Cryptosporidium and Giardia are relatively common. One possible transmission route is consumption of contaminated fresh produce; this study investigated the occurrence of Cryptosporidium and Giardia in fresh produce in four districts of rural Tigray in Ethiopia. Fresh produce samples (n = 55) were analysed using standard laboratory procedures. Overall, 15% (8/55) of samples were found to be contaminated. Although contamination levels were mostly low, a few samples had high numbers of Giardia cysts (up to around 70 cysts per 30 g sample). Molecular analyses were largely unsuccessful, but Giardia Assemblage A was identified in one sample. Contamination with these parasites was identified in two of the four districts, but, although a similar pattern has already been described for water contamination, this may be at least partially explained by sampling bias. Nevertheless, we speculate that access to clean water sources may be an important factor for reducing the occurrence of these pathogens. Given the public health and veterinary burden associated with both parasites, the factors which are of importance for their circulation in the communities and environments deserve further investigation.In rural Ethiopia, where people often share their homes with their livestock, infections of humans and animals with  Foodborne parasitic diseases are an important public health concern globally  resultinAccording to WHO estimates of disease burden, foodborne parasitic diseases, excluding enteric protozoa, caused an estimated 23.2 million cases and 45,927 deaths in 2010, resulting in an estimated 6.64 million Disability Adjusted Life Years (DALYs) [Cryptosporidium and Giardia [Fresh produce can be contaminated by enteric protozoan parasites, including  Giardia . Contami Giardia  during c Giardia ,6, or du Giardia .Cryptosporidium oocysts and Giardia cysts in moist and refrigerated environments means that fresh produce is a suitable transmission vehicle [Cryptosporidium oocysts are more robust than Giardia cysts, and oocysts can survive under less favourable storage conditions for more extended periods [Cryptosporidium oocysts that have contaminated fresh produce during harvesting may be still infective at the marketplace where it is sold for human consumption. Although, Giardia cysts are more fragile and may not survive less-favourable environmental conditions [Cryptosporidium and Giardia from contaminated fruits like berries can be significantly decreased by simple washing before consumption [The survival capabilities of  vehicle . Cryptos periods . Cryptosnditions , contamisumption . Howeversumption ,8,9, andsumption .Cryptosporidium and Giardia, are common [In developing countries, like Ethiopia, enteric protozoan infections, including e common . Both pae common . Diarrhoe common . In addie common , or those common ; given te common . The proCryptosporidium oocysts has been reported to range from 4.7–12.8% and with Giardia cysts from 1.3–18.5% indicating relatively high occurrence of contamination [Cryptosporidium and Giardia is relatively common in Ethiopia, the importance of this contamination to human health remains unknown.Investigations of parasitic contamination of vegetables and fruits collected from local markets have been conducted in various towns in Ethiopia ,20,21,22mination ,20,21,22In most of Ethiopia, fruits and vegetables are sold in open-air marketplaces and streets, and often this produce is eaten directly after purchase without washing . Fresh produce displayed for sale without washing has been reported to be more likely to be contaminated with parasites than washed fresh produce . Fresh pAs part of a larger One Health study on the epidemiology of cryptosporidiosis and giardiasis among humans and animals in rural areas of Tigray, Ethiopia, we investigated contamination of vegetables and fruits collected from farmers’ backyards, irrigated farmlands, and open-air local markets at four locations. In the study areas, family labour was used for all the tasks, from land preparation to harvesting and transporting of the products to the market, organic manure  used to improve production.Cryptosporidium and Giardia in fresh produce at four locations in Tigray, Ethiopia, to assess the species and genotypes of these parasites in order to provide information on transmission pathways and epidemiology, and to compare these data with the occurrence of the parasites in water samples previously reported from the four sampling districts.Our aim was to determine the occurrence of Between October 2018 and January 2019, samples of fresh produce (vegetables and fruits commonly eaten raw) were collected from local open-air markets, backyards of farmers, and irrigated farmlands from four selected districts of Tigray Region, Ethiopia, namely: Enderta (south-eastern zone of the region), Kilte Awulaelo (eastern zone), Hintalo Wejirat (south-eastern zone), and Raya Azebo (south zone).2 of which 30,062 ha is cultivable land [Enderta (Location 1) is located at 13°14′ N and 39°40′ E with an altitude ranging from 1500 to 2300 metres above sea level (masl). The district covers a total area of 89,812 kmble land . Locatioble land . A minorble land .Hintalo Wejirat (Location 2) is at latitudes between 12°55′ N and 13°20′ N and longitudes 39°20′ E and 39°55′ E with an elevation area ranging from 1400 to 2850 masl. The district covers an area of 193,309 ha with an approximate total population of 153,500 ,26. The Kilte Awulaelo (Location 3) is located between 13°45′ N–14°00′ N and 39°30′ E–39°45′ E. The elevation ranges from 1980 to 2500 masl. The average daily air temperature ranges between 15 °C and 30 °C. The mean annual rainfall is 601 mm. The district covers an area of 101,758 hectares, of which 21,620 hectares are farmlands ,28. The Raya Azebo (Location 4) is located at 12°39′ N latitude and 39°44′ E longitude. The altitude ranges from 930 to 2300 masl. The area has two rainy seasons, with light rains between February to April period and heavy rains between July–September. The mean annual rainfall is 724 mm, with mean daily maximum and minimum temperatures of 18 °C and 14 °C, respectively for the western highlands and 23 °C and 20 °C, respectively in the valleys . The disThe majority of households do not cultivate vegetables, rather relying on local markets to purchase fresh produce. In Tigray, for example, production of vegetables and fruits is reported in only 14% and 8% of households, respectively . HistoriOf the vegetables and fruits available for raw consumption in the regions studied, the most common ones are cabbage, lettuce, carrot, pepper, tomato, and guava. These are the fresh produce types included in our study. The samples were collected from the four districts which are subdivided into ‘tabias’ (villages). Twenty-four samples were analysed from Location 1 (4 tabias), five samples from Location 2 (1 tabia), twenty-one samples from Location 3 (4 tabias), and five samples from Location 4 (1 tabia) . The numStudy participants were interviewed regarding their sources of vegetables and fruits for consumption. Based on responses, samples of fresh produce were collected from the relevant sources. The participants reporting that they ate vegetables produced in their own backyards provided vegetable samples from this location. Some participants reported that their vegetables and fruits sources came from their own irrigated fields, and these samples were obtained from their fields. In all cases, the collected samples were put in a plastic bag, coded with date, district, and site of collection, and transported to the parasitology laboratory, College of Veterinary Medicine, Mekelle, Ethiopia.The samples were refrigerated at 4 °C for a maximum of 24 h at the parasitology laboratory and processed on the following day. For leafy vegetables (lettuce and cabbage) and peppers, 30 g were weighed into stomacher bags (Seward BA6041/STRfilter bag), 200 mL of 1 M glycine buffer was added to immerse the sample, and the samples were processed by stomacher/paddle blender for one minute. For carrots, tomatoes, and guava one or two were selected on the basis of their size and put into filtered stomacher bags and washed for 5 min by hand in 200 mL of 1 M glycine buffer [The eluate was transferred into five 50 mL centrifuge tubes, the bag washed with distilled water, and the wash water transferred to the tubes. The tubes were transported to Ayder Referral Hospital, Department of Microbiology, Mekelle on the same day, where they were centrifuged for 10 min at 1550 relative centrifugal force. Following aspiration of the supernatant, the pellets were vortexed and combined in a single tube per sample and refrigerated at 4 °C before being transported to Norway for further analysis.Cryptosporidium oocysts and Giardia cysts. Although 16 samples from Location 1 district were processed according to the ISO 18 744 [IMS was performed using Dynabeads GC-combo kit for isolation of O 18 744  protocolO 18 744 .Cryptosporidium oocyst walls and Giardia cyst walls  and 4′,6′ diamidino-2-phenylindole (DAPI) was used to stain the DNA in the nuclei of these organisms. Samples were mounted with M101 No-Fade Mounting Medium then each slide was covered by a glass coverslip and viewed immediately.Single-well slides of air-dried sample concentrates were fixed with methanol and stained with fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies (mABs) against A Leica DCMB fluorescence microscope equipped with Nomarski differential interference contrast (DIC) optics was used for examination of the slides. A blue filter block  was used to visualize FITC conjugated mABS labelled cysts and oocysts and a UV filter block  was used to visualize the presence or absence of DAPI-stained sporozoite nuclei. All observations were at 200× or 400× magnification.Cryptosporidium oocysts and Giardia cysts; preliminary identification was based on reactivity with the monoclonal antibody and appropriate shape and size. Ovoid or spherical objects with brilliant apple-green fluorescence of the appropriate size and shape were examined under the UV filter to determine the presence of nuclei, both to support identification (if present) and for assessing the sample suitability for further investigation by molecular methods. The number of Cryptosporidium oocysts and Giardia cysts observed per sample was recorded.The entire well of the stained slides was examined for the presence or absence of Cryptosporidium and 55% for Giardia, regardless of whether the ISO 18744 [The recovery efficiencies of both the methods used were estimated using spiked samples in which known numbers of flow cytometry-sorted oocysts and cysts  were spiked onto 30 g lettuce and dried at room temperature for 2 h before analysis as described. Recovery efficiencies were estimated to be approximately 30% for SO 18744  protocolSO 18744 . This isCryptosporidium and Giardia from artificially contaminated berries.DNA extraction was conducted on positive samples using DNeasy PowerSoil Kit  protocol, with some modifications. The fresh produce sample post IMS (250 µL) and 60 µL of the lysis solution (solution C1) were added to the PowerBead Tubes and vortexed together to mix. This was then subjected to bead beating to release the DNA by breaking the oo(cyst) walls using a FastPrep-24 5G  in two cycles of 4 metre/s for 60 s with a 45 s pause between the cycles. In the end, the DNA was eluted in 40 µL of the elution solution (solution C6) and stored at −20 °C. A recent study by Temesgen et al., 2021  reportedGiardia investigation, PCR targeting the glutamate dehydrogenase gene, beta-giardin gene, and SSU gene were conducted according to published protocols [Cryptosporidium, PCR