in the previous year), 2. chair stands (inability to rise from a chair 5 times without using hands) and 3. reduced energy levels detected by the question: “Do you feel full of energy?” from the Geriatric Depression Scale. Patients with the presence of 0, 1, and 2 or 3 components were defined as Robust, Prefrail, and Frail, respectively. To assess frailty-related physical activity parameters, we evaluated hand grip strength, walking speed and skeletal muscle mass. We measured hand grip strength using a JAMAR digital hand dynamometer , and evaluated 6-m walking speed by a portable gait rhythmogram . Skeletal muscle mass was assessed by bioelectrical impedance Analysis . Skeletal muscle index (SMI) was calculated as the skeletal muscle mass in kilograms divided by height in meters squared. We used self-reported questionnaire forms for a history of falls and bone fractures in the previous year.We assessed frailty status of the subjects using the Study of Osteoporotic Fractures (SOF) criteria as previously reported18. This questionnaire consisted of 20 items about the intake frequency of 1. staple food  for breakfast, 2. staple food for lunch, 3. staple food for dinner, 4. meat, 5. fish, 6. tofu (soybean curd), 7. eggs, 8. milk, 9. vegetables, 10. fruits, 11. deep-fried foods, 12. cakes, 13. juice or isotonic drinks, 14. junk foods, 15. sweets like candies and chocolates, 16. frozen foods, 17. pickles, 18. ham, sausage or kamaboko (boiled fish paste), 19. miso soup (fermented soybean paste) and by the participants. alcohol. The intake frequency was collected for each item on the basis of the following 8 categories: 1 =  < 1 time/month, 2 = 1–3 times/month, 3 = 1–2 times/week, 4 = 3–4 times/week, 5 = 5–6 times/week, 6 = 1 time/day, 7 = 2 times/day, 8 = 3 times/day.Dietary habits of RA patients were obtained from the self-reported questionnaire as described previouslyThe disease activity and physical disability of RA were evaluated by the 28-Joint RA Disease Activity Score (DAS28-ESR), Steinbrocker’s stage and the health assessment questionnaire disability index (HAQ). Baseline laboratory data were assessed including hemoglobin (Hb), albumin (Alb), and C-reactive protein (CRP). The data on Current RA medications including Methotrexate (MTX), prednisolone (PSL), Tumor necrosis factor-α (TNF-α) inhibitors, Interleukin-6 (IL-6) receptor inhibitors and cytotoxic T-lymphocyte antigen 4 (CTLA4) immunoglobulin were collected from the KURAMA database.Data on continuous variables, dietary habits and categorical variables are expressed using median (interquartile), median (range) and numbers (%), respectively. To compare characteristics of RA patients divided into three groups by frailty status, we carried out a Steel–Dwass test for continuous variables or a Fisher’s exact test for categorical variables. To analyze the relationship between the frequency of each food intake and the prevalence of prefrail or frail, we used the Cochran-Armitage trend test.For identification of RA-related contributing factors to frailty, we first adopted univariate logistic regression analysis. We constructed a dummy variable as follows: 0 = robust and 1 = prefrailty or frailty, and following analyses were carried out with its frailty variable as a dependent variable. After selecting variables detected as significant variables in the univariate analysis that were considered to be clinically relevant, we conducted multivariate logistic regression analysis. To determine the dietary factors that contribute to frailty status, further analysis was carried out. A dummy variable was constructed with 0 and 1: fish and meat dish, 0 = low frequency (≤ 2 times/weeks) and 1 = high frequency (3 times/week ≤); milk, vegetable and fruits, 0 = low frequency (≤ 6 times/week) and 1 = high frequency (1 time/day ≤). As the distribution of intake frequency of each food differed greatly, we redistributed intake frequency into two categories based on the medians. We then adopted multivariate logistic regression analysis using these variables as well as RA-related contributing factors. Furthermore, the same analysis was conducted in elderly RA patients aged 65 years and older (n = 147) because clinical definitions of frailty are generally used for the elderly who are over 65 years of age. In addition, we also constructed another logistic regression model, a forward stepwise logistic regression model, to confirm the independent variables . Statistical significance was analyzed using JMP14  and P values < 0.05 were considered significant.20. Regarding the current RA medications being used by the participants in this study, 72.5%, 28.6%, and 43.8% of patients were treated by MTX, PSL, and biological agents, respectively.A total of 306 female patients with RA were enrolled in the following analyses. Characteristics of the patients are described in Table To characterize differences among the frailty stages, three groups were compared: robust, prefrail, and frail Table . The aveTo learn which factors contribute to prefrail and frail status, we carried out logistic analysis. In univariate analysis, age, duration of RA, DAS28-ESR, HAQ, and PSL use were positively associated with the presence of prefrailty or frailty Table  left. InBecause little is known regarding nutrition therapy for frailty in RA in contrast to the general population, we performed analysis to determine the relationship between frailty with RA and food intake habits. Trend analysis revealed that the frequency of food intake was inversely correlated with the prevalence of prefrailty or frailty in 5 of the 20 groups on the questionnaire: fish Fig. A, meat B, milk C, vegeta21, and was significantly correlated with current disease activity (DAS28-ESR) in multivariate logistic analysis. These results accord with previous reports that RA patients have higher prevalence of sarcopenia than the community-dwelling elderly, and that pro-inflammatory cytokines such as IL-6 and TNF-α participate not only in RA progression but also in frailty development due to catabolic effects on muscles11. Inflammatory mediators are also associated with the development of several age-related diseases 22, and may be candidates for therapeutic targets of frail progression.To the best of our knowledge, the present study is the first to reveal statistical correlations between dietary habits and frailty in an RA population. The prevalence of frailty was higher compared to that in general7.In univariate logistic analysis, other variables were also associated with the presence of frailty such as age, physical activity parameters  and PSL/MTX use. Age-related decline in physical activity including hand grip strength and walking speed is the very definition of frailty, and these results suggest the validity of the SOF index for assessing frailty status in RA populations as well as in general. PSL use induces proximal muscle wasting and weakness, and is an indicator of thigh muscle area and density in RA patients. MTX use was negatively associated with the prevalence of frailty in the analysis, which might reflect a limitation of therapeutic options for frail patients23, and fish oil–derived n–3 PUFA therapy slows the normal decline in muscle mass and function24. Serum levels of n-3 PUFA parallel dietary fish consumption26 and correlate with a 27% reduction in all-cause mortality risk of elderly27. Marine-derived n-3 PUFAs also have anti-inflammatory effects on RA progression28. In prospective, double-blind, randomized controlled studies by Kremer et al.30, dietary supplementation of n-3 PUFAs dose-dependently decreases RA activity parameters  and serum levels of IL-1β. n-3 PUFAs also protect against joint inflammation in some animal models of RA33, and modulate a range of immunological reactions in RA patients such as reduced production of leukotriene B4 by neutrophils29, IL-1 by macrophage29 and prostaglandin E2 by mononuclear cells34. In addition, our group has previously shown a negative correlation between seafood intake frequency and RA disease activity14. Based on our results and these knowledge, habitual fish intake may contribute to the prevention of frailty through anti-inflammatory effects via n-3 fatty acid and partly through protein intake.In additional multivariate analyses, we also found that fish intake frequency rather than meat intake frequency was negatively correlated with the presence of frailty in RA patients. Fish is well known as an important source of potentially beneficial nutrients for preventing frailty such as protein and n-3 polyunsaturated fatty acids (n-3 PUFAs). High protein intake is associated with low prevalence of frailty35. FFQ is a straightforward self-reported questionnaire previously used for quantitative evaluation of dietary habits in RA patients as well as in general18. Both are simple and versatile methods, and are potentially useful for investigation of correlations between frailty and dietary habits in various clinical populations.In the present study, we applied a combination of simplified frailty index (SOF criteria) and food frequency questionnaire (FFQ) to assess frailty and dietary habits. SOF criteria have only three components , and efficiently predict risk of adverse outcomes and mortalityThere are several major limitations in the present study. Although the FFQ in the current study can determine the intake frequency of each type of foods, we cannot estimate the actual total amount of protein intake from the FFQ data and cannot determine whether the benefit of fish consumption is due to increased protein intake or other beneficial compounds. Our cross-sectional study does not imply causation, and the long-term relationship of dietary habits and frailty is still unknown. It is not known whether our findings can be generalized because of clinical factors that may differ in each study, such as epidemiological backgrounds of our participants, a dietary culture based on fish, and criteria of frailty. In addition, only female participants were included in the present study. Moreover, RA patients treated in a university hospital may have uncommon clinical characteristics compared to those in other medical institutions.In conclusion, frailty has a positive correlation with RA disease activity and a negative correlation with fish intake frequency. Habitual fish intake may thus play a key role in nutritional intervention for preventing progression of frailty and RA.Supplementary Information