targeting the SSU-rRNA gene was conducted [For rotocols ,35. For Reactions were carried out in a total volume of 25 µL that included 2 µL of template DNA, 0.4 µM of each primer and 12.5 µL of DreamTaq PCR Master Mix (2X) . Bovine serum albumin (BSA) 0.2 µL of (20 mg/mL) was used in the reaction targeting the beta giardin and GDH genes.The PCR products were examined following separation on a 2% agarose gel, stained with SYBR Safe DNA gel stain and visualized under UV illumination. A ready-to-use DNA ladder  of 100 bp was used for fragment size determination.Purification of the positive products was carried out using ExoSAP-IT PCR product clean-up reagent  and sent to a commercial company  for sequencing in both directions. Sequences were checked using Geneious prime software and compared with sequences in GenBank using NCBI BLAST.Results were collected in a database in Excel. Data analysis was made using simple descriptive statistics and frequency using STATA version 15. Fisher’s exact test was used to determine associations with sample location and detection of parasites and also between sources of samples and detection of parasites.Cryptosporidium oocysts and/or Giardia cysts. DAPI staining was not observed in any of the oocysts or cysts, indicating that the oocysts or cysts had ruptured and there was no longer nuclear material within the oocysts or cysts. Of these 8 samples, 2 were contaminated with Cryptosporidium only, 5 were contaminated with Giardia only, and 1 was contaminated with both Giardia and Cryptosporidium. All 8 positive samples were from Location 1 and Location 3 districts, which were also the districts from which most samples were collected.Out of 55 fresh produce samples processed, 8 (15%) were found to be contaminated with The contamination rate of different types of fresh produce is depicted in Giardia cysts were counted and one lettuce sample where 28 Giardia cysts were counted; all other positive samples had fewer than 20 oo(cysts).The number of contaminating oo(cysts) per sample of vegetable and fruits are described in Giardia positive) was obtained from a backyard , 3 from farmers’ fields , and 4 from markets (2 Giardia positive and 2 Cryptosporidium positive of 36 market samples). No source was more or less likely to be contaminated with these parasites than another.Of the positive samples, 1 , in the latter of which Cryptosporidium oocysts were also detected, and also from the two other Cryptosporidium positive samples.DNA was isolated from the three samples with the highest number of Giardia SSU gene from the sample containing 28 cysts . DNA sequencing of this PCR product was successful, and revealed it to be Assemblage A, with 99% similarity to sequences at GenBank Accession numbers MH047247.1 and MH047246.1. All other PCR attempts for the other Giardia and Cryptosporidium positive samples were unsuccessful despite multiple attempts at several genes.PCR resulted in amplification at the Cryptosporidium and Giardia appeared to occur frequently in Locations 1 (Enderta) and 3 (Kilte Awulaelo). These are also the two locations where contamination was found in fresh produce  was also observed in one water sample from this location [In our previous article , contami produce . In addilocation .Cryptosporidium and Giardia in four districts of rural Tigray, northern Ethiopia. The most important finding from this study is that contamination of fresh produce with these parasites occurs relatively frequently in this area, with 8 out of 55 samples (15%) positive for Cryptosporidium and/or Giardia. Contamination was only found in two districts, Location 1 and Location 3, with a district-specific occurrence of 4/24 (17%) and 4/21 (19%), respectively. However, it should be noted that considerably more samples were analysed from these districts . This sampling bias means that we cannot reach a conclusion on whether the contamination risk was higher in Locations 1 and 3 than in Locations 2 and 4.This study investigated the extent of contamination of fresh produce with the protozoan parasites Cryptosporidium spp. and Giardia contamination, respectively, from vegetable and fruit samples collected from local markets in Bahirdar city, northwest Ethiopia. Similarly, Bekele et al., 2017 [Cryptosporidium spp. and Giardia of 4.7% and 10%, respectively, from vegetables and fruits collected from local markets in Arba Minch town, southern Ethiopia. Moreover, Endale et al., 2018 [Cryptosporidium spp. and Giardia, respectively, from vegetables and fruits collected from local markets in Dire Dawa, Eastern Ethiopia. Furthermore, Tefera et al., 2014 [Cryptosporidium spp. (12.8%) and Giardia (7.5%) from fruits and vegetables collected from selected local markets of Jimma town, Southwest Ethiopia. Nevertheless, the only similar study that we could find from Tigray reported a higher occurrence than we found with 7.3% and 18.5% occurrence of Cryptosporidium and Giardia on fresh produce from markets in the town of Aksum [Other reports from Ethiopia on contamination of fresh produce have produced similar results regarding the proportion of contaminated fresh produce. For example, Alemu et al., 2020)  reported20  reporl., 2018  reportedl., 2014 , reporteof Aksum . DiscrepGiardia cysts. Reports from other parts of the world also generally indicate low numbers of parasites among contaminated fresh produce. For example, a study from Norway reported 1–8 Cryptosporidium oocysts or Giardia cysts in 100 g samples of various types of fruits and vegetables [Cryptosporidium and Giardia) counts were usually below 10 per 30 g sample [Cryptosporidium oocysts rather than Giardia cysts. Another variable of interest is the type of fresh produce examined. In this study we analysed samples of vegetables commonly eaten raw  and guava. Although only a few guava samples were analysed, it was found to be the fresh produce most likely to be contaminated, with Cryptosporidium in 33% (2/6) and Giardia in 17% (1/6) of guava samples, followed by cabbage, lettuce, and pepper. Neither Cryptosporidium nor Giardia were detected on tomatoes and carrots. The reason for this may reflect factors associated with cultivation (carrots are below the surface of the soil and therefore less likely to be directly contaminated) or other factors, such as transport. As tomatoes are more likely to be damaged than the other fresh produces, they may be transported in containers and are therefore less likely to be contaminated during transport. Cabbage and lettuce may be more likely to be contaminated due to their high surface area, and their rough surfaces [As previously noted, none of these other studies from Ethiopia quantified the extent of contamination. Although in our study the number of parasites detected per sample was usually low (mostly fewer than 5 (oo)cysts per 30 g sample), a few of the samples were quite heavily contaminated, with one sample harbouring over 70 getables . More reg sample . Howeverg sample , althougsurfaces . The higCryptosporidium) or genotype (both Cryptosporidium and Giardia) contaminating fresh produce. This information may provide clues on the source of contamination, and indicate the potential public health significance. Although we were only able to obtain data from one sample of Giardia, the finding of Assemblage A, which is known to infect humans and animals, opens for the possibility of multiple different transmission routes leading to contaminated produce, including zoonotic transmission.None of the other studies from Ethiopia have investigated the species , but not at the other two locations . HoweverGiardia cysts that are not as robust as Cryptosporidium oocysts [Giardia positive sample corroborates this suggestion, as DNA amplification was possible. It is not possible to determine whether the parasites contaminating the fresh produce were viable or infectious at the time of sample collection, and therefore presenting a public health risk to consumers. The lack of oo(cyst) nuclei at detection suggests that the parasites were non-infective. However, it is possible that damage to the oo(cyst)s occurred after sample collection, such as during transport, storage, or processing, and were indeed infective at the time of sampling.As with many studies, there are some important limitations in this study. The total number of fresh produce samples that we analysed was relatively low 55) compared to other studies, including those from Ethiopia where reported sample sizes were between 108 to 384 5 compare,21,22,37Cryptosporidium oocysts and Giardia cysts was relatively common in Tigray region. Despite the fact that contamination was only detected in two of the four districts in the study, this could reflect sampling bias rather than a lack of contamination at Locations 2 and 4. Although contamination levels in positive samples were generally low, a few samples were more heavily contaminated with Giardia cysts. Similar results have already been reported regarding contamination of water supply, with the same two districts apparently having cleaner water. Although our data may suggest certain factors in specific locations may contribute to the circulation of these parasites within the environment, sampling bias also has a role. Given the public health and veterinary burden associated with both parasites, trying to pinpoint the factors of importance in reducing the circulation of these parasites in the communities and environments deserves further investigation.In conclusion, our study found that contamination of fresh produce with


The aim of the present study was to study the effects of cycling and pure concentric and pure eccentric high-intensity interval exercise (HIIE) on skeletal muscle  and cerebral oxygenation. Twelve healthy males  performed, in a random order, cycling exercise and isokinetic concentric and eccentric exercise. The isokinetic exercises were performed on each randomly selected leg. The muscle and the cerebral oxygenation were assessed by measuring oxyhemoglobin, deoxyhemoglobin, total hemoglobin, and tissue saturation index. During the cycling exercise, participants performed seven sets of seven seconds maximal intensity using a load equal to 7.5% of their body mass while, during isokinetic concentric and eccentric exercise, they were performed seven sets of five maximal muscle contractions. In all conditions, a 15 s rest was adopted between sets. The cycling HIIE caused greater fatigue  compared to pure concentric and pure eccentric isokinetic exercise. Muscle oxygenation was significantly reduced during HIIE in the three exercise modes, with no difference between them. Cerebral oxygenation was affected only marginally during cycling exercise, while no difference was observed between conditions. It is concluded that a greater volume of either concentric or eccentric isokinetic maximal intensity exercise is needed to cause exhaustion which, in turn, may cause greater alterations in skeletal muscle and cerebral oxygenation. High-intensity interval training is an exercise modality that consists of repeated short periods of high, maximal, or/and supramaximal intensities, separated by intervals of either low-intensity exercise or rest ,2. DurinHigh-intensity interval training can be performed using a wide range of exercise activities such as cycling, running, rowing, swimming, and resistance training . In thes2Hb, O2 delivery), deoxyhemoglobin , total hemoglobin , and tissue saturation index   ,54.The HIIE during cycling exercise was performed using a cycle ergometer  and consisted of 7 sets of 7 s maximal intensity efforts with 15 s interval between sets. Specifically, during every set, the participants were pedaling with no brake on the flywheel as fast as they could and, when the pedaling rate reached 100 rpm, a load equal to 7.5% of their body mass was dropped instantly on the flywheel . The stop-clock was starting automatically at the 100 rpm and the participants had to maintain maximal pedaling rate for 7 s while they remained seated throughout the trial. Participants were verbally encouraged to maintain their maximal effort during each set.The HIIE during the isokinetic concentric and eccentric exercises was performed using an isokinetic dynamometer , and consisted of 7 sets of 5 repetitions with 15 s interval between sets, while the angular velocity was set at 60°/s. The isokinetic dynamometer was calibrated before each exercise session according to the manufacturer’s instructions. Subjects were seated (120° hip angle) with the lateral femoral condyle aligned with the axis of rotation of the dynamometer, and were coupled to the dynamometer by an ankle cuff attached proximally to the lateral malleolus. Each subject’s functional range of motion was set electronically between 20° and 100° of knee flexion (full extension 0°). Each isokinetic concentric or eccentric repetition lasted 1.34 s  or 6.7 s/set, which was almost equal to the duration of each set during cycling exercise . Gravitational corrections were made to account for the effect of limb weight on torque measurements. Participants were verbally encouraged to maximally activate their knee extensors throughout each exercise bout.2Hb]) and deoxyhemoglobin (Δ[HHb]), which reflect the dynamic balance between muscle oxygen delivery and extraction in the underlying tissue  were related to microvascular blood volume changes [2Hb], [HHb], [THb], and TSI were assessed from the resting values recorded over the last 30 s of a 5 min resting period preceding the start of the test. The measurements were, therefore, normalized based on these recordings (arbitrarily defined as 0 μM) and the data are presented in Δ values.Skeletal muscle and cerebral oxygenation were assessed simultaneously and non-invasively using the continuous wave NIRS technique, with two wavelengths of near-infrared light (760 and 850 nm) . The NIRS units  consisted of multi-distance optical probes, configured with one optical receiver and three optical source emitters in order to monitor three separate regions of the tissue simultaneously. The three source emitters were on the same line as the detector. The inter-optode spacings between emitters and receiver were 30, 35, and 40 mm, and the penetration depth was approximately one-half of the distance between the emitter and the receiver  . The NIRg tissue ,38. Moreficients . Changes changes . The chaRegarding muscle oxygenation, the NIRS unit (PortaMon) was placed at the lower third of vastus lateralis muscle , after the site had been shaved and cleaned using an alcohol swab. The skinfold thickness over the vastus lateralis of both legs at the site of PortaMon placement was measured using a skinfold caliper  to determine adipose tissue thickness , since it may influence the amplitude of the NIRS signal . In the Rate of perceived exertion (RPE) was assessed during high-intensity interval exercise protocols at baseline and after the 4th and the 7th set .p > 0.05). A two-way ANOVA test was used to compare the three exercise modes in regard to performance, as well as in muscle and cerebral oxygenation [group (3 exercise modes) × time (7 sets) or (3 sets) in the case of rate of perceived exertion]. When a significant interaction was obtained, pairwise comparisons were performed through the Sidak test. When sphericity was violated, the Greenhouse–Geisser correction was applied. Data are presented as mean ± standard error of the mean (SEM) and the level of significance was set at α = 0.05. The SPSS version 21.0 was used for all analyses .The normality of all dependent variables was examined by the Shapiro–Wilk test and no significant violence of normality distribution was found (p < 0.001) compared to isokinetic concentric (118.1 ± 2.3 W) and eccentric (124.7 ± 4.2 W) exercise protocols. Cycling performance was gradually decreased, as expressed by fatigue index . Specifically, the magnitude of the fatigue index reduction was greater in cycling compared to concentric exercise between the third and the seventh bout while, compared to eccentric exercise, the difference was greater between the second and the seventh bout. The magnitude of fatigue index between the two isokinetic modes was greater in concentric compared to eccentric exercise. Regarding RPE, there was a significant main effect of exercise protocol (p < 0.001), with the mean values being higher in cycling (13.5 ± 1.0) compared to concentric (12.0 ± 0.7) and eccentric (12.1 ± 0.7) exercise, whereas no difference was observed between concentric and eccentric protocols  or exercise mode by time interaction (p > 0.05) were observed for muscle Δ[O2Hb], Δ[HHb], and Δ[THb] among HIIE protocols. However, the main effect of time was significant (p < 0.01) in all HIIE regarding muscles Δ[O2Hb], Δ[HHb], and Δ[THb]. In particular, muscle Δ[O2Hb] rapidly decreased at the initiation of exercise and remained low until the end of the exercise (p = 0.01) on muscle ΔTSI, with the mean values being higher in cycling (2.14 ± 0.45%) than in concentric exercise . Muscle TSI significant increased only during the cycling exercise . In particular, cerebral Δ[O2Hb] rapidly increased at the initiation of exercise, followed by a decrease at the fourth set (p > 0.05)  B.2Hb] and the Δ[HHb] were significantly reduced and increased, respectively, while no difference was observed between exercise modes. On the contrary, the tissue saturation index was significantly greater during cycling compared to concentric and eccentric exercises. Cerebral oxygenation was not different among the three exercise modes, based on the measured parameters .In the present study, we investigated whether skeletal muscle  and cerebral oxygenation would be affected differently during HIIE of different muscle contraction modalities, such as cycling , pure concentric, and pure eccentric exercise. It was found that the mean power output was significantly greater during cycling compared to both isokinetic concentric and eccentric exercise protocols, which could be related to the greater level of fatigue that was found during the cycling HIIE compared to concentric and eccentric exercises. Regarding muscle oxygenation, in the three modes of exercise, the Δ and an increase in muscle Δ[HHb] in response to acute high-intensity interval exercise when performed in a wide range of exercise durations (6–30 s), recovery periods (12 s–2 min), and exercise types (cycling and running) [2Hb] and significant increases in Δ[HHb], whereas this response  is an indication of decreased muscle oxygenation , Δ[HHb], Δ[THb], and TSI) during the eccentric and concentric muscle actions. However, regarding cycling exercise, there was a tendency for increased Δ[O2Hb] during the first two sets of HIIE, which could be attributed to increased nervous activation [2Hb] was marginally decreased. Moreover, during the cycling exercise, the cerebral Δ[HHb] gradually increased, reaching significance at the last two sets of HIIE. No significant difference was observed among the three modes of muscle action. It is clear that neither the different performance magnitude between conditions nor the different type of muscle action were adequate to affect cerebral oxygenation. The present results are partially in line with previous investigations where HIIE of either running or cycling caused decreases in cerebral [O2Hb] and tissue saturation index while also causing increases in cerebral [HHb]  and Δ[HHb], was significantly reduced during high-intensity interval exercise in the three conditions; however, no difference was observed between them; (iii) the tissue saturation index was greater during cycling compared to concentric and eccentric exercises; (iv) cerebral oxygenation  was only marginally affected during the cycling exercise, while no difference was observed between conditions. It is clear that a greater volume of isokinetic concentric or eccentric maximal intensity is needed to cause exhaustion which, in turn, may cause greater alterations in skeletal muscle and cerebral oxygenation.The aim of the present study was to collect muscle and cerebral oxygenation data during exercise and to uncover the interaction between different physiological systems in response to different types of muscle contraction. To the best of our knowledge, this is the first study that compares skeletal muscle and cerebral oxygenation during HIIE for different modes of muscle contraction, i.e., cycling , pure concentric, and pure eccentric exercise. The main findings of the present investigation are: (i) HIIE caused different responses in the fatigue index between the cycling exercise and pure concentric and pure eccentric isokinetic exercises; (ii) muscle oxygenation, as measured via Δ[O


The imbalance of osteogenic, adipogenic, and chondrogenic differentiation in bone marrow mesenchymal stem cells (BMSCs) occurred in multiple age-related degenerative diseases such as osteoporosis and osteoarthritis. In order to improve our understanding and control of multi-directional differentiation of BMSCs in rats, using high-throughput sequencing, we identified key gene regulatory events in the early stages of lineage commitment. Data analysis revealed two transcription factors  with elevated expression throughout the initiation of differentiation (3 h), lineage acquisition (12 h), and early lineage progression (72 h) of three-directional differentiation. For osteogenic differentiation, 792, 1,042, and 638 differentially expressed genes including 48, 59, and 34 TFs were identified at three time points, respectively. Moreover, the functional analysis demonstrated that 4, 12, and 5 TFs were only differentially expressed during osteogenic differentiation at 3, 12, and 72 h, respectively, and not during other two-directional differentiation. Hopx showed enhanced expression throughout three early phases during the osteogenic differentiation but no significant change in other two-directional differentiation. A similar pattern of Gbx2 expression occurred in chondrogenic differentiation. Thus, Hopx and other early responder TFs may control the osteogenic cell fate of BMSCs and participate in the development of osteoporosis. Gbx2 and other early responder TFs should be considered in mechanistic models that clarify cartilage-anabolic changes in the clinical progression of osteoarthritis. Systemaineering , but it ineering .in vitro differentiation process is unable to produce cartilage similar to articular cartilage formed under normal physiological conditions  and maturation (from progenitors to specific cell types) . The linAlthough the enriched biological processes and TFs are mostly identical between these lineages within the first 4 days after induction , the earin vivo differentiation of rat BMSCs from a two-dimensional culture system was applied, and by using high-throughput transcriptome sequencing technique and multiple bioinformatics methods, we systematically investigated genes, especially TFs being regulated upon lineage commitment of rat BMSCs into adipocytes, chondrocytes, and osteocytes. One of our key findings was the characterization of genes and regulatory programs controlling the early stages of lineage commitment of rat BMSCs. Progress in this area will be of great value to understand age- and pathology-related fractures and metabolic syndromes, and to guide a better application of BMSCs in tissue engineering and regenerative medicine.BMSCs from 4 different species  fail to elicit a