To investigate the decentration and tilt of plate-haptic multifocal intraocular lenses (MfIOLs) in myopic eyes.Myopic  and non-myopic (21.0 mm < AXL ≤ 24.5 mm) cataract eyes were enrolled in this prospective study and randomly assigned to receive implantation of Zeiss AT LISA tri 839MP lenses (Group A) or Tecnis ZMB00 lenses (Group B). In total, 122 eyes of 122 patients were available for analysis. Decentration and tilt of MfIOLs, high-order aberrations (HOAs), and modulation transfer functions (MTFs) were evaluated using the OPD-Scan III aberrometer 3 months postoperatively. Subjective symptoms were assessed with a Quality of Vision questionnaire.Near and distance visual acuities, tilt and horizontal decentration did not differ between the two groups, postoperatively. However, myopic eyes of Group B showed greater vertical decentration than those of Group A , particularly when the MfIOLs were placed horizontally or obliquely. Overall decentration of myopic eyes was greater in Group B than in Group A . In Group B, AXL was negatively correlated with vertical decentration and positively correlated with overall decentration. No such correlations were found in Group A. Intraocular total HOAs, coma, trefoil and spherical aberrations were lower in Group A than in Group B for a 6.0 mm pupil among myopic eyes. Generally, Group A had better MTFs and fewer subjective symptoms than Group B among myopic eyes.Plate-haptic design of MfIOLs may be a suggested option for myopic cataract eyes due to the less inferior decentration and better visual quality postoperatively. Driven by the increasing expectations and visual demands of cataract patients, cataract surgery has now developed into a form of refractive surgery , 2, withDespite increased use, the implantation of MfIOLs in myopic cataract eyes remains controversial. In addition to retinal abnormalities , the comRecently, we observed that the plate-haptic MfIOLs appeared to show better stability in myopic eyes, in that they seldom spin or migrate to one corner of the capsular bag after gentle manipulation with the nucleus chopper during surgery. Unlike the C-loop haptic design with a gap between the haptics and the optic, the plate-haptic design of MfIOLs achieves direct support from the capsular bag through the four corners of the IOL. Thus, we speculated that this design may present better capsular stability than the C-loop haptic MfIOLs, providing a better option for spectacle independence among myopic eyes undergoing phacoemulsification and IOL implantation.The aim of the current study was to compare the capsular stability outcomes of plate-haptic and C-loop haptic MfIOLs in myopic eyes by evaluating the decentration and tilt with the OPD-Scan III aberrometer . Visual quality and patient symptoms were also compared.www.clinicaltrials.gov, accession number NCT03062085).Patients undergoing lens phacoemulsification of cataract and IOL implantation were enrolled in this prospective, randomized, controlled study over a period of 2 years. The study was conducted in accordance with the ethical principles originating from the Declaration of Helsinki and its amendments, consistent with Good Clinical Practices and local regulatory requirements. Written informed consent was obtained from all study subjects prior to enrollment, and the protocols were reviewed and approved by the institutional review board of the Eye and Ear, Nose, and Throat (EENT) Hospital of Fudan University, Shanghai, China, where the study was conducted. The study was affiliated to Shanghai High Myopia Study  and kappa angle < 0.4 mm. Patients were excluded if they had diabetes or if their eyes had zonular weakness, strabismus, retinal pathology, uveitis, glaucoma, or previous intraocular procedures or trauma. During February 1, 2017, and February 1, 2019, at the EENT Hospital of Fudan University, 68 myopic (AXL > 24.5 mm) and 62 non-myopic (21.0 mm < AXL ≤ 24.5 mm) cataract eyes were enrolled and randomly assigned to receive implantation of the Zeiss AT LISA tri 839MP lens  or the Tecnis ZMB00 lens  according to the random number table. Eyes with severe intraoperative or postoperative complications or lost to follow-up were excluded from data analysis. A total of 122 eyes of 122 patients completed the study and were available for analysis .Prior to surgery, all the patients underwent complete ophthalmic examinations which included assessment of visual acuity, slit lamp examination, corneal topography , AXL measurements , fundoscopy, and B-scan ultrasonography.All the surgeries were performed by a single, experienced surgeon (Prof. Y.L.) using a standard procedure. A 2.6 mm clear corneal incision was made temporally before a 5.5 mm continuous curvilinear capsulorhexis, hydrodissection and phacoemulsification. The IOL was implanted in the capsular bag and adjusted to the center. After thorough removal of the viscoelastic, the incision was hydrated. Following surgery, all patients received topical prednisolone acetate , levofloxacin , and pranoprofen eye-drops .Three months after surgery, all patients underwent complete ophthalmic examinations. Uncorrected distance visual acuities , corrected distance visual acuities , and uncorrected near visual acuities  were assessed.The tilt of the MfIOLs was obtained directly from the intraocular tilt data in the wavefront mode of the OPD-Scan III aberrometer, and decentration of the MfIOLs was measured using the same methodology as reported in our previous study . OPD-ScaDysphotopsia symptoms were assessed by a validated Rasch-adjusted Quality of Vision (QoV) questionnaire which included 10 dysphotopsia items . The que2 test. Pearson’s correlation analyses were used to analyze relationships between continuous variables. All P values were 2-sided, and P values less than 0.05 were considered statistically significant. Statistical analysis was performed using SPSS version 22 .A sample size of 22 per group was calculated to detect a 0.15 mm between-group difference in IOL decentration with an intended power of 90% and a significance level of 5%. A minimum size of 30 patients was enrolled in each group, assuming that some would be lost to follow-up. All continuous data were presented as mean ± standard deviation. Between-group differences for continuous data were assessed using Student’s t-test, and categorical variables were compared using the χ2 tests for sex and operated eye, all P > 0.05). Postoperative UDVA, CDVA and UNVA did not demonstrate significant differences between the two groups .Characteristics of all study patients are shown in Table P > 0.05). Group B presented significantly greater vertical and overall decentration than Group A . The comparisons of the two MfIOLs among non-myopic and myopic eyes in terms of the postoperative decentration and tilt are shown in Table P < 0.001), while no such differences were found for horizontal decentration and tilt either among myopic or non-myopic eyes .There were no significant differences between the two groups for tilt and horizontal decentration . Among non-myopic eyes, vertical decentration showed no difference between the 2 types of MfIOLs, regardless of IOL placement . However, in myopic eyes, the C-loop haptic MfIOLs showed significantly greater vertical decentration than the plate-haptic MfIOL when placed horizontally or obliquely .The influence of MfIOL in-bag placements on vertical decentration are shown in Table P = 0.003; Fig. P < 0.001; Fig. P > 0.05).In Group B, AXL was negatively correlated with vertical decentration . Among myopic eyes, total HOAs, coma and spherical aberrations were significantly lower in Group A than in Group B either for 6.0 mm or 4.0 mm pupil diameter . Myopic eyes of Group A showed lower total HOAs than those of Group B either for 6.0 mm or 4.0 mm pupil diameter, and lower coma, trefoil and spherical aberrations were also found in myopic eyes of Group A, as compared to those of Group B for a 6.0 mm pupil diameter . Myopic eyes of Group A presented a significantly higher total ocular MTF than those of Group B for a 6.0 mm pupil diameter . For both the 6.0 mm and 4.0 mm pupil diameter, ocular MTFs were higher in Group A than in Group B, especially among myopic eyes  was significantly higher in eyes of Group A than those of Group B for a 6.0 mm pupil diameter . Comparisons of the two MfIOLs among non-myopic and myopic eyes in terms of QoV questionnaire scores are shown in Table P < 0.05).In terms of subjective symptoms assessed by the QoV questionnaire, Group A had lower Rasch-adjusted QoV scores for frequency and severity of dysphotopsia symptoms than Group B (Group A vs. Group B: frequency: 4.7 ± 5.1 vs. 7.3 ± 6.9, respectively, Student’s t-test, Though MfIOLs have been widely utilized in order to meet patients’ demands on both near and distance vision , the appThe implantation of MfIOLs in myopic eyes is a relatively controversial issue , 14. RisIn our previous study , we founIn addition to the haptic design, the material and overall diameter of IOLs may also affect the capsular stability of IOLs. David F. Chang found that the C-loop acrylic toric IOL had better rotational stability than the plate-haptic silicone toric IOL, due to the fact that the hydrophobic acrylic material could better adhere to capsules than silicone . HoweverMoreover, the influence of capsulorhexis and capsular fibrosis on IOL decentration cannot be neglected. Eyes with a severe eccentric capsulorhexis are more likely to have a decentered IOL . Small cCapsular stability of IOLs is crucial to their optical performance , 23. IOLIn this study, the following details also need to be clarified. Though postoperative visual acuities did not differ between the two IOLs in myopic eyes, the tendency of IOL decentration had significant influence on visual quality. Closely correlated with patient satisfaction and quality of life, visual quality is a crucial factor to consider in multifocal IOL implantation . BesidesIn conclusion, our study found that the plate-haptic MfIOLs presented less inferior decentration and better visual quality than C-loop haptic MfIOLs in myopic eyes, which may provide a preferred option for myopic cataract patients with a strong desire of spectacle independence.


Fetal superior vena cava (SVC) is essentially the single vessel returning blood from the upper body to the heart. With approximately 80-85% of SVC blood flow representing cerebral venous return, its interrogation may provide clinically relevant information about fetal brain circulation. However, normal reference values for fetal SVC Doppler velocities and pulsatility index are lacking. Our aim was to establish longitudinal reference intervals for blood flow velocities and pulsatility index of the SVC during the second half of pregnancy.This was a prospective study of low-risk singleton pregnancies. Serial Doppler examinations were performed approximately every 4 weeks to obtain fetal SVC blood velocity waveforms during 20–41 weeks. Peak systolic (S) velocity, diastolic (D) velocity, time-averaged maximum velocity (TAMxV), time-averaged intensity-weighted mean velocity (TAMeanV), and end-diastolic velocity during atrial contraction (A-velocity) were measured. Pulsatility index for vein (PIV) was calculated.SVC blood flow velocities were successfully recorded in the 134 fetuses yielding 510 sets of observations. The velocities increased significantly with advancing gestation: mean S-velocity increased from 24.0 to 39.8 cm/s, D-velocity from 13.0 to 19.0 cm/s, and A-velocity from 4.8 to 7.1 cm/s. Mean TAMxV increased from 12.7 to 23.1 cm/s, and TAMeanV from 6.9 to 11.2 cm/s. The PIV remained stable at 1.5 throughout the second half of pregnancy.Longitudinal reference intervals of SVC blood flow velocities and PIV were established for the second half of pregnancy. The SVC velocities increased with advancing gestation, while the PIV remained stable from 20 weeks to term. Assessment of fetal well-being is important in obstetrics for optimizing clinical management, and for that purpose, Doppler ultrasonography is one of the most commonly used methods . It has In 1990, Reed et al. described the blood flow velocity waveform patterns of the SVC and IVC in normal and growth restricted fetuses, and in fetuses with cardiac arrhythmia demonstrating that the time-velocity integral of diastolic waveform was lower in severly growth restricted fetuses, and that the reversed end-diastolic velocity was augmented during atrial contraction in fetal tachyarrythmias . A decadAccordingly, our aim for the present study was to establish longitudinal reference intervals for the fetal SVC blood velocities and PIV for the second half of pregnancy.This was part of a prospective observational study on fetal cardiovacular function conducted at the Department of Obstetrics and Gynecology, University Hospital of North Norway, Tromsø, Norway, during 2009–2012. Pregnant women were informed about the study and recruited at the time of routine ultrasound screening at 18–20 gestational weeks. A total of 142 women with uncomplicated singleton pregnancies were enrolled in the study after obtaining written consent, and examined approximately every 4 weeks until term. Some data on impedance based and volume blood flow based cerebro-placental and umbilico-cerebral ratios from this study population have been reported previously , 17.All participants had their routine second trimester scan when their gestational age was confirmed by the measurement of biparietal diameter or head circumference. Inclusion criteria were: age ≥ 18 years, uncomplicated singleton pregnancy, and gestational age ≥ 18 and < 24 weeks at enrollment. Exclusion criteria were: preeclampsia in previous pregnancy, gestational diabetes or history of preexisting maternal diseases complicating the pregnancy, such as chronic hypertension, diabetes mellitus or autoimmune disease. Fetuses with growth restriction , major congenital malformations or chromosomal abnormality were also excluded.The ultrasound examinations were performed transabdominally with the pregnant woman in a supine semirecumbent position. Three specialist obstetricinas experienced in ultrasonography performed the Doppler ultrasonographic measurements. Ultrasonography was performed using a Vivid 7 Dimension ultrasound system  equipped with a M4S sector transducer with frequencies of 1.5–4.3 MHz. At each study visit, after confirming the presence of fetal heart activity, fetal biometry was perfomed to estimate the fetal weight and umbilical artery and middle cerebral artery (MCA) Doppler pulsatility indices were measured as reported previously , 17.The SVC was assessed in a long axis view where it enters the right atrium. The Doppler sample gate was placed at the point where the vessel enters the right atrium without overlapping the ascending aorta, using the method described previously , 17. VelThe maximum velocity envelope of the SVC waveforms was automatically traced using the software of the ultrasound machine. The trace was visually inspected and sensitivity adjusted if required. Manual tracing was used if automated tracing was not appropriate. The peak systolic (S) velocity, diastolic (D) velocity, time-averaged maximum velocity (TAMxV), time-averaged intensity weighted mean velocity (TAMeanV) and end-diastolic velocity during the atrial contraction (A-velocity) were measured Fig. . An averAll pregnant women participating in the study had regular antenatal follow-up according to clinical routine. Data on the course of pregnancy, birth, and neonatal outcome were collected from the electronic medical records.The sample size was calculated based on the formula proposed by Royston and Altman according to which the sample size of a longitudinal study is equivalent to the number of participants required to construct gestational age-specific reference ranges in a cross-sectional study divided by a design factor D  that equals to 2.3 , 21. Ass10 transformed except the A-velocity that required square (∧2) transformation, and the SVC PIV needed 1/SQRT transformation. Best fitting fractional polynomials were chosen to construct gestational age specific mean curves for each variable from a list of 44 regression models based on R2 value. Multilevel regression modeling was used to calculate the mean and percentiles of SVC velocities and PIV in relation to gestational age taking into account the repeated measures design of the study [P-value < 0.05 was considered as statistically significant.Data analysis was performed using IBM SPSS Statistics for Windows. Version 24.0. (IBM Corp. Armonk. NY) and MATLAB R2019a (Matworks. Inc. Natick. MA). Data distribution for normality was checked by using the Shapiro-Wilk test. Logarithmic or power transformations were used to achieve normal distribution of the data. The best transformation was chosen based on the Box-Cox transformation lambda values (λ) calculated for each dependent variable. All SVC velocities were loghe study . Associa+ 4 weeeks by emergency cesarean section due to fetal distress associated with abruptio placenta, had intraventricular bleeding leading to hydrocephalus requiring shunting. All other babies were healthy on discharge from the hospital.Of the 142 low-risk pregnant woman enrolled, one was excluded from the study due to missing follow-up data, and another seven because SVC Doppler was not recorded. This resulted in a study population of 134 women and a total of 575 obervations with 510 (88.7%) successful recordings of adequate quality SVC blood flow velocity waveforms. The baseline characteristics of the study population and pregnancy outcomes are presented in Table All the SVC velocities were postive (antegrade), but the A-velocity was zero in 5.7% and negative (retrograde) in 8.9% of observations. The mean fetal heart rate was 137–140 beats/min.Gestational age-specific reference values for each SVC velocity variables and PIV with their corresponding 2.5th, 5th, 10th, 50th, 90th, 95th and 97.5th percentiles, are presented in Tables P < 0.0001) and estimated fetal weight . The A-velocity had a weaker but significant correlation with head circumference , but no significant correlation with estimated fetal weight . However, the SVC PIV did not correlate significantly either with fetal head circumference  or with estimated fetal weight . Similary, we found no significant correlation between SVC PIV and MCA PIV . However, we found a significant correlation between SVC TAMxV and MCA TAMxV , and SVC S-velocity with MCA peak systolic velocity .The mean peak S-velocity increased with advancing gestation from 24.0 cm/s at 20 weeks to 39.9 cm/s at 40 weeks, D-velocity from 13.0 at 20 weeks to 19.1 cm/s at 40 weeks, TAMxV from 12.7 cm/s at 20 weeks to 23.2 cm/s at 40 weeks, TAMeanV from 7.0 cm/s at 20 weeks to 11.3 cm/s at 40 weeks, and A-velocity from 4.8 cm/s at 20 weeks to 7.2 cm/s at 40 weeks. The SVC PIV did not change significantly with gestational age and remained stable at 1.50 from 20 to 40 gestational weeks  compared with normal controls (n = 15) [The SVC PIV did not change signicantly as both the systolic and end-diastolic velocities as well as the TAMxV increased proportionately during 20–40 weeks of gestation. In a previous study by Fouron et al., the SVC S-velocity was reported to be slightly higher and A-velocity deflection during arterial contraction insignificantly deeper in fetuses with absent umbilical artery end-diastolic flow (n = 15) . This su(n = 15) . This is(n = 15) . We foun . This sOne of the strengths of our study is its longitudinal design. Longitudinal studies are preferred for studying serial changes in physiological parameters with advancing gestation compared with cross-sectional studies. Besides, they require only about a half to a third of the sample size needed in a cross-sectional study to estimate the gestational age specific percentiles with the same precision . FurrtheThe quality of Doppler blood flow veocimetry is operator dependent, and not reporting intra- and inter-observer variability could be considered as a limitation of our study. However, acceptable reproducibility of SVC velocity measurements has been documented previously . One mayConsidering that the pulsatile precordial veins tend to have a partially blunted rather than a parabolic spatial blood flow velocity profile , our resOur study population was relatively homogenous and consisted mainly of White European women. As fetal growth and size vary significantly in different societies  and blooLongitudinal reference intervals of SVC blood flow velocities and PIV were established for the second half of pregnancy. The SVC velocities increased with advancing gestation, while the PIV remained stable from 20 weeks to term. We believe these measurements have a role in the fetal assessment as they represent the fetal brain circulation.