proliferative response from allogeneic lymphocytes and therefore can function across the species barrier . Given t2 and 95% humidity at 37°C. BMSCs at passage 2 were seeded in 12-well cell culture plates and after 2 days, cells were induced to differentiate into osteoblasts using mesenchymal stem cell osteogenic differentiation medium , or to adipocytes using mesenchymal stem cell adipogenic differentiation medium , or to chondrocytes using mesenchymal stem cell chondrogenic differentiation medium . Differentiation media were replaced every 3–4 day.This study was approved by the Animal Ethics Committee of Shandong Provincial Hospital affiliated to Shandong First Medical University. BMSCs were collected from femurs and tibia of 3 female Sprague Dawley (SD) rats (3 weeks old) by washing the marrow cavity using high glucose Dulbecco’s Modified Eagle Medium (DMEM)  until bones appear white. Obtained cell clumps were dispersed to cell suspension by using 21-gauge needles. The cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) in an incubator of 5% COProtein concentration assay, Elisa assay, alizarin red staining, Oil Red O staining and Alcian blue staining were performed as previously described .To obtain enough RNA for the gene expression profiling analyses, we pooled three individual cultures in TRIzol (Life Technologies), resulting in a total of three experimental samples per time point  per lineage  to be used for gene expression profiling analyses. Plus, the control samples, there were a total of 30 samples. RNA was isolated as previously described .TM One . An mRNA library was constructed by NEBNext® UltraTM II mRNA Library Prep Kit for Illumina® . QubitTM dsDNA HS Assay Kit , D1000 Screen Tape , and KAPA Library Quant kit  universal qPCR Mix were used to assess the quality and yield of the constructed library. Finally, the mRNA was sequenced by NovaSeq 6000 .The quality of RNA was analyzed using NanoDropp-value < 0.05 was used as the threshold for determining gene upregulation or downregulation. Differentially expressed TFs were identified by using the AnimalTFDB 3.0 database.1 Gene Ontology (GO) enrichment2 and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment3 analysis was utilized to determine the biological function of the differentially expressed TFs and investigate its possible involvement in the lineage-commitment of rat BMSCs. The protein-protein interaction (PPI) network of identified genes was predicted using the multiple protein online tool in STRING database . Relationships between critical TFs and their differentially expressed target genes were predicted to construct the TF-target regulatory network based on the previous method | in mRNA expression ≥ 1 with an adjusted database , a searcs method .p-values were < 0.05.Statistical analyses were conducted by using SPSS Software, version 17.0 . One-way analysis of variance (ANOVA) followed by LSD method was used to compare the protein concentration and ALP expression between different groups, which are expressed as the means ± standard deviation. Differences between means were considered statistically significant when On day 21 after induced differentiation, histological staining for calcium in the osteogenic differentiation group showed that extracellular matrix (ECM) was mineralized , Oil RedData cross−comparability was evaluated via principal component analysis (PCA) for confirming biological variability between different samples. All samples were grouped separately , indicatThe volcano plot and heatmap of differentially expressed TFs at 3, 12, and 72 h of osteogenesis were shown in In osteogenesis of BMSCs, there were 13, 20, and 14 TFs being identified to be differentially expressed only at 3, 12, and 72 h after inductive differentiation . In adipAt 3 h after induction of differentiation, it was noticed that there were 15, 24, and 17 TFs only differentially expressed in osteogenesis, adipogenesis, and chondrogenesis, respectively . At 12 hGO and KEGG pathway enrichment analysis of differentially expressed TFs at different time points of three directions of differentiation was performed to identify the most relevant biological processes (BPs), molecular functions (MFs), cellular components (CCs), and pathways. The top enriched terms in BP, CC, MF, and KEGG at 3 h after osteogenesis were presented in The differentially expressed TFs between osteogenesis and adipogenesis at 3 h after inductive differentiation were also significantly enriched in BPs terms such as “negative regulation of transcription from RNA polymerase II promoter,” “regulation of transcription, DNA-templated.” Results of functional enrichment analysis of the differentially expressed TFs between every two lineage-commitment at 3 h of inductive differentiation were presented in The interactions between the proteins expressed from differentially expressed TFs at 3 h of osteogenesis consisted of 17 nodes and 13 edges, with Klf4 gene being identified to have a relatively high connectivity degree . The PPIThe interactions between the proteins expressed from differentially expressed TFs between osteogenesis and adipogenesis at 3 h after inductive differentiation consisted of 6 nodes and 6 edges, with the Sox9 gene being identified to have a relatively high connectivity degree . The intBased on the integrated analysis between TFs and DEGs, it could be noticed that during osteogenesis, the differentially expressed TFs at 3 h were able to regulate 111, 92, and 46 DEGs at 3, 12, and 72 h after differentiation . Four diThe regulatory networks between TFs and DEGs at 12 and 72 h after differentiation were presented in Most previous studies investigating the differentiation process of BMSCs used primary cells isolated from humans. BMSCs isolated from rat functions across barriers and could be used as a source of regenerative therapy for bone metabolic diseases and bone defects. In the first phase of differentiation, gene expression analyses revealed a high number of differentially expressed TFs in all three directions of differentiation. In the regulatory model derived from our data, the first phase represents the initiation stage of the differentiation program and is characterized by expression changes of many transcription-related genes that regulate lineage commitment and set the stage for further differentiation toward a stable phenotype. Downstream analyses of the differentially expressed TFs identified at 3 h of osteogenic differentiation showed that they were capable of regulating 92 and 46 DEGs in the second and third phases, respectively. As for the second phase, differentiating lineages begin to deviate, as reflected by the fact that many transcriptional changes are direct targets of the differentially expressed TFs in the first phases. In the current study, TFs that changed per hour decreased as time extended, which suggested that the differentiating cells had reached a stable phenotype. This illustrates the transition from proliferation to differentiation and reflects the inverse correlation between these processes as has been described in various other differentiating cells.Interestingly, we identified two TFs (Tsc22d3 and Epas1) that were upregulated in three lineages at all three time points, which may play an important role in the initiation of osteogenesis, adipogenesis, and chondrogenesis. Tsc22d3, also known as glucocorticoid−induced leucine zipper, is a glucocorticoid−responsive anti−inflammatory molecule that could regulate intracellular signaling pathways via hypoxia-induced factor-1α (HIF−1α) as well as AP−1 . Tsc22d3Then we used a variety of bioinformatics analysis methods to analyze these differentially expressed TFs to obtain a comprehensive understanding. As a result, GO terms of the differentially expressed TFs at 3 h of osteogenesis, adipogenesis, and chondrogenesis for biological process categories included “negative regulation of transcription from RNA polymerase II promoter,” “transcription, DNA-templated,” and “regulation of transcription, DNA-templated.” Furthermore, the KEGG pathway enrichment analysis revealed that the differentially expressed TFs at 3 h of osteogenesis and adipogenesis were predominantly associated with the “signaling pathways regulating pluripotency of stem cells” . Id2 and Id3 were downregulated in three lineages while Klf4 was upregulated in the process of adipogenesis and osteogenesis. This suggests that the initiation of BMSCs differentiation is similarly activated in both lineages, and changes of these signaling pathways and TFs are necessary to exit the immature multi-potent cell stage and acquire a specialized mesenchymal phenotype. While in the second phase, the enriched KEGG pathways begin to deviate. For osteogenesis, “signaling pathways regulating pluripotency of stem cells” was still significantly enriched, while for adipogenesis, “PI3K-Akt signaling pathway” and “MAPK signaling pathway” were significantly enriched. For chondrogenesis, “cAMP signaling pathway” was significantly enriched.The regulatory network provided potential connections between identified DEGs and TFs. There were four, six, and six differentially expressed TFs identified at 3 h during osteogenesis, adipogenesis, and chondrogenesis possessing targets among DEGs identified at all three time points. It could be noticed that Cenpa and Epas1 overlap in osteogenesis and adipogenesis while Sox9 overlaps in adipogenesis and chondrogenesis. Like Epas1, Cenpa was also upregulated in both osteogenesis and adipogenesis at 3 h. Cenpa is a functionally conserved molecule about 17kDa that forms a centromere-specific nucleosome with H2A, H2B, and H4, whose overexpression is crucial for multiple kinds of cancers . The rolin vitro experiment based on isolated rat BMSCs. The differentiation processes may possess different characteristics in vivo. Findings obtained from in vitro experiments cannot simply be transposed to predict the reaction of an entire organism in vivo. Second, only three time points were selected in this study. These time points were separately locating in three phases and were used as delegates. Third, further in-depth studies are needed to establish whether MSCs from adipose tissue or other anatomical locations undergo a similar cascade of transcriptional events. Adipose-derived MSCs may respond very differently with respect to their clonogenic potential and doubling time in comparison with BMSCs  can be found below: The animal study was reviewed and approved by the Animal Ethics Committee of Shandong Provincial Hospital affiliated to Shandong First Medical University.FL and QZ conceived and designed the study. FL, QZ, JD, and DZ analyzed RNA sequencing data. FL, QZ, and PZ performed the basic experiment. FL and QZ wrote the manuscript, which was commented on by all authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Patellofemoral instability (PFI) encompasses symptomatic patellar instability, patella subluxations, and frank dislocations. Previous studies have estimated the incidence of acute patellar dislocation at 43 per 100,000 children younger than age 16 years. The medial patellofemoral ligament (MPFL) complex is a static soft tissue constraint that stabilizes the patellofemoral joint serving as a checkrein to prevent lateral displacement. The causes of PFI are multifactorial and not attributed solely to anatomic features within the knee joint proper. Specific anatomic features to consider include patella alta, increased tibial tubercle–trochlear groove distance, genu valgum, external tibial torsion, femoral anteversion, and ligamentous laxity. The purpose of this paper is to provide a review of the evaluation of PFI in the pediatric and adolescent patient with a