Background: Objective assessment of shoulder joint active range of motion (AROM) is critical to monitor patient progress after conservative or surgical intervention. Advancements in miniature devices have led researchers to validate inertial sensors to capture human movement. This study investigated the construct validity as well as intra- and inter-rater reliability of active shoulder mobility measurements using a coupled system of inertial sensors and the Microsoft Kinect (HumanTrak). Methods: 50 healthy participants with no history of shoulder pathology were tested bilaterally for fixed and free ROM: (1) shoulder flexion, and (2) abduction using HumanTrak and goniometry. The repeat testing of the standardised protocol was completed after seven days by two physiotherapists. Results: All HumanTrak shoulder movements demonstrated adequate reliability (intra-class correlation (ICC) ≥ 0.70). HumanTrak demonstrated higher intra-rater reliability (ICCs: 0.93 and 0.85) than goniometry (ICCs: 0.75 and 0.53) for measuring free shoulder flexion and abduction AROM, respectively. Similarly, HumanTrak demonstrated higher intra-rater reliability (ICCs: 0.81 and 0.94) than goniometry (ICCs: 0.70 and 0.93) for fixed flexion and abduction AROM, respectively. Construct validity between HumanTrak and goniometry was adequate except for free abduction. The differences between raters were predominately acceptable and below ±10°. Conclusions: These results indicated that the HumanTrak system is an objective, valid and reliable way to assess and track shoulder ROM. Measuring the active range of motion (AROM) of the shoulder joint is a fundamental component in any physical examination to diagnose disease, identify functional limitations and monitor patient progress after conservative therapy or surgical intervention ,2. ThereUniversal goniometry (UG) is the most widely used instrument to measure the range of motion (ROM) of body joints in clinical settings ,4. It coThe recent development of small, portable, and wearable electromechanical sensors can bridge the gap between the practicality of commonly used clinical tools and more accurate, yet expensive, optical systems. Inertial motion units (IMUs) comprise 3-axis accelerometers, 3-axis magnetometers and 3-axis gyroscopes. IMUs, when paired with data fusion software, can define motion, heading and orientation . The accPicerno et al.  reportedHowever, one limitation of IMUs is its potential to underestimate large-angle movements and overestimate small-angle movements .The Microsoft Kinect  is a low-cost, marker-less system with a depth-sensing camera utilising infrared (IR) laser projection and an IR camera for real-time body tracking in three dimensions (3D). Developers have paired the device with software for the purpose of assessing and measuring human movement. The Kinect has been adapted and evaluated to assess 3-D shoulder kinematics during computer use , provider) = 0.993, the limits of agreement (LoA) ±11°, 95% CI: 8.7–12.6°) and abduction  [The Kinect, when compared against the gold-standard Vicon motion capture system for shoulder movements, has demonstrated higher levels of correlation and more acceptable levels of accuracy for forward flexion (Pearson’s correlation (7–12.8°) . Additio7–12.8°) ,34,35,367–12.8°) ,38. The More recently, the integration of IMUs and the Kinect can potentially address the limitations of inertial sensor drift , noncaliThe HumanTrak system  is the coupling of inertial sensors with the updated Microsoft Kinect (v2). Prior studies have validated the Kinect (v2) for the assessment of postural control  and for HumanTrak has been validated against the Vicon motion capture system and AMTI force plate system with linear regression results demonstrating excellent correlation for shoulder flexion/extension (average ± standard deviation (SD): 0.907 ± 0.023; minimum: 0.862; maximum: 0.9444) .To our knowledge, no studies have investigated the coupling of inertial sensors with the Kinect to measure shoulder AROM or assess the accuracy of fixed target reaching. Furthermore, previous reliability and validity studies of either of the devices were statistically underpowered due to small sample sizes ,48,49,50r: >0.75) and reliability (ICC: ≥0.70) for measuring free and fixed shoulder AROMs in healthy individuals.Prior to coupled devices being used in clinical practice, they must be assessed for their measurement properties . Therefore, this study was designed to determine the intra- and inter-reliability of HumanTrak to measure active free and fixed shoulder motions. To establish construct validity, obtained results were compared with UG, which was chosen for its wide acceptance and applicability in clinical settings ,55,56,57The aims of the study were threefold: (1) to establish HumanTrak intra- and inter-rater reliability, (2) to determine HumanTrak’s validity with goniometry, and (3) to compare HumanTrak results under two test conditions—free AROM and fixed AROM using consistent targets to reach.Fifty asymptomatic adults ) with no history of shoulder pathology were recruited from a public hospital in Sydney, Australia . Participants from the hospital database were invited to participate via email. All participants gave their written informed consent before participation. The study was approved by the local hospital Ethics Committee (ref no. 17/005) and preregistered in the Australian New Zealand Clinical Registry (ACTRN12617000266369).The sample size calculation was based on the data from a previous pilot study . The calRater A and Rater B were registered Physiotherapists with 10 years and 2 years of clinical experience, respectively. Both raters were responsible for body sensor placement, initiating the protocol instructions and executing the inertial motion system with the Kinect. Rater A was blinded to goniometric measurements between assessments with independent rater-recording results. HumanTrak data were automatically uploaded to a secure cloud-based server, and all ROM values were not visible to raters during or between assessments. The third independent unblinded rater collated and analysed all measurements.The HumanTrak system integrated four wireless inertial sensors A worn onThe miniature inertial sensor (dimension: 23 mm × 32.5 mm × 7.6 mm) consisted of a 3-axis gyroscope and a 3-axis accelerometer. Each sensor was applied bilaterally on the dorsal side of the wrist on the ulnar styloid and around the lower leg above the lateral malleolus. The sensor was securely attached to a plastic-covered slap bracelet with an interior metal coil spring.A high-speed radio frequency (RF) protocol transferred the inertial data to a receiving computer. The automatic calibration of the system was established, and the Kinect was positioned at a height of 1.4 m with a tilt of −2.0°. Feet markers were placed 1.5 m away from the Kinect to ensure consistency with the participant placement.®, Boise, ID, USA), was used by Rater A for all shoulder measurements.A single 12 inch plastic goniometer, model 12-1000  Model 2, k was used for the inter-rater analysis to determine if HumanTrak can be used with confidence and reliability among equally trained clinicians ,61.ICCs for absolute agreement were reported, and the mean value from each testing session was used for the analysis.Given that ICCs may be affected by a limited range of data or intersubject variability, absolute measures of reliability were determined . The stant error . Inter-rnt error  with 95%nt error ,64.Reliability and construct validity were assessed using the criteria by Terwee et al. . ReliabiThe results of intra-rater reliability with ICC, SEM and MDC values are presented in For free AROM, the highest intra-rater reliability was observed for forward flexion. For fixed AROM in the cage, the highest intra-rater reliability was observed for abduction. All SEM and MDC values were relatively low, indicating good absolute reliability.r ≥ 0.75) between week 1 and week 2 for HumanTrak were demonstrated for free forward flexion and fixed abduction  and abduction (ICC = 0.85 and 0.94) than UG. HumanTrak demonstrated lower SEM and MDC values than goniometry with small measurement errors (2–4°), indicating good absolute reliability. Similar to our results, an initial study utilising the BioCap system (the beta version of HumanTrak) reported adequate intra-rater reliability for free shoulder forward flexion (ICC = 0.93 and 0.83) and abduction (ICC = 0.82) . PreviouThis study examined the inter-rater reliability of inertial sensors coupled with the Kinect. All free and fixed AROM movements demonstrated adequate inter-rater reliability (ICCs = 0.88–0.98) with the exception of fixed forward flexion (ICC = 0.65). The Bland–Altman plots demonstrated acceptable levels of agreement between raters, and the 95% LoA were below ±10° for all movements, except for fixed forward flexion. By constructing a freestanding cage with suspended ropes, we used a novel approach to assess HumanTrak’s accuracy in detecting the same fixed point. Despite the best efforts to ensure the same targets were reached within and between trial sessions, human error from the participant cannot be discounted. Therefore, the higher SEM and MDC values for fixed forward flexion were likely due to experimental errors from participants not reaching the same target between raters.r = 0.77). Similarly, Lee et al. [This study established adequate reliability and construct validity between HumanTrak and goniometry only for free forward flexion (ICC = 0.84, e et al.  reportedThe Bland–Altman plots demonstrated low mean differences and wide 95% LoA above the clinically significant threshold of ±5°. For agreement, clinicians should recognise differences between HumanTrak and goniometry can be expected to vary by up to ±15°. Compared to our results, Huber et al.  reportedExperimental limitations may explain why the highest intra- and inter-rater reliability values were achieved for free forward flexion and fixed abduction. Participants performing free forward flexion without a target were more likely to consistently move their arm in the single sagittal plane. In contrast, it was observed that reaching forward for a target in the cage elicited substitute movements involving excessive shoulder abduction. It was observed that fixed abduction demonstrated higher reliability than free abduction. An explanation for this result is that unrestricted abduction can be performed in the scapular or coronal plane, resulting in a less consistent measurement. These findings highlight that regardless of HumanTrak’s accuracy and reliability, careful positioning and rater observation are essential to avoid compensatory or unwanted movements.We acknowledge this study had several limitations. First, we used goniometry as a “gold standard” rather than a motion capture system. Authors chose to not replicate findings that have previously validated HumanTrak against the Vicon system . While tParticipants in this study were healthy, predominately young, and not stratified for age. However, this population was appropriate, since we eliminated functional deficits and pain as potential confounding variables to establishing reliability. For practical reasons, no attempts were made to measure participants at the same time of day on each occasion; hence, we did not control for any potential variations in AROM at different times of the day. Finally, caution should be used in interpreting the HumanTrak inter-rater reliability results due to the smaller sample size of 15 participants.Current system limitations reside in the HumanTrak system calibration process where each subject is required to stand straight to allow adjustments for any tilt in camera orientation. Slight errors in tilt calibration can occur in cases where the participant is unable to completely stand straight. However, this can be avoided if a healthy participant is initially used for calibration prior to tracking the rest of the participants, since the tilt calibration is only camera-dependent and will not change between participants.Shoulder AROM was calculated into specific planes: sagittal plane for flexion/extension and coronal plane for adduction/abduction. These planes are defined from the camera sensor’s reference. As such, a current limitation is that the subject is required to perfectly face the camera to maximise the accuracy of the ROM measurements. Errors in measurement might occur, if the subject is facing the camera at an angle, with the amount of errors increasing as the angle increases.HumanTrak is a reliable and valid tool for surgeons and physiotherapists to deliver a quick and functional evaluation of the shoulder. As a marker-less, nonintrusive system for ROM evaluation, HumanTrak demonstrates potential as feasible adjunct to clinical observation in rehabilitative and outpatient clinical settings. Inertial sensor technology coupled with optical-based systems can provide accurate and objective data to support clinical decision-making and facilitate better clinical outcomes.


BRCA1 and BRCA2 genes, the genetic testing result has become critical in their care. With the recent FDA approval of alpelisib for the treatment of PIK3CA‐mutated hormone‐receptor positive metastatic breast cancer, tumor molecular profiling to identify somatic mutations and potential molecularly targeted agents is increasingly utilized in the treatment of advanced breast cancer.Genetic testing for cancer predisposition is recommended to women with breast cancer who meet the criteria for such testing. After the FDA approvals of the poly ADP ribose polymerase (PARP) inhibitors, olaparib and talazoparib, for treatment of metastatic breast cancer, carrying germline mutations in Combining germline and somatic sequencing (paired testing) offers an advantage over a single technique approach. Our study evaluates the role of paired testing on the management of breast cancer patients.CHEK2 (2) and ATM (1) genes. 95% of tumors had somatic mutations. Seventy‐seven percent of tumors had genomic alterations targetable with agents approved for breast cancer and 88% had molecular targets for agents approved for other cancers. Clinical examples of such use are described and potential future directions of tumor and paired testing are discussed.Forty‐three breast cancer patients treated at Rush University Medical Center underwent paired germline and somatic variant testing in 2015 to 2017. A retrospective chart review was conducted with the analysis of demographic, clinical, and genomic data. Three actionable germline variants were found in the Germline variants were present in a relatively small patient group not routinely tested for inherited alterations. Potentially targetable somatic alterations were identified in the majority of breast cancers. Paired testing is a feasible and efficient approach that delivers valuable information for the care of breast cancer patients and eliminates serial testing. Tumor molecular profiling (TMP) is usually employed in advanced BC when all standard treatments have been exhausted.BRCA1/2 mutation in a patient with metastatic BC who may benefit from treatment with poly ADP ribose polymerase (PARP) inhibitors. Combining germline testing with TMP (paired testing or PT) may provide a time and resource efficient option to reveal molecular targets for therapy and genetic predisposition to cancer.Recent advances in precision oncology allow for the administration of more effective and less toxic treatments, leading to improved patient outcomes. Molecularly targeted agents (MTAs), such as imatinib in chronic myelogenous leukemia or trastuzumab in Her‐2/neu‐positive breast cancer, have shown significant prolongation of overall survival in the corresponding malignancies and sometimes, cures.We performed a pilot study of PT in a series of 43 BC patients to assess the feasibility and clinical impact of this testing approach at a single academic medical center. We discuss the implications of PT results for treatment selection, subsequent cancer surveillance, and screening of family members. We also discuss the efficiency of the PT approach.2We performed a retrospective chart review of women with the diagnosis of BC who underwent PT when deemed appropriate by their oncologists in the context of routine clinical care at Rush University Medical Center from November 2015 to February 2017. The following data were collected: patient age, ethnic background, clinical stage at diagnosis, histology, ER/PR/HER2 receptor status, BC treatment history, and PT results. All patients received pre‐test risk assessment and genetic counseling to inform them about PT. Post‐test genetic counseling was provided for patients and/or their families who tested positive for a pathogenic or likely pathogenic germline variant. The study was approved by the Rush University Institutional Review Board (IRB# 17041703‐IRB01). Participants were de‐identified before group statistical analyses.2.1DNA was extracted from a primary tumor for the majority of subjects while a metastatic lesion was analyzed when the primary tumor was not available. Blood or saliva was also submitted for each patient for germline DNA extraction. Samples were processed as previously described.APC, BRCA1, BRAC2, MLH1, MSH2, MSH6, MUTYH, PMS2, PTEN, RB1, RET, SDHAF2, SDHB, SDHC, SDHD, STK11, TP53, TSC1, TSC2, and VHL. In some cases, germline testing was performed on additional cancer susceptibility genes as clinically indicated based on genetic risk assessment. Actionable germline alterations were defined as those alterations associated with a currently available option for cancer surveillance, prevention, or treatment in either the patient or their close family members.Alterations identified in TMP and associations with MTAs were reported based on peer‐reviewed studies and other publicly available resources. Germline variants when identified in blood or tumor were reported for the following genes: 3Age at diagnosis, ethnicity, stage at the time of testing, histologic type, and tumor markers of breast cancer for 43 patients are reported in Table Somatic genomic alterations in the tumors tested were highly prevalent with 95% 42/43) of tumors having at least one reported mutation with an average of 6.5 (range 0‐17) mutations per tumor. Overall, 77% of tumors contained at least one target for BC therapy, and 88% contained an FDA‐approved targeted therapy for another type of cancer, potentially available off‐label for BC.  accounted for 40% of alterations. Twenty‐two genes, altered in this cohort of patients, had an associated MTA for breast cancer while alterations in additional genes added to the number of patients who were potentially eligible for an MTA , a likely pathogenic variant in the CHEK2 gene (c.1427C > T), and a likely pathogenic splice site variant in the ATM gene (2466 + 1G > C). None of these three patients met the then current National Comprehensive Cancer Network (NCCN) germline genetic testing criteria.Three patients were found to have actionable germline variants that were not previously suspected including one pathogenic mutation in 3.1Because 47% of our sample (20 patients) consisted of patients with early stage breast cancer, real‐time clinical care was usually not impacted by PT but rather, adjuvant or neoadjuvant treatment was based on stage, pathologic characteristics of the tumor including tumor markers, and, in some cases, on the results of molecular prognostic assays such as Oncotype DX and Mammaprint. In one patient (Patient C), an MTA was considered in adjuvant setting and in two women (Patient A and Patient B) MTAs were administered when BC recurred.KRAS amplification, BARD1 p.A724V, TP53 p. K139_P142del, and TP53 copy neutral LOH. Based on these findings, she was prescribed a suggested MTA off‐label, Trametinib, a mitogen‐activated protein kinase (MEK) inhibitor, which targets KRAS amplification. Although it does not directly inhibit MEK, trametinib has been shown to limit tumor progression via CD8 T‐cell mediated factors by altering signaling along the RAS‐ERK pathway in cancers with KRAS amplification.Patient 404 122  was diagPIK3R1 p.R461_E462delinsQ, FGF3 amplification, FGF4 amplification, and HER2‐Neu (ERBB2) mutation, p. S310F. Given the finding of HER2‐Neu mutation, she was offered participation in phase II study with neratinib.HER2‐Neu mutations activate the HER2‐Neu oncogene without its amplification, Trastuzumab and other commonly used HER2‐Neu directed agents are usually ineffective in HER2‐Neu‐mutated BC. Neratinib is a small molecule, which irreversibly inhibits both HER2‐Neu amplifications and mutations.Patient 435 327  was diagTP53 and a hemizygous loss of NF2, yielding four possible therapies, including an mTOR inhibitor, everolimus, approved in combination with anti‐hormonal therapy for metastatic BC. Given the significant amount of residual disease and this molecular alteration, she was evaluated for the adjuvant trial with everolimus.Patient 487 820  was diag4TP53 gene, followed by CDH1 and PIK3CA. This correlates with other studies, which have described TP53 as the most commonly mutated gene in human cancers.TP53 was less than 12% and that in PIK3CA was less than 6%, which are lower than reported results for breast cancer. Those studies, however, include non‐coding regions with high mutation frequencies, not all of which can be classified as driver mutations. Mutational signatures extended to genome rearrangements, characterized by tandem duplications or deletions, appear to be associated with defective homologous recombination‐based DNA repair.TP53 are more difficult to target than mutations in oncogenes (gain of function mutations).BRCA1 and BRCA2). Preliminary data suggest that these agents can also elicit synthetic lethality in the presence of mutations in other tumor suppressor, such as TP53 and PTEN, which are mutated in 37% and 3% of all BCs, respectively, and represented 11.8% and 5.4% of mutations in our sample.In our study, 95% of all tumors analyzed demonstrated at least one somatic mutation presumed to be involved in tumorigenesis. The most commonly detected mutation was in PIK3CA, which allow unregulated cell proliferation, had associated MTAs  while others, such as a loss‐of‐function mutations in the tumor suppressor gene, CDH1, were not targetable in our patients (ESR1 (estrogen receptor 1). This may be explained by the number of primary, non‐metastatic breast tumor tissue in our sample, as the incidence of ESR1 mutations rises to 15% to 20% in metastatic ER‐positive tumors after prior endocrine treatments, suggesting the development of resistance.Gain‐of‐function mutations in genes such as patients . NotablyA molecular target for breast cancer treatment was available for a majority (33/43) of patients. However, we did not observe responses in two patients who received MTAs. This is consistent with the initial results of clinical trials looking at the use of MTAs in prospective fashion.When analyzing the results of MTA clinical trials, it is important to remember that they depend on target identification in tumor tissue and availability of matching MTAs. In the initial phase of the NCI sponsored MATCH  trial, only 9% of patients had a matched targeted therapy, when there were only 10 MTAs and 10 corresponding arms in the study. However, the percentage of patients increased to 23% when the number of MTAs and the respective arms in the study went up to 24%.BRCA1/2 somatic mutations found 55.7% were positive for pathogenic BRCA1/2 germline mutations, confirming “a second hit” hypothesis.CHEK2 and ATM genes, which are consistent with these data.Previous studies have revealed the prevalence of germline variants discovered in paired testing. A retrospective study of Stanford patients with CHEK2 and ATM can impact cancer surveillance and cancer screening, as well as prevention for blood relatives.ATM and CHEK2 mutation carriers) and more frequent colonoscopies for colon cancer surveillance (for CHEK2 mutation carriers only).BRCA1/2 mutations based on age at breast cancer diagnosis and burden of breast, ovarian, and other related cancers in the family. While these guidelines are useful to identify individuals who should be considered for genetic testing, germline mutations in the BRCA1/2 and other BC susceptibility genes may be missed in individuals who do not meet these criteria as their sensitivity is limited.BRCA 1/2 mutations in the tumors of BRCA 1/2 mutation carriers can cause functional reversal of the germline mutation and restoration of the wild‐type BRCA, which results in resistance to carboplatin and PARP‐inhibitors in these patients. As opposed to “a second‐hit” mutation, which affects a normal allele, a “reversing” mutation usually affects the allele with a germline mutation.Variants in BRCA1/2 mutations as well as germline and somatic mutations in other genes in homologous recombination (HR) pathway, including ATM and CHEK2. In a Phase 2 study of olaparib monotherapy in metastatic breast cancer patients with germline or somatic mutations in DNA repair genes (Olaparib Expanded), the response to olaparib is studied in patients with metastatic BC and germline or somatic mutations in the following genes: ATM, ATR, BARD1, BRIP1 (FANCJ), CHEK2, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCM, MRE11A, NBN, PALB2, RAD50, RAD51C, and RAD51D.Ongoing studies evaluate a potential benefit of PARP‐inhibitors in the presence of somatic BRCA1/2 mutations.The same genomic alterations can be a target for a chemotherapy agent carboplatin, which is known to double the objective tumor response rate in women with metastatic BC and germline CHEK2 and ATM genes). In addition, the somatic alterations in two of these three tumors also included many genes in HR pathway   Table . It is lBRCA1/2 mutation carriers. Moreover, clinical trials are looking at a potential benefit of PARP‐inhibitors in adjuvant and neoadjuvant treatment of high‐risk BC patients with these mutations.PIK3CA mutations derived significantly greater benefit from letrozole over tamoxifen in BIG 1‐98 adjuvant trial.As discussed earlier, TMP and PT data are usually not incorporated in treatment decisions for early BC. However, this may change with the increased use of platinum agents for adjuvant and neoadjuvant treatment of BC in Our study showed that the use of PT is feasible in an actual clinical setting and a single academic institution. Even though TMP can potentially identify germline mutations, their ascertainment as such is difficult without a paired germline testing.5With the rising demand for targeted therapies and the rapidly changing landscape of MTAs, it is obvious that tumor sequencing holds promise for BC. Outcomes of the three patients in this study, treated with MTA, based on somatic alterations, demonstrate the limitations that remain for their current use. In the years to come, cancer treatment may be dictated more by tumor mutations than tumor type. Yet, before BC tumor sequencing and paired testing become routine, active targets must be validated and more MTAs developed, including those that aim at targets currently deemed “not‐druggable.” Achieving these goals is likely to improve insurance reimbursement for BC sequencing and PT, which, at present, is often lacking.Clinical trials are desperately needed to evaluate the efficacy and safety of MTAs. However, enrollment into these trials can be challenging as they are increasingly conducted in smaller biomarker‐enriched patient populations selected by the presence of the target. In addition, since <1% to 20% of tumors harbor individually rare somatic mutations, collecting and reporting individual responses to MTAs as described in this paper (“N‐of‐one experiences”) are crucial for future success of MTAs.Analysis of cancer‐related genes in paired germline and tumor DNA samples can lead to increased detection of clinically significant heritable mutations compared to the predicted yield of targeted germline testing based on current clinical guidelines. Identification of germline variants can help guide therapeutic and preventive interventions. Drawing from our experience in this study, we predict expanding indications for paired somatic and germline testing in the future for BC, as well as other types of cancer.At the time research was conducted, Virginia Speare, Holly LaDuca, Carin Espenschied, Amal Yussuf, and Philip Gray were full time employees of Ambry Genetics. Carin Espenschied is a current employee and stock holder at Guardant Health. Otherwise we have no other conflicts of interest to report.Conceptualization, L.U., C.E., PG; Methodology, E.E., V.S, K.B., P.G., C.E., L.U.; Investigation, E.E., M.C., R.R., K.B., H.L., A.Y., L.U.; Formal Analysis, P.G., A.Y., V.S.; Resources, M.C., R.R., P.G., L.U; Writing ‐ Original Draft, E.E., J.C., J.P., K.B., and L.U.; Writing ‐ Review & Editing, E.E., J.C., J.P., K.B., V.S., C.E., H.L., A.Y., P.G., T.K., L.B., H.L., M.C., R.R, and L.U.; Visualization, C.E., A.Y., V.S, J.C.; Supervision, C.E., L.U., V.S.; Funding Acquisition, L.U., H.L., P.G., V.S.All authors had full access to the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. The study was approved by the Rush University Institutional Review Board (IRB# 17041703‐IRB01).Data S1. Supporting information.Click here for additional data file.