specific focus on the contributions of coronal and transverse plane deformities. Moreover, a framework will be provided for the incorporation of bony procedures to address these issues. Patellofemoral instability (PFI) encompasses symptomatic patellar instability, patella subluxations, and frank dislocations. Dislocations are almost always to the lateral side of the femoral trochlea. Patellar subluxation occurs when the patella partially dislocates but does not fully dislocate out of the trochlear groove . In ordeHevesi et al. developed a scoring system known as the recurrent instability of the patella score that focuses on four factors, including age <25 years (2 points), skeletal immaturity (1 pt), trochlear dysplasia (1 pt), and the tibial tubercle–trochlear groove to patellar length ratio (TT–TG/PL) (1 pt). Patients were stratified into three groups of low- (0–1), intermediate- (2–3), and high-risk (4–5) groups based on these factors. Instability-free survival was provided at 1, 2, 5 and 10 years; for the high-risk group, this was 84.4% at 1 year, 62.5% at 2 years, 34.4% at 5 years, and 20.8% at 10 years . Wi. Wi16]. With the patient sitting, active knee flexion and extension are examined to evaluate the patellar tracking and determine the presence of a J-sign. The patient sits on the edge of the table and the knee is allowed to flex from full extension. The examiner can hold the limb in terminal extension to allow for the quadriceps to relax in order to visualize the resting position of the patella before the patient begins to actively flex. As the knee begins to flex, the examiner releases enough support to stimulate the patient to contract the quadriceps. In the patient with a J-sign, the lateralized patella shifts medially just as the knee begins to flex. ,20. The Next, with the patient laying in a supine position with the quadriceps relaxed, patellar glide is quantified by placing a medial translation force followed by a lateral one, using the width of the patella divided into quadrants as reference based on percentage of the patella translated ; in addition to this, the patellar endpoint, whether firm or soft, is recorded. The Fairbank patellar apprehension sign is evaluated by placing a laterally directed force on the patella with the knee in 30° of flexion and is positive if the patient indicates discomfort or apprehension . The patStaheli et al. reviewed 500 lower-extremity rotational profiles and provided reference values: hip internal rotation for males 50° (25–65°) and for females 40° (15–60°); hip external rotation 45° (25–65°) . Th. Th54]. Alternatively, Rosskopf et al. demonstrated that low-dose biplanar radiographs are reliable when compared to CT, and subsequently MRI, when obtaining a lower-extremity rotational profile ,56. LimbTibial torsion is defined as the physiologic rotation of the tibia from the proximal to the distal articular axis of the tibia in the transverse plane and historical attempts have been made to measure this angle using both radiographic and later CT imaging ,60. An eBernhold et al. found that the TT–TG could be measured in 82% of their radiographic patellofemoral view study cohort and that this measured 5–8 mm smaller than MRI TT–TG. Studies have noted consistently smaller TT–TG values by MRI (approximately 4 mm) compared with CT .,64.63,64 with CT ,66,67,68 with CT .Seitlinger et al. proposed an alternative method for quantifying a lateralized patellar tendon insertion utilizing the tibial tubercle-posterior cruciate ligament distance, which is measured from the midpoint of the insertion of the patellar tendon to the medial border of the posterior cruciate ligament  when compared to a control group (5.8° +/− 3.6°) and correlated with the TT–TG measurement (r = 0.87) .Recognition and treatment of PFI have undergone considerable evolution in recent years, with more than 100 different surgical techniques reported in the literature ,74,75,76Once coronal plane malalignment has been identified, several treatment options are available. Two primary factors for determining the appropriate corrective procedure include growth remaining determined by bone age and the deformity location. In patients with open physes, growth modulation is a treatment option with a relatively low morbidity and low complication rates for addressing PFI in patients with genu valgum . TensionIn skeletally mature patients, both opening and closing wedge osteotomies about the distal femur are reliable treatment options for coronal plane malalignment. In a retrospective cohort study of 18 patients (20 knees) with genu valgum and PFI, Frings et al. demonstrated that the utilization of a closing wedge distal femoral osteotomy in combination with MPFL reconstruction and tibial tubercle osteotomy (TTO) eliminated recurrence of re-dislocation with median follow up of 16 (12–64) months . In a skTransverse plane deformities have been recognized by previous studies as a potential driver of PFI and anterior knee pain with historical interest in femoral torsion as a potential driver of developmental dysplasia and osteoarthritis of the hip ,101,102.The effect of transverse plane correction on other planes is an important consideration ,111. UsiStaheli indicated surgical intervention for patients older than 8 years of age with tibial torsion greater than 30° [Krengel and Staheli reported the results of 52 tibial rotational osteotomies  and reported five (13%) serious complications with proximal tibial osteotomies including compartment syndrome, peroneal nerve palsy and deep infection while there were no significant complications in the distal osteotomy group . Delgadothan 30° .The author’s preferred technique is the utilization of distal tibia and fibula osteotomy with gradual correction using a circular external fixator with surgeon discretion with regard to implementing acute correction at the time of surgery. Intraoperative neuromonitoring is used at our institution which we have found allows for safe acute intraoperative correction. Additional benefits of gradual correction include early weight bearing, no residual implants and the ability for shared decision making with the patient and family to determine final rotational alignment. Although the authors consider rotational malalignment when treating recurrent patellofemoral instability, a medial patellofemoral ligament reconstruction is often times first-line treatment even with significant rotational malalignment. Rotational osteotomies are considered in combination with a MPFL reconstruction in a revision PFI with severe deformity  .The importance of limb alignment and rotational profile considerations in the treatment of PFI cannot be overstated. Eckhoff wisely surmised, “The static and dynamic relationships of the underlying tibia and femur determine the patellar tracking pattern.” . Thus, a


Key findings with the manufacturer-supplied gel showed reduced turbidity in broth-dilution tests by 50% against Candida albicans and Candida glabrata at RBG concentrations 0.2–0.4% of neat product, respectively. A 50% reduction in turbidity of Escherichia coli, Streptococcus agalactiae, Enterococcus faecalis ranged from 1.6–2.2% and Gardnerella vaginalis was shown by flow cytometry counts to undergo a 50% reduction at 0.3% RBG. Propidium iodide staining indicated a rapid reduction of cell integrity of G. vaginalis almost immediately while after 4 h 45% of E. coli cells were stained. The lactic acid in BHI inhibited bacteria and yeast at concentrations ranging from 0.2–1.8% but inhibition was not solely due to pH since a 1:4 dilution of RBG resulted in a pH near neutral (6.75). Other findings showed biofilm accumulation assessed after 10-days exposure of Candida spp. to RBG and was reduced by an average of one-third (community strains) to one-half (drug-resistant strains). One excipient of the RBG, disodium EDTA, inhibited the growth of bacteria and yeast at concentrations below those present in RBG and may accentuate the activity of the host defense factor, lactoferrin. We conclude that RBG is a potent inhibitor of vaginal microorganisms relevant to vaginitis or intrapartum infections and contains excipients that may contribute to its antimicrobial activity.Many non-prescription preparations intended to treat or alleviate symptoms of vaginal infection are available in American and European markets, but many have scant preclinical or clinical research underpinning. Respecta For decades infectious diseases have been addressed largely through the application of antibiotics to specific conditions. In the case of gynecologic and obstetrical infections, antibiotics have served both for therapy and prophylaxis. This primary reliance on antibiotics engendered antibiotic guidelines and standards of care that inform medical practice worldwide ,2,3. In Infections encountered in obstetrics and gynecology have generated more scientific interest contemporaneously with a better understanding of the microbiome of the lower female genital tract ,12,13 anWomen with vaginal symptoms often turn to over-the-counter (OTC) preparations, nutraceuticals, and supplements or non-prescription medical devices to treat conditions that may or may not be due to the most common infectious causes including VVC  or BV 88,15. The® Balance Gel (RBG) produced by Giellepi SpA, Milan Italy, has demonstrable biological activities appropriate for its intended use as an adjuvant in treating or preventing symptomatic vaginal conditions [Candida spp. and Gardnerella vaginalis.In this paper, we present studies performed to determine if a proprietary topical product for vaginal health, Respectanditions . This genditions . The for2 cfu/mL). Optical density (OD) readings were normalized with growth in BHI representing 100% to facilitate interspecies comparison with dose-response data plotted in Primary among properties of proposed therapeutics for vaginal infections or dysbiosis is the ability to inhibit growth or induce damage to relevant microbial species. Initially, doubling dilutions of RBG in brain-heart infusion broth (BHI) were inoculated with a multi-taxon set of organisms including non-fastidious bacteria and yeast for broth-dilution growth measurement. Turbidity was measured at 450 nm in each well before and after overnight incubation at 37 °C. Because the baseline turbidity of the undiluted gel was high, practical evaluation of turbidity was possible at dilutions ≥ 1:32. Plating 10 μL aliquots from dilutions from 1:2 through 1:32 revealed no growth for any of the test organisms  was estimated. ED50, based on the data in E. coli, 0.34% RBG; E. faecalis, 0.56%; S. agalactiae, 0.9%; C. albicans 1, 0.12%; C. albicans A, 0.08% and C. glabrata, 0.03%.Gardnerella vaginalis has particular relevance to symptomatic vaginal conditions. This organism is not in and of itself the single cause of bacterial vaginosis (BV) but is present in most symptomatic occurrences and when in abundance represents one of the sentinel organisms for BV. Because the organism is fastidious and of relatively small size compared to other bacteria, turbidity measures are less successful. Accordingly, we employed flow cytometry to obtain a dose-response of RBG to this organism. Gardnerella vaginalis strain AMD (see methods). Bacteria recovered from V-agar plates were incubated with dilutions of RBG overnight at 37 °C in 5% CO2 and resulting cultures were counted by flow cytometry.Gardnerella vaginalis strain AMD appears highly susceptible to the antibacterial activity of RBG. The lowest dilutions representing 2.5% and 1.25% RBG showed complete inhibition of Gardnerella vaginalis strain AMD. The ED50 was estimated as 0.34% RBG. As indicated by this experiment, E. coli and