Plasmodium and Toxoplasma. It is believed that agamococcidians are not capable of sexual reproduction, which is essential for life cycle completion in all well studied parasitic apicomplexans. Here, we describe three new species of agamococcidians belonging to the genus Rhytidocystis. We examined their cell morphology and ultrastructure, resolved their phylogenetic position by using near-complete rRNA operon sequences, and searched for genes associated with meiosis and oocyst wall formation in two rhytidocystid transcriptomes. Phylogenetic analyses consistently recovered rhytidocystids as basal coccidiomorphs and away from the corallicolids, demonstrating that the order Agamococcidiorida Levine, 1979 is polyphyletic. Light and transmission electron microscopy revealed that the development of rhytidocystids begins inside the gut epithelial cells, a characteristic which links them specifically with other coccidiomorphs to the exclusion of gregarines and suggests that intracellular invasion evolved early in the coccidiomorphs. We propose a new superorder Eococcidia for early coccidiomorphs. Transcriptomic analysis demonstrated that both the meiotic machinery and oocyst wall proteins are preserved in rhytidocystids. The conservation of meiotic genes and ultrastructural similarity of rhytidocystid trophozoites to macrogamonts of true coccidians point to an undescribed, cryptic sexual process in the group.Agamococcidians are enigmatic and poorly studied parasites of marine invertebrates with unexplored diversity and unclear relationships to other sporozoans such as the human pathogens  Their life cycles are enigmatic. The Leuckart's triad  typical of the sporozoan life cycle2 seems to be broken in rhytidocystids: no gametogony  and merogony  have been documented to date. Only sporozoites, large trophozoites embedded in the host intestinal epithelium, and coccidian-like oocysts with numerous sporocysts have been observed. Originally identified as gregarines from the family Monocystidae, rhytidocystids were later classified as coccidians4. Currently, the Agamococcidiorida comprises two families: Rhytidocystidae with five named species and Gemmocystidae with a single species Gemmocystis cylindrus originally described from eight species of Caribbean scleractinian corals. This parasite has been preliminarily assigned to agamococcidians because gamonts and other developmental stages beside sporozoites and oocysts  are not known5.The order Agamococcidiorida Levine, 1979 is a small group of Apicomplexa initially established for rhytidocystids—insufficiently investigated coccidian-like parasites of polychaetes7. No molecular data on Gemmocystis are available but, based on similarities in cell size, coccidian-like morphology, and localization in their hosts , it was suggested that G. cylindrus belongs to corallicolids8. The corallicolids, previously called “genotype N” or “apicomplexans Type-N” in nuclear ribosomal DNA (rDNA) phylogenies10 and ARL-V in plastid rDNA phylogenies12, are related to eucoccidians10. In the most recent higher rank classification of protists13, the agamococcidians are assigned to the Apicomplexa incertae sedis. Lately, transcriptomes of two unclassified rhytidocystids were sequenced14, one of which corresponds to Rhytidocystis pertsovi sp. n. described in the present paper. The multiprotein phylogeny showed a basal position of rhytidocystids in the coccidiomorph clade14 but included only sparse species sampling without coralicollids and closest known relatives of Rhytidocystis such as Margolisiella islandica15. Clarifying the position of rhytidocystids in densely sampled phylogenies is therefore essential for the further development of apicomplexan systematics. In this study, we sequenced three new species of rhytidocystids from the White Sea and created phylogenies of Apicomplexa based on SSU rDNA alone and concatenated SSU, 5.8S, and LSU rDNAs. We here describe these new species as Rh. nekhoroshkovae sp. n. and Rh. dobrovolskiji sp. n. with light and scanning electron microscopy and Rh. pertsovi sp. n. with light, scanning and transmission electron microscopy. To assess whether the “asexual” rhytidocystids potentially retain the capacity for meiotic recombination, we surveyed the available transcriptomic data of Rh. pertsovi and yet undescribed Rhytidocystis sp. from Travisia forbesii for meiosis-specific genes. We also searched for the homologs of the coccidian oocyst wall proteins (OWPs) in rhytidocystids.Until recently, only a couple of studies on molecular phylogeny of rhytidocystids have been published. Those phylogenies were based on 18S ribosomal RNA gene (rDNA) and did not conclusively resolve the rhytidocystid positionPectinaria (Cistenides) hyperborea collected in the vicinity of Educational and Research Station “Belomorskaya” of Saint Petersburg State University . Infected midguts showed plenty of white dots on the outside surface, which corresponded to rhytidocystids, generally located at the basal part of the midgut epithelium. The polychaetes were usually heavily infected (hundreds of parasites per host).Parasites were found in all 30 examined polychaetes Spindle-shaped zoites were observed inside the host enterocytes Fig. A. As zoiOphelia limacina collected in the vicinity of ERS SPbU (see Methods for details). Parasites were embedded in the host midgut epithelium and visible as white dots from the inside and outside the gut. The hosts contained between several and several dozens of parasites.Parasites were found in 34 out of 70 (48.6%) examined polychaetes The early trophozoite stages were crescent-shaped and measured 21.6–41.0 µm long  . Similar to Rh. dobrovolskiji, parasites were located in the host midgut epithelium. The infected polychaetes contained from a few up to hundreds of parasites.Parasites were found in 73 out of 106 (68.9%) examined polychaetes Intracellular crescent-shaped trophozoites measured 14.3–25.3 µm long  were found inside the host enterocytes , Rh. dobrovolskiji , and Rh. pertsovi . Despite parasitizing the same hosts and being very similar in appearance, Rh. dobrovolskiji and Rh. pertsovi were different genetically.The near-complete sequences of rRNA operon  were obtained for Both Bayesian (BI) and Maximum Likelihood (ML) analyses produced almost identical tree topologies except for the position of blastogregarines, which were either the sister lineage to coccidiomorphs  or placed among gregarines BI; Fig. . The BayRhytidocystis species had relatively short branches and grouped into a common rhytidocystid clade with the other representatives of the genus (Rh. cyamus and Rh. polygordiae), an undescribed parasite of the European oyster Ostrea edulis, and several environmental sequences from oceanic sediments with PP = 1 and BP = 88%. The rhytidocystids then formed a robust higher-order clade (PP = 1.0 and BP = 100%) with an unidentified parasite of Tridacna croecia, the coccidians Margolisiella islandica and Pseudoklossia pectinis, and an environmental sequence from a sulfidic karst spring in Slovenia (KT072247). The robust Rhytidocystis-Pseudoklossia-Margolisiella clade was the most early-branching lineage of coccidiomorphs, including coccidians and haematozoans . We suggested the name “Eococcidia” for this clade  is homologous to the more recently described TgOWP8-1218. Similarly to the TgOWP1-7 family, the proteins contain periodical cysteine residues and a signal peptide. The TgOWP8-12 family was significantly expanded in rhytidocystids compared to the coccidians , which were located within projected apical ends of host enterocytes, similar to Rh. pertsovi. Apparently, most of the real biodiversity of rhytidocystids remains undiscovered.Previously described ade Fig.  were der7, making it unclear whether they could be related to Gemmocystis cylindrus, the other agammococcidian now represented by the “genotype-N” and coralicollid sequences. In some phylogenies, Rh. cyamus and Rh. polygordiae were the sister group of the coccidians Margolisiella islandica and Pseudoklossia pectinis23, but their common clade was never strongly affiliated with gregarines, cryptosporidians, or coccidiomorphs. Reasons for the lack of resolution might be two-fold: Rh. cyamus and Rh. polygordiae form long tree branches which may cause the long branch attraction artifact, and the earlier studies used SSU rDNA phylogenies only, which have inferior resolution to the whole rRNA operon27. In the current study, we enlarged a broadly and evenly sampled alveolate dataset with environmental sequences and short-branching sequences of our new species. We also sequenced the first rhytidocystid LSU rDNA and analyzed their concatenated rDNA phylogeny in both Maximum likelihood and Bayesian frameworks. The resulting phylogenies have a resolution superior to earlier studies and resolve rhytidocystid and sporozoan relationships in two important ways. Firstly, rhytidocystids are unrelated to corallicolids which include Gemmocystis cylindrus , and the whole clade lacks obvious shared morphological characteristics (synapomorphies). Nevertheless, since this robust clade has been recovered in several analyses and discovered as basal group of all coccidiomorphs, including coccidians and haematozoans, we suggest establishing the new taxon Eococcidia for basal coccidiomorphs mainly parasitizing marine invertebrates. The term “Eococcidia” refers to Eos—the goddess of the dawn in Greek mythology, who rose in the morning from the Oceanus; the prefix “eo-” is used in geology and biology for the designation of something to be early (Eococcidia—early coccidians). Coccidian genera Merocystis and Aggregata are also candidate members to Eococcidia since they were recovered as close relatives of Rhytidocystis, Pseudoklossia, and Margolisiella29.Previous phylogenetic studies of SSU rDNA sequences did not resolve the position of rhytidocystidsrus Fig. , demonstcle Fig. 15,22,23.31. Four out of five previously described rhytidocystids have trophozoites in the host intestinal epithelium7, but their intracellular stages were never detected. Here we provide evidence of intracellular localization of three new rhytidocystid species. The findings of putative sporozoites inside host cells and young trophozoites within parasitophorous vacuoles in Rh. nekhoroshkovae, and trophozoites tightly packed inside host cells in Rh. dobrovolskiji suggest that the development of both species begins intracellularly. The trophozoites of Rh. pertsovi on squash preparations of the host enterocytes and in TEM sections were undoubtedly located under the plasma membrane of the host cell. Perhaps, the development of other rhytidocystid may also start with intracellular forms. Rh. polygordiae was the only species previously described with TEM, and its trophozoites were observed in the interstitial space between adjacent host enterocytes6. However, the structure purported to be “the plasma membrane of the adjacent epithelial cell”  has the thickness (a little more than 20 nm) and appearance of two membranes with the intercellular space between them, and most likely represents two plasma membranes of two adjoining enterocytes. Furthermore, “the interstitial space”  is filled with structures resembling endoplasmic reticulum and apparently is the host cell cytoplasm. We therefore suppose that at least some trophozoites of Rh. polygordiae were located intracellularly. Overall, evidence for intracellular development strongly links rhytidocystids with other coccidiomorphs such as coccidians and haematozoans to the exclusion of gregarines and cryptosporidians, which are chiefly extracellular or epicellular. Congruent with their phylogeny  made of components of host origin or host and parasite origin, or be in direct contact with the host cell cytoplasmRhytidocystis species varies from oblong Rh. polygordiae and bean-shaped Rh. cyamus to flat oval cells of Rh. opheliae and Rh. henneguyi7. In the case of Rh. pertsovi and Rh. dobrovolskiji, it seems to be that crescent-shaped, bean-shaped, irregular, roundish and spherical forms represent successive stages of development. A zoite invades the host cell and transforms into a trophozoite. Presumably, during the growth inside the limited space of the host cell, the young trophozoite bends and becomes crescent-shaped, then it loses peaked cell poles and becomes bean-shaped; over time, it undergoes marked growth and becomes tightly packed under the host cell membrane. Released from the host cell to the interstitial space of the tissue, a trophozoite unbends, becomes irregular, then roundish and spherical eventually. Spherical trophozoites, apparently, represent the transitional form to the oocyst stage. Young oocysts Rh. dobrovolskiji look like spherical trophozoites covered by a thick transparent envelope and resemble young oocysts of Rh. sthenelais37.The trophozoite cell shape of earlier described Rh. pertsovi looks typical of sporozoans and possesses all general structures. Rh. pertsovi retains an active apicoplast14 but none was observed in this TEM study. Instead, we found numerous dense bodies of oval or round shape, which could be homologous to wall-forming bodies (WFBs) in coccidian and cryptosporidian macrogamonts41. The WFBs mediate the formation of the oocyst wall, which more than 90% is made up of proteins42. The presence of oocysts and putative WFBs has prompted us to search in rhytidocystid transcriptomes for homologs of oocyst wall proteins (OWPs), some of which are proven to be located in WFBs43. The analysis revealed an astounding diversity of transcripts related to COWP, TgOWP1-7 and TgOWP8-12 family proteins, supporting the presence of WFBs in rhytidocystids and a common mechanism for their oocyst wall formation with coccidia and cryptosporidia.The cytoplasm of intracellular trophozoites M. islandica, which is an eucoccidian-like protist, whose life cycle includes all three types of sporozoan reproduction: gametogony, merogony, and sporogony (Leuckart's triad), and which, hence, produces sexual gamonts15, the supposed absence of sexual life stages in rhytidocystids raises the possibility that they recently lost sexual reproduction. Generally conserved across the eukaryotes, the core meiosis machinery includes nine proteins , with functions spanning sister chromatid cohesion, induction of double-strand breaks, heteroduplex DNA and synaptonemal complex formation, and Holliday junction resolution45. In rhytidocystids, the core meiotic gene set is short of two genes for Msh4 and Msh5, unlike the chrompodellid Vitrella brassicaformis, which retains a full set of core meiosis-specific genes and where sexual process has been proposed46. The absence of the heterodimer-forming Msh4 and Msh5 in rhytidocystids is consistent with their absence in other coccidiomorphs’ genomes  in our phylogeny. However, we classify Eococcidia into Conoidasida and Coccidia because of the findings of the apical complex in their zoites51. We suggest the order Pseudoklossiida for Pseudoklossia and Margolisiella (former Eimeriorina) as P. pectinis and M. islandica were recovered strongly within the eococcidians, but not within the eimeriids ; life cycle—complete Leuckart’s triad; development and merogony intracellular, sexuality intracellular or extracellular, fecundation intracellular or extracellular. Agamococcidiida  emend.Order Rhytidocystis.Eococcidia. Merogony, microgamonts and microgametes not reported. Rhytidocystidae Levine, 1979.Family Agamococcidiida. Early development intracellular; adult trophozoites extracellular in the host intestinal epithelium or coelom; large oocysts with many tens of sporocysts in the host intestinal epithelium or coelom; in annelids.Rhytidocystis Henneguy, 1907.Genus Rhytidocystis nekhoroshkovaesp. n. Miroliubova, Simdyanov, Janouškovec, Paskerova, 2020.Description. Trophozoites 13.0–68.0 µm, flattened, almost round or irregular; immotile; young stages intracellular, adults extracellular. Spherical centric nucleus 6.6–19.2 µm with a spherical centric nucleolus. Cell surface of adult trophozoites rugose with longitudinal and transverse grooves, little creases and small depressions and numerous micropores in the curved rows, which merged with each other.Molecular data. Partial rDNA , GenBank accession number MT231950.Type locality. White Sea, Kandalaksha Gulf, Chupa Bay, Podpakhta Strait , a depth of ~ 20 m.Type habitat. Marine.Type host. Pectinaria (Cistenides) hyperborea Malmgren, 1866 (Polychaeta: Pectinariidae).Location in host. Midgut epithelium.Type (syntype) material. A platinum sputter-coated SEM stub with several protists, slides with histological sections, specimen of parasite cells and host material fixed in ethanol have been deposited in the collection of Department of Invertebrate Zoology, Saint Petersburg State University; extracted DNA used for obtaining of rDNA sequences deposited in the collection of Department of evolutionary biochemistry, Belozersky Institute for Physico-Chemical Biology, Lomonosov Moscow State University; Fig. Etymology. This species was named in honor of Svetlana Nekhoroschkova, PhD, an inspiring ecology lecturer in the Ecological and Biological Lyceum , dear teacher of the author Tatiana Miroliubova.Rhytidocystis dobrovolskijisp. n. Miroliubova, Simdyanov, Janouškovec, Paskerova, 2020.Description. Young intracellular trophozoites 21.6–41.0 µm, crescent-shaped. Larger trophozoites 36.3–67.0 µm; irregular, roundish then spherical and smooth; extracellular. Spherical centric nucleus with relatively big eccentric or centric nucleolus 3.6–10.0 µm in young and 12.1–25.0 µm in larger forms. Young oocyst within thick transparent envelope.Molecular data. Partial rDNA , GenBank accession number MT231947.Type locality. White Sea, Kandalaksha Gulf, Chupa Bay, Yakovleva Inlet  at a depth of ~ 1–15 m.Type habitat. Marine.Type host. Ophelia limacina Rathke, 1843 (Polychaeta: Opheliidae).Location in host. The midgut epithelium.Type (syntype) material. A platinum sputter-coated SEM stub with several protists, specimen of parasite cells and host material fixed in ethanol have been deposited in the collection of Department of Invertebrate Zoology, Saint Petersburg State University; extracted DNA used for obtaining of rDNA sequences deposited in the collection of Department of evolutionary biochemistry, Belozersky Institute for Physico-Chemical Biology, Lomonosov Moscow State University; Fig. Etymology. This species was named after Dr Andrej Alexandrovitch Dobrovolskij (1939–2019), Professor of the Department of Invertebrate Zoology at Saint Petersburg State University, a recognized authority in the field of Invertebrate Zoology and Parasitology, the Great Teacher of many generations of Russian zoologists, who devoted himself utterly to science and students. In particular, he taught and inspired the authors TSM, TGS, and GGP.Rhytidocystis pertsovisp. n. Miroliubova, Simdyanov, Janouškovec, Belova, Paskerova, 2020.Description. Young intracellular trophozoites 14.3–25.3 µm; crescent-shaped then bean-shaped with a spherical centric nucleus 4–6.6.0 µm and a spherical eccentric nucleolus. Larger trophozoites extracellular, 21.3–59.2 µm; irregular, roundish then spherical; with a spherical centric nucleus 8.0–20.0 µm and a relatively big eccentric or centric nucleolus. The cell surface of spherical trophozoites has no folds.Molecular data. Partial rDNA , GenBank accession number MT231948; transcriptome shotgun assembly, GenBank accession number GHVQ00000000.1.Type locality. White Sea, Kandalaksha Gulf , a depth of ~ 1–15 m.Type habitat. Marine.Type host. Ophelia limacina Rathke, 1843 (Polychaeta: Opheliidae).Location in host. The midgut epithelium.Type (syntype) material. A platinum