Gardnerella vaginalis strain AMD underwent membrane damage upon exposure to the RBG, and the effect was almost instantaneous for Gardnerella vaginalis . E. coli was also included as a less fastidious comparison organism, and it also increased PI staining over 4 h exposure to 2.5% RBG. Gardnerella vaginalis strain AMD appeared more susceptible to this treatment compared to E. coli, but E. coli despite being more tolerant of RBG, increased in propidium staining over the time course of the experiment.Propidium iodide (PI) staining, a marker of non-viability, was used to indicate damage to the bacterial membrane. N NaOH and 10 μL samples were sub-cultured using a loop onto BHI agar. When no growth was seen after 24 h the plates were held for an additional 7 days, and no growth was observed from either the neat or pH adjusted RBG. Taken together, these pH-related studies suggested that RBG has antibacterial activity due to components in addition to lactic acid and its pH effect.RBG is manufactured to a pH between 3.5 and 4 (manufacturer information)  with a sEDTA is one of the excipients in RBG that may contribute to antimicrobial activity, and preliminary evidence of lactic acid-EDTA interaction was sought. The multi-taxon set of test organisms was challenged with broth dilutions of 0.2% w:v disodium EDTA in BHI, lactic acid at 5% v:v, and dilutions of a combination of equal parts of 0.2% EDTA and 5% lactic acid and turbidity was measured after overnight incubation. As in previous experiments, ED50 for individual and combination dilutions was evaluated. Candida strains from the MP8 panel and 12 non-drug-resistant community strains. Because this experiment employed 1 h exposure at 37 °C, involved a single concentration of the test articles , and derived results by flow cytometry, it was feasible to use a larger number of fungal strains. PI staining was employed to determine the viability of the test organisms. Each organism was dispensed in order so analysis could be performed in a “first-in-first-out manner”. As indicated by the data . The test organisms used for this experiment included 12 drug-resistant the data , in the Candida (drug-resistant and non-resistant community strains) for in vitro biofilm production and assessment of inter-strain variation. All test organisms were grown in BHI to prepare an inoculum (100 μL directly dispensed from the starter culture) for the experiment. Bacterial isolates were not evaluated. Biofilm was developed in the presence of 2.5% in half-strength BHI and compared to half-strength BHI by safranin staining of adherent biomass . Since biofilm involves stress responses, it was decided to incubate well plates for 10 days . Control levels of biofilm for each strain were determined in half-strength BHI and compared to the same strain incubated with 2.5% RBG in half-strength BHI. Because half of the test organisms were drug-resistant and the other half from community isolates, these were evaluated as separate groups with the results presented in Biofilm plays an important role in mucosal infections and specifically, in this present context, vulvovaginal candidiasis, and bacterial vaginosis. Therefore, preliminary information on biofilm generation in the presence of RBG was evaluated using strains of Candida albicans and Candida glabrata,) and bacteria (including Gram-negative and positive organisms) for testing we did a broth-dilution experiment with turbidity as an indicator of growth. Because of the viscosity of the formulated product, it was initially diluted on a w:v and because of its optical density of dilutions < 1:32 were too high to allow meaningful turbidity measures therefore plating (10 μL) on BHI agar confirmed a lack of growth in lower dilutions (see methods). Internal validity for this experiment was supported by the OD of each well being measured before and after incubation with all conditions set up in triplicate. OD as a percent of controls is plotted in Candida albicans A is multi-resistant to antifungal drugs. Interestingly, growth inhibition by RBG was greater for yeast isolates than bacteria . The literature is replete with studies showing lactic acid bacteria having an inimical effect on Gardnerella vaginalis which is not derived solely from lactic acid. Less information is available on the direct effect of lactic acid on Gardnerella vaginalis, although the maximum concentration of lactic acid produced in cultured Lactobacillus was 2.5 mg/mL [Gardnerella vaginalis [The initial demonstration of antimicrobial activity against our multi-taxon panel (bacteria and yeast) was limited to six organisms, and additional microbial species relevant to vaginal infection would be useful in future work. Lactic acid-based products other than RBG have been tested against es Virus ,23 which.5 mg/mL  and a reWhile larger test organism panels might discover strains or species resistant to RBG, the limited panel of organisms suggested that RBG diluted to 5% or 10% would be expected (based on subculture experiments) to inhibit all test panel organisms. Applying this information to intravaginal use, we estimate that a 3–5 mL vaginal application of RBG would result in a dilution of approximately 1:2  and woulE. coli and Gardnerella vaginalis. In this time-based experiment , compromised membrane integrity of periment , rapid iembranes . Howeverembranes , so we dNeisseria gonorrhea [Chlamydia trachomatis [Candida albicans and Candida glabrata inhibition was more dependent on acetic than lactic acid [Microbiologic observations on lactic acid appear commonly in the food science literature but fewer in vitro studies are related to BV associated bacteria , Neisseronorrhea , Chlamydchomatis , or Herpchomatis  and one tic acid . PerhapsA systematic review of the literature regarding the use of lactic acid products for BV and microbiota regulation  reportedSince RBG contains EDTA which may contribute to antimicrobial activity, we showed EDTA at concentrations present in RBG inhibited six test organisms  and the In addition to augmenting antimicrobial agents, EDTA has also been reported to enhance the activity of host defense factors including lysozyme and lactoferrin,  both of Candida biofilm in the presence of a single concentration (2.5%) of RBG against 24 Candida albicans strains (12 community isolates and 12 drug-resistant isolates described in Methods). In terms of incubation time, the times reported in the literature differ widely, but one report which observed biofilms over 140 days, referred to 2–12 days to be short term incubation [Biofilm research has grown in scope and complexity in recent years and the preliminary, and admittedly, basic effort to see if RBG may reduce biofilm was useful mainly as a first step to the subsequent investigation. Biofilm technologies, both in terms of methods and specialized apparatus are impressive, but subject to scrutiny as numerous variables in organisms, media, growth conditions have limited the ability to compare results between studies . A simplcubation  while, fcubation . UndoubtCandida albicans [Because we demonstrated that RBG has antimicrobial activity, it is possible biofilm may have been blocked by the initial antimicrobial effect, although the presence of small amounts of biofilm would argue against this. It is possible to dissect the biofilm development with more detailed experiments. For example, while lactic acid and EDTA may prevent initial growth, farnesol, a component of RBG is a quorum-sensing molecule and may play a role in decreased biofilm formation . While falbicans . Anotheralbicans . LikewisWhile the goal of demonstrating antibacterial activity of RBG and its components was foremost in our research, the intent of the manufacturer to use RBG as an adjunctive treatment to rebalance the vaginal microbiota remains a problem for future research. In addition to in vitro studies of interactions among individual components of RBG a study of the possible antimicrobial effect of RBG on lactobacillus indigenous to the vagina or used as probiotics should be undertaken and in a clinical investigation of the effect of RBG on vaginal lactobacillus pursued. Such studies would round out the findings reported here.2 confirming the absence of detectable microorganisms in the material provided. RBG was viscous and adherent to volumetric equipment and had a milky appearance, requiring some initial steps before use, detailed below , to our laboratory in Indianapolis  and was immediately placed at 4 °C prior to use. Upon arrival, samples of RBG were applied with a sterile swab to blood agar plates incubated at 37 °C in 5% COed below . 2 cfu/mL) of viable organisms.Visible cloudiness of dilutions remained up to a 1:32 dilution which obviated the use of turbidity readings for the dilutions below 1:32. However, all dilutions from 1:2–1:1028 were added to microtiter wells and inoculated with test organism, but OD readings below 1:32 were not used. Instead, 10 μL samples were spotted on BHI agar to determine the presence  indicated that the formulated RBG contained in addition to water, disodium EDTA, polydextrose, glycogen, xanthan gum, sodium hyaluronate, propylene glycol, farnesol, decylene glycol, hydroxyacetophenone, PEG-40, hydrogenated castor oil, tocopherol acetate, and lactic acid (sufficient 80% lactic acid) to achieve a final pH of 3.5–4. The exact quantities are proprietary information, but we are authorized to state the concentration of EDTA is 0.2% which informed experiments with EDTA.Gardnerella vaginalis. BHI agar was made by adding 1.5% bacteriologic grade agar before autoclaving and pouring into plates.Blood agar plates were obtained from BD BBL  for general testing of sterility of RBG and V-agar (BD BBL) was used for propagation of Bacteria and yeast were grown in BHI broth  which was prepared and autoclaved according to manufacturer instructions. BHI was also used for the dilution of RBG. Lactic acid 85% and disodium EDTA were ACS grade. Propidium iodide  was made as a 100 mg/mL stock solution and added to samples to be stained for flow cytometry at 10 μL/mL of the sample.Crystalline bovine lactoferrin was supplied by Giellepi SpA , dissolved in sterile water at a concentration of 2%, and stored at 4 °C until use. Gardnerella vaginalis strain AMD. Gardnerella vaginalis was obtained from BEI Resources  and sub-cultured from frozen stock directly onto V agar and grown for 48 h at 37 °C in 5% CO2 and colonies lifted directly from the plate and sub-cultured again on V agar and colonies were picked and suspended into BHI for immediate use.For this research, 31 test organisms, described below, were available as frozen (−80 °C) stocks that prior to use, were allowed to thaw and inoculated into BHI, grown overnight at 37 °C before further use, except E. coli ATCC 11775, Streptococcus agalactiae ATCC 13813, and Enterococcus faecalis ATCC 19433. The three fungal strains included one Candida albicans (abbreviated CA 1) obtained from a local clinical laboratory as further explained below. The other two strains were Candida glabrata ATCC 2001 and Candida albicans ATCC 64124 selected from the ATCC MP8™ multi-drug resistance pattern of resistance to 3 echinocandins, 3 triazoles and intermediate resistance to 5-flucytosine. For convenience, this organism is abbreviated here as CA A.Scouting general antimicrobial activity employed a multi-taxon selection of organisms that included 3 bacterial and three fungal species. Bacteria were Candida isolates, a set of organisms which included 12 community isolates (abbreviated CA 1 mentioned in the multi-taxon set above was included with 11 others designated as CA 2-12). The clinical isolates, provided by a local clinical laboratory, had been