sputter-coated SEM stub with several protists, specimen of parasite cells and host material fixed in ethanol have been deposited in the collection of Department of Invertebrate Zoology, Saint Petersburg State University. Resin blocks and fixed slides containing pieces of infected host intestine deposited in the collection of the author TGS, Department of Invertebrate Zoology, Lomonosov Moscow State University; extracted DNA used for obtaining of rDNA sequences deposited in the collection of Department of evolutionary biochemistry, Belozersky Institute for Physico-Chemical Biology, Lomonosov Moscow State University; Figs. Etymology. This species was named after Nickolay Pertsov (1924–1987), a long-time director and an eminent innovator at the White Sea Biological Station of Lomonosov Moscow State University, where this species was found and sampled.Pectinaria (Cistenides) hyperborea Malmgren, 1866 (Polychaeta: Pectinariidae) were found in the vicinity of Educational and Research station “Belomorskaya” of the Saint Petersburg State University (ERS SPbU), Chupa Bay, Podpakhta Strait , at a depth of ~ 20 m, under the thermocline. Bristle worms Ophelia limacina Rathke, 1843 (Polychaeta: Ophellidae) were collected from two distant locations: White Sea Biological Station Lomonosov Moscow State University (WSBS MSU), Velikaja Salma, Kandalaksha Bay, White Sea  and in the vicinity of ERS SPbU, Yakovleva Inlet, Keret’ Archipelago, Chupa Bay, Kandalaksha Bay, White Sea  at a depth of ~ 1–15 m. The O. limacina worms from these different locations were stored and processed independently. The coccidian individuals and pieces of the infected host intestine were isolated with fine tip needles under Olympus SZ40  or MBS-10  stereomicroscopes.Polychaete hosts were collected from the sublittoral zone in different sites in Kandalaksha Gulf, White Sea in 2015–2018. The bristle worms 52 4 μm thick sections were made with the Leica RM-2265 microtome and stained with Ehrlich's Hematoxylin. Separate alive parasites and squash preparations of the host intestine fragments containing parasites as well as histological sections were photographed with the help of Leica DM 2500 light microscopes equipped with differential interference contrast (DIC) optics and Plan-Apo objective lenses and connected to a Leica DFC295 or a Nikon DS-Fi1 digital cameras . All in vivo microphotographs were taken with the use of DIC technique, microphotographs of histological sections—by means of bright-field microscopy (LM). Maximal dimensions of protist cells were measured with the ImageJ program (rsb.info.nih.gov/ij/). Average values and standard errors were calculated.Fragments of the infected host intestine were fixed with Bouin solution, rinsed two times with distilled water and dehydrated in ethanol series. Using paraffin-celloidin methodRhytidocystis pertsovi were also studied with transmission electron microscopy (TEM). For both methods, the individual trophozoites or small fragments of the infected host intestine were fixed with 2.5% (v/v) glutaraldehyde in 0.05 M cacodylate buffer (pH 7.4) containing 1.28% (w/v) NaCl in an ice bath in the dark. The fixative was once replaced with fresh fixative after 1 h, and the total fixation time was 2 h. The fixed samples were rinsed three times with cacodylate buffer and post-fixed with 2% (w/v) osmium tetroxide in the cacodylate buffer . After fixation, samples were dehydrated in an ethanol series.The cell surface of the isolated parasites was studied with scanning electron microscopy (SEM). Trophozoites For SEM dehydrated samples were critical point dried in liquid CO2 and then sputter-coated with platinum. The samples were investigated with a Tescan MIRA3 LMU scanning electron microscope , and FEI Quanta 250 .53 and examined under a JEM 1011 electron microscope .For TEM study dehydrated samples were transferred to an ethanol/acetone mixture 1:1 (v/v), rinsed twice in pure acetone, and embedded in Epon resin using a standard procedure. Ultrathin sections obtained using LKB-III  or Leica EM UC6  ultramicrotomes were contrasted with uranyl acetate and lead citrateRh. nekhoroshkovae and Rh. pertsovi were fixed with 96% ethanol, the sample of Rh. dobrovolskiji was fixed with RNA-later . Extraction of DNA from fixed cells was performed using the NucleoSpin Tissue kit . Whole Genome Amplification (WGA) was performed for Rh. nekhoroshkovae and Rh. dobrovolskiji samples using REPLI-g Midi Kit . The contiguous nucleotide sequences  were assembled from a series of overlapping fragments obtained by PCR with different pairs of primers, followed by Sanger sequencing . The rDNA fragments were amplified with Encyclo PCR kit  in a total volume of 20 µl using a DNA Engine Dyad thermocycler (Bio-Rad) and a T100 Thermal Cycler (Bio-Rad). General scheme of PCR protocol as follows: lid temperature 100 °C; initial denaturation at 95 °C for 2.5 min; 40 cycles of 95 °C for 30 s (denaturation); 48–55 °C for 30 s ; 72 °C for 1.5 (elongation) and a final extension at 72 °C for 10 min. Table Isolated trophozoites (up to 50 cells from each location) were washed three times in filtered sea water and deposited into 1.5 ml microcentrifuge tubes. Samples of Pterospora schizosoma, Monocystis agilis, Lecudina tuzetae, and Heliospora caprellae were assembled from the transcriptome sequencing data generated by Mathur et al.56. The rDNAs of Lankesteria abbotti were assembled from the transcriptome sequencing data of The Marine Microbial Eukaryote Transcriptome Sequencing Project57. The sequencing data were obtained from the NCBI Sequence Read Archive , and the assemblies were performed with SPAdes58 utilizing k-mer size 127 and Trinity59 programs. Assembled rDNA contigs were aligned using MAFFT60 and inspected by eye for assembly errors and chimeric sequences.The rDNAs of Rhytidocystis spp., their closest relatives , two adeleid coccidians , and major gregarine lineages not represented in 5.8S and LSU rDNA databases . The missing nucleotide sites of 5.8S and LSU rDNAs were marked as "N" in the concatenated dataset. The rDNAs' alignments were generated in MUSCLE 3.661 under default parameters and then manually adjusted and concatenated with BioEdit 7.0.9.062; columns containing few nucleotides or hypervariable regions were removed. Representatives of stramenopiles and Rhizaria were used as outgroups. The final dataset included 99 taxa with 4,517 unambiguously aligned sites.The alignment of concatenated SSU, 5.8S, and LSU rDNAs  was prepared for phylogenetic analyses. The taxon sampling was designed in order to maximize the phylogenetic diversity of Apicomplexa and completeness of sequences in alignments, by preferentially selecting taxa having both SSU and LSU rDNA sequences. However, since the taxon samplings of 5.8S and LSU rDNAs are limited by available sequences, we then expanded the dataset by adding SSU rDNA-alone sequences of previously published 63 under the GTR + Г model and CAT approximation (25 rate categories per site). The procedure included 100 alternative runs of the ML analysis and 1,000 replicates of multiparametric bootstrap. Bootstrap percentages were merged on the user trees (both ML and BI) with the same program. Bayesian inference (BI) analyses were done in MrBayes 3.2.664 under GTR + Г + I model with 12 discrete categories of gamma distribution. The following parameters were used: nst = 6, ngammacat = 8, rates = invgamma; parameters of Metropolis Coupling Markov Chains Monte Carlo (mcmc): nruns = 2, nchains = 4, temp = 0.2, ngen = 10,000,000, samplefreq = 1,000, burninfrac = 0.5. The average standard deviation of split frequencies at the end of computations was 0.001441.Maximum-likelihood (ML) analyses were performed with RAxML 8.2.9Rhytidocystis species were carried out with HMMER65 using profiles constructed from protein family alignments. The corresponding protein families were identified with OrthoFinder66 clustering with predicted protein sequences in a set of 70 eukaryotic genomes; the protein family alignments were generated using MAFFT60. We utilized the following sources for genomic data: Genome database of NCBI (https://www.ncbi.nlm.nih.gov/genome), Genome Portal of DOE JGI , Ensembl Protists resources (https://protists.ensembl.org/), genome projects of Marine Genomics Unit (https://marinegenomics.oist.jp/), and genomic resources of multicellgenome lab (https://multicellgenome.com/). The transcriptomic data for Rhytidocystis pertsovi and Rhytidocystis sp. ex Travisia forbesii were obtained from GenBank transcriptome shotgun assembly projects GHVQ00000000.1 and GHVS00000000.1. The transcriptome assemblies of Rhytidocystis species were processed with TransDecoder67 utilizing BLAST68 and HMMER searches against the UniProtKB/Swiss-Prot69 and Pfam70 databases for ORF prediction. Orthology of proteins discovered by HMMER profile searches was verified by reciprocal BLAST searches against OrthoFinder orthogroups: proteins with best hit outside of the queried protein family were excluded from the set of findings. The findings satisfying reciprocal BLAST search criterion were added to the protein family alignments using MAFFT60, and the family membership was further inspected by reconstructing phylogenies. The trees for meiosis-specific protein families were reconstructed by IQ-TREE71 using the LG + C10 + F + G4 profile mixture model, and ultrafast bootstrap approximation72 with 1,000 replicates for estimation of branch support; IQ-TREE reconstructions for oocyst wall protein families utilized ModelFinder73 to automatically select the best-fit model. The trees were visualized using MEGA74 and iTOL75. The completeness estimates for genomic and transcriptomic data were performed with BUSCO76 using the eukaryota_odb9 dataset.Searches for meiosis-specific and oocyst wall protein families in the transcriptomes of Supplementary file1