isolated and characterized by standard methods and supplied to us without patient information. Drug-resistant Candida albicans were from the ATCC MP8™ panel included the following: ATCC 64124 (mentioned above as part of the multi-taxon set), ATCC 10231, ATCC 76485, ATCC 28121, ATCC 90819, ATCC MYA-1023, ATCC MYA-427, ATCC MYA-574, ATCC 38289, ATCC 11651, ATCC 96901, ATCC 90029 . Additional information including the antifungal drug resistance pattern infection site and the geographic location of origin for each strain are available on the ATCC product information documents (ATCC.org).For experiments that employed a range of 4–1 × 105 organisms/mL. After setting up the plates, OD was measured at 450 nm to establish baseline readings for each well. The same procedure was used for measuring the antimicrobial potency of lactic acid and EDTA.To accommodate the rheologic and optical properties of RBG the following steps were taken before its use in experiments. Due to its viscosity, RBG was not readily measured with volumetric equipment and was first diluted 1:2 with sterile water by aseptic transfer to pre-weighed sterile tubes, re-weighed and an equal weight of sterile water added. After vigorous mixing, further dilutions were made into a brain-heart infusion (BHI) medium with volumetric equipment. In the dilution process, care was taken to thoroughly mix (exhaustively pipetting up and down) each dilution before transferring to the next dilution blank. For each organism tested, a total of 11 serial two-fold dilutions, beginning with 1:2 RBG in BHI were dispensed in 200 μL volumes in 96 well plates and an equal volume of BHI in the 12th well served as the growth control. Sufficient wells were prepared to allow triplicate determinations for each of the six test organisms in the multi-taxon set. Starter cultures of test organisms were prepared by a subculture of growing cultures overnight in fresh BHI and turbid starter cultures were diluted 100-fold in sterile water and 10 μL of this initial dilution was used to inoculate wells of test plates. Prior use of these organisms indicated that this inoculum procedure provided 1 × 102 viable organisms/mL were present. Turbidity was documented by a microplate reader with a 450 nm filter. The starting OD for each well was subtracted from the corresponding OD after incubation and the change was reported as the ratio of growth in the treated well to the growth in BHI controls. The 50% effective dose (ED50) was used to indicate the concentration of inhibitor at 50% of the difference between maximum growth (BHI) control and starting absorbance of the culture. It was calculated by linear interpolation of the concentration of inhibitor using OD values below 50% and above 50%.Low dilutions of RBG had high background turbidity which precluded the use of OD readings for dilutions < 1:32 consequently, after overnight incubation, 10 μL aliquots from each well were spotted on BHI agar. The absence of growth on BHI agar indicated fewer than 1 × 10E. coli growth in rich medium produces a strong signal at 450 nm with OD 1 = 4.09 × 109 cfu/mL [Measurement of OD at 450 nm has been used previously by us and although higher wavelengths are more commonly used, precedence in the literature for this wavelength exists ,49,50,51Gardnerella vaginalis, which provided little turbidity in BHI, and where minimal amounts of RBG (which contains mucoadhesive components) would contact the flow cell to reduce the risk of fouling the flow cell. Propidium iodide 10 μL (from 100 mg/mL stock) was added 10 min before applying the sample to the cytometer. After the addition of the propidium iodide, samples were placed in the dark at room temperature (drawer below the cytometer) and applied one-by-one to the instrument. Scatter plots of FSC-h versus FL2-h with the FL2 marker set at FL2-h = 1 × 103 provided the number and percent of stained and unstained events. The fluorescence threshold was established previously by comparing heated to unheated propidium iodide-stained bacterial samples.Flow cytometry provides a precise measure of microbial cells and when coupled with propidium iodide staining, also permits estimation of the proportion of cells that are damaged. Our laboratory uses the Accuri C6 cytometer which features a peristaltic pump sample injection which allows accurate quantitation of events/μL, unlike pneumatically applied samples. However, we chose to limit the use of flow cytometry to those experiments where propidium iodide staining was the endpoint, experiments with Candida albicans and 12 antifungal drug-resistant strains from the ATCC MP8 were grown overnight in BHI broth. Biofilm estimates were based on 2 mL cultures prepared in 24 well plates with diluted RBG and compared to wells with BHI control medium. Cultures were held for 10 days at 37 °C, and the plates remained undisturbed; refeeding did not occur. After incubation, the contents of each well were aspirated off, washed three times with water to remove planktonic organisms, and stained with 1% safranin for 1 h at room temperature. The unbound stain was aspirated off and wells were again washed three times with water and allowed to dry. The stain remaining in the wells represents the adherent biofilm and the color was mobilized into reagent alcohol and read spectrophotometrically at 560 nm. Absorbance due to biofilm in test wells was divided by absorbance in control (BHI) wells and reported as percent of control.While a variety of sophisticated methods and apparatus are employed in biofilm studies, we intended to pursue a simple polystyrene plate method  to deterp < 0.05. Where needed, evidence of normal distribution of data was supported by measures of skew and kurtosis as well as Kolmogorov-Smirnov calculations.In addition to descriptive statistics (mean and standard deviation), comparison of means by paired or unpaired t-test were calculated using Microsoft XL. Significance was set at Gardnerella vaginalis and Candida albicans that resists the effect of dilution that would be expected on intravaginal application. Antimicrobial activity is provided by lactic acid, although our results suggest that additional components of the RBG formulation may contribute to antimicrobial activity including EDTA. Bacterial and yeast membrane integrity was compromised by exposure to RBG, and artificial biofilm generation was diminished. The findings of possible interactions among components of RBG, though limited, may be valuable in hypothesis generation for additional mechanistic or possible interactions that may occur when RBG encounters vaginal fluid. Based on demonstrated antimicrobial effects, further clinical evaluations seem appropriate, particularly to determine if RBG can restore a healthy vaginal microbiome in women affected by dysbiotic vaginal conditions and if it affects probiotic lactobacilli.RBG is a formulation of components generally recognized as safe in the marketed formulation. This research proved RBG inhibitory toward vaginal microorganisms relevant to vaginal infection, dysbiosis and prematurity. Data were provided that showed the activity of lactic acid and EDTA contribute to the antimicrobial activity. The work presented indicates a robust antimicrobial effect of RBG toward bacteria including


N = 517) and 2D (N = 845) ultrasound methods showed that 2D techniques yield significantly lower estimates of FUPR than 3D (p < 0.0001). A power law function was shown to best capture the change in FUPR with fetal age (FA) for both 2D (R2 = 0.95) to measured values of amniotic creatinine concentration (N = 664). The FUPR3D data together with creatinine levels in the fetal urine and serum resulted in median predicted fetal GFR values of 0.47, 1.2, 2.5, and 4.9 ml/min at 23, 28, 33, and 38 weeks of fetal age (50% CV), respectively. These values are in good agreement with neonatal values observed immediately at birth. The derived FUPR and creatinine functions can be utilized to assess fetal renal maturation and predict fetal renal clearance.Adequate prediction of fetal exposure of drugs excreted by the kidney requires the incorporation of time-varying renal function parameters into a pharmacokinetic model. Published data on measurements of fetal urinary production rate (FUPR) and creatinine at various gestational ages were collected and integrated for prediction of the fetal glomerular filtration rate (GFR). The predicted GFR values were then compared to neonatal values recorded at birth. Collected data for FUPR across different gestational ages using both 3D ( The kidney is the main organ responsible for urine formation and excretion of drugs from the body. The kidneys develop between the 5th and 12th week of fetal life and increase significantly during the second half of pregnancy, owing to intense nephrogenesis (nephron formation) and a significant increase in the number of nephrons , 4. The Postnatal renal function can be quantified by many different methods. However, limited options are available for assessing fetal renal function. The options available include amniotic fluid volume, ultrasonographic appearance of kidneys, urine production and the biochemical composition of fetal urine . AlthougDue to clinical and ethical constraints, few studies have reported GFR values, either from pre-clinical species –12 or frMeasuring fetal GFR has its place in pharmacokinetics (PK) for predicting the fetal renal clearance of xenobiotics and their level in the amniotic fluid. Determination of fetal GFR requires quantifications of its variables, including urine flow, and concentration of the markers in the fetus, and in the fetal urine at different gestational weeks. Since inulin cannot be injected to the fetus for measuring GFR, the use of endogenous creatinine or Cystatin C is an alternative method.The first direct measurement of fetal urinary production/flow rate was demonstrated by Campbell et al. . The meavia undertaking a thorough analysis of the published literature on FUPR and fetal creatinine concentration in the fetal serum (SerCr), urine (UrCr), and in the amniotic fluid during fetal development.The aim of this paper was to assess the fetal GFR https://pubmed.ncbi.nlm.nih.gov/) and Google Scholar (https://scholar.google.com/) in April 2021. The key words utilized in the searches include “fetal urine/urinary production”, “fetal urine/urinary flow”, “fetal glomerular filtration” and “fetal GFR”. An additional search was carried out to identify published articles with measured values of creatinine in the fetal urine, serum and amniotic compartments at different gestational ages using for example the following search terms for creatinine- “amniotic fluid constituents”, “amniotic fluid creatinine”, “fetal amniotic creatinine”, “fetal urine creatinine”, “fetal urine biochemistry”, and “fetal urinary creatinine”. References within the individual search results were also reviewed to identify any additional articles that may have been missed in the initial searches.Searches for published literature articles relating to fetal urinary flow rate were carried out using PubMed  the subjects were healthy females with no known pregnancy-related complications, (2) the pregnancy was described as normal, and were not characterized by any known fetal abnormalities, including kidney abnormalities (3) only singleton pregnancies and (4) an explicit mention of gestational age at which fetal urine production rate or creatinine concentration was measured. Control data from subjects meeting the above-mentioned criteria for studies that were conducted specifically to look at complicated pregnancies were also included. One of the initial selection criteria was to only include studies carried out in a Caucasian population. However, due to the paucity of published data where only Caucasian population were considered, as well as evidence from the data suggesting there are no significant ethnic differences , this waFrom each individual study, the mean and standard deviation (SD) for both the fetal urinary production rate and the concentration of creatinine in the amniotic fluid, urine and fetal serum at the different gestational ages in weeks (GA) were calculated. The fetal age in weeks (FA) was calculated from the GA (FA= GA-2). The weighted mean (μ) from the different studies was then calculated using Equation 1:nj is the number of observations in the jth study, xj is the mean value from the jth study and N is the total number of studies for the current fetal age.where *) was calculated using Equation 2:The weighted standard deviation .Where To quantify the fit to data of the regression analyses, a weighted mean squared error (MSE) was used, given by Equation 3:W is the total number of sampling times for the different fetal ages; ni is the total number of observations across all studies in the ith week; yi is the model predicted FUPR in the ith week, and μi is the weighted mean of the FUPR of all subjects in the ith week across all studies.Where R2) value was then calculated for each of the three functions.To identify which function best fits the data, weighted regression analyses was carried out for different growth functions including, linear, exponential, polynomial, and power law. Each function's parameters were optimized using the Microsoft built-in Excel's LINEST function. The weighted mean squared error and coefficient of determination  were summarized for the changes in the parameter values using constant coefficient of variations (CV), based on the observed data, of 30% for FUPR, 25% for UrCr, and 25% for SerCr. Due to the absence of fetal GFR measurements, data from preterm and term neonates observed immediately after birth were compared against the predicted fetal GFR using Equation 4.Thirteen different original research articles, which altogether provided over 1,300 measured values of FUPR from pregnant women across different gestational ages and met the pre-defined criteria, were identified in the literature . The FUPThe FUPR from these studies ranged from a minimum reported value of 0.5 ml/hr at 19 weeks GA , to 118.t-test assuming unequal variances, with alpha = 0.05, was carried out. Data from the 44 data points using 2D measurements  and the 20 data points using 3D measurements  from 35 to 40 GAs were significantly different (p < 0.0001).A plot of the measured FUPR in ml/min against GA (weeks) from the different studies is shown in The weighted mean for the collated data measured by 3D ultrasound indicated that the FUPR (mean ± SD) increases from 0.10 ± 0.08 ml/min at 21 GAs to 0.33 ± 0.15 ml/min at 30 GAs, reaching about 1.39 ± 0.64 ml/min at term. Since the 3D ultrasound data gives more accurate measurements of fetal bladder volume , the 3D Where FA is fetal age in weeks.The weighted mean for the collated data measured by 2D ultrasound indicated that the FUPR (mean ± SD) increases from 0.12 ± 0.1 ml/min at 21 GAs to 0.21 ± 0.11 ml/min at 30 GAs, reaching about 0.42 ± 0.19 ml/min at term. A power law function was selected among the tested functions , for thePerformance of these functions together with the data are shown in Since 2D ultrasound methods are more commonly used for fetal assessments, it would be useful to have a function that can transform 2D-derived estimates of fetal urine production rates into values that would have been obtained using the 3D technique. To this end, we derived the following analytical function by dividing the 3D model by the 2D model as shown in Equation 7.Where FUPR_2D is the estimate of FUPR as calculated using the 2D method and FUPR_3D is an estimate of fetal urine production rate that would have been obtained if the 3D technique had been employed instead. This equation can be applied as a correction function to transform 2D measurements to 3D estimates. The effect of applying this scaling function on the 2D data is shown in N = 1,319) across various gestational ages, meeting the pre-defined inclusion criteria were identified :Collected data on changes of amniotic creatinine concentration indicate a rapid increase in amniotic fluid creatinine concentration (mg/L) from a mean (SD) of 4.5 (1.1) at 10 GAs to 7.5 (1.8) at the middle of pregnancy reaching a value of 22 (5.4) at term . The lonR2 = 0.9527) between these two variables . Data from two studies , 48 wereThe term (10/88.42) is for unit conversion from μmol/L to mg/L. Since this equation was derived from fetal data between 16 and 33 GAs, an assumption was made that this equation can be used to predict fetal UrCr at term (Scenario B).n = 522), serum samples were simultaneously measured, and the maternal and fetal SerCr levels showed an equilibrium  from 16 GAs until term (R2 = 0.9543)  :Fetal SerCr = Maternal SerCr = neonatal SerCr at birth).Data were generated using this function assuming 25% CV and then compared against 4,509 SerCr measurements obtained at birth (23–42 GAs) from 1,181 newborns . Due to Predicted fetal GFR values at different gestational weeks are given in This study quantifies the longitudinal increase of FUPR with respect to fetal age and the associated changes in creatinine levels in the fetal urine and amniotic fluid in uncomplicated pregnancies. These findings have potential applications in the assessment of normal fetal GFR maturation. Different studies have previously measured FUPR as an assessment of fetal renal function and perinatal outcomes in both normal and complicated pregnancies , 22, 30.The introduction of 3D ultrasonography offers more accurate volume measurements of irregular shapes  compared to the 2D ultrasonography, including fetal bladder volume as it quantifies the volume from the three spatial dimensions. When 3D ultrasonography is coupled with the rotational multiplanar technique VOCAL, a detailed and quick acquisition of the bladder volume can be obtained with lower inter- and intra-observer variability  and more2 value indicating the best fit to the observed data, however, it predicted negative values for FUPR before the 19th fetal week  at 40 weeks GA. This value is ~3-fold less than the mean estimated value of 1.41 ml/min (0.085 L/h) at term using the FUPR3D technique. A correction function for the FUPR2D data has therefore been derived to facilitate FUPR measurement conversion at different fetal or gestational ages.The current study utilized FUPR values measured using 2D or 3D ultrasound to derive a function to predict changes of FUPR with fetal age. Previous studies derived various functions  and proptal week . Since ire small , the powAssessment of fetal renal maturation has been attempted by different methods, including the measurement of amniotic fluid volume, clearance of endogenous or exogenous filtration markers, urine production and urinary analyte levels, which can be measured in either fetal urine or in amniotic fluid , 15. TheR2 = 0.95) implying the role of fetal urinary contribution.Creatinine (MWt of 113 g/mol) is commonly used for assessing postnatal renal function. Its amniotic fluid levels increase rapidly between 8 and 16 GAs, whilst its level in maternal serum remains almost unchanged, which indicates increasing excretion by the fetus into the amniotic fluid . CollectCollected data on fetal UrCr at different fetal ages  were limn = 522), SerCr levels were measured simultaneously and it was found that both maternal and fetal SerCr level were in equilibrium from 16 GAs until term in one study , we have compared the maternal SerCr using a previously developed gestational age-dependent maternal SerCr function  with neoThe calculated fetal GFR based on FUPR, and creatinine data are given in Quantifying the maturation of fetal renal function has its place in PBPK modeling, especially for renally cleared compounds after maternal intake such as most anti-infective drugs including gentamicin, amikacin, penicillin, cephalosporin, tenofovir after maternal intake. Given the difficulty in measuring fetal GFR, quantification of an alternative method can facilitate PK assessment of drug exposure within the feto-placental unit during pregnancy. Providing a continuous equation to describe the fetal renal maturation process also increases the applicability of PBPK at different weeks of fetal age.Finally, this work has different limitations. This work quantifies the increase in the fetal renal function, based on changes in FUPR obtained from different studies carried out in different ethnic groups to construct the observed changes during fetal development. The impact of ethnicity, if it exists, was not considered due to data limitation. However, the overlap of FUPR measurements in Asians and Caucasians  suggestsAnother limitation is that some studies reported results as summary statistics, while others reported individual values. Integration of these data were performed after calculating summary statistics from individual data, which can result in an under-estimation of interindividual variability of FUPR. Fetal and maternal covariates were not available to enable inclusion of physiological covariates in the FUPR functions.Finally, it is not known to what degree the fetal FUPR is affected by maternal illness, for example maternal renal impairment. Therefore, in this work, only data from healthy pregnancies were selected. This is because the understanding of normal fetal renal physiology is a prerequisite when quantifying the impact of different disease states on fetal renal function. Some of the studies which we used in our analysis compared FUPR in healthy pregnancies with FUPR in medically complicated pregnancies, and showed that FUPR is significantly different only when the pregnancy complication affects the growth of the fetus , 30. ExaIn addition, we did not attempt to assess other renal function biomarkers or amniotic fluid composition during fetal development. The amniotic level of β2-microglobulin increases from 7.9 (1.0–24.7) mg/L at 16–17 GAs to 11.6 (3.4–14.1) mg/L at 24–27 GAs  indicatiIn conclusion, age-dependent functions for FUPR, amniotic creatinine, and fetal urine creatinine have been derived in this study by integrating data from multiple studies to predict fetal urine flow during development and can be used for assessing the fetal GFR at different fetal ages. The absence of fetal GFR measurements, due to ethical reasons, challenges the verification of the predicted values. A possible way to assess the suitability of the presented approach of predicting fetal GFR is by integrating it within a fetal PBPK model to predict fetal and amniotic exposure of renally eliminated compounds at different GAs. The fetal GFR results reflect the current knowledge and data availability. Additional studies are required to establish reference values for normal fetal urine and serum creatinine levels from sufficient subjects at different gestational weeks to build the confidence in the normal fetuses as a reference population so the revision of the fetal GFR can be continued and the impact of diseases on fetal renal function mechanisms can be quantified.The original contributions presented in the study are included in the article/UE, AP, and KA collected the data. KA, UE, AP, and AB analyzed the data. UE, AB, and KA wrote the manuscript. All authors reviewed the manuscript and contributed to the article and approved the submitted version.All authors are paid employees of Certara UK Limited (Simcyp Division) and may hold shares in Certara.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.