The pace of decline in fertility rates in sub-Saharan Africa appears to have slowed or stalled in the last few decades. This study examines the socioeconomic associated with cumulative fertility in Ghana. Negative binomial regression models were used to estimate determinants of cumulative fertility using data from the Ghana Demographic and Health Surveys of 2003, 2008, and 2014. The composition of children ever born by women did not change considerably over the study periods. Socioeconomic disparities in educational attainment, household wealth, employment, and employer status are significantly associated with cumulative fertility risks in Ghana. The current age of women, age at sexual debut, and marital status, among others, are also linked to cumulative fertility levels. Place and region of residence are also linked to fertility in Ghana. Cumulative fertility levels in Ghana are underscored by considerable socioeconomic disparities among women of reproductive age. Fertility regulation policies should hinge on improving the socio-economic wellbeing of women in Ghana. Fertility is one of the three main components of population dynamics that shape the size, structure, and composition of the human population. For the past few decades, world fertility decline has gained momentum, reaching historically low rates between 2010 and 2015 ,4. As a Similarly, in Ghana, fertility has declined slowly from 6.4 children per woman in 1988 to 4.2 children per woman in 2014 , a drop The potential effects of higher fertility on the human population may be diverse. Fertility combined with mortality and migration affects the structure of the human population and has negative implications for child and maternal health, child schooling, the demographic dividend as well as the natural environment . The sloThe data source for this research is the 2003, 2008, and 2014 waves of the Ghana Demographic and Health Surveys (GDHS). These are three consecutive surveys conducted by the Ghana Statistical Service in collaboration with the Noguchi Memorial Institute for Medical Research (NMIMR), Ghana Health Service, and ICF Macro, among others , as partCumulative fertility (number of children ever born) of women, which is a count outcome, was used as the outcome variable for this study. The main independent variables considered in this study included socioeconomic characteristics such as education , wealth status , work status , employment period  and employer status . These variables comprise the main interest of the current study by serving as the foundation of the analysis.The control variables–potential confounding factors–considered in this analysis included demographic characteristics such as age group , age at first sex , marital status , ethnicity , and religion . Spatio-temporal factors such as place of residence , region of residence as well as survey year  were as well considered for the control variables. The inclusion of these variables in the current study was mainly informed by the significant associations observed between these variables and fertility by several previous studies –25.The data processing and analysis were done with the R statistical software (version 3.5.1) . Descripi) = β0+ β1X1+ β2X2+β3X3; where the log of children born to a woman ‘i’ is equal to the intercept (β0) plus the predictor variables (β1X1+ β2X2+β3X3) to be estimated. Three negative binomial regression models were fitted to estimate the population parameters, such as the betas and confidence intervals.The negative binomial regression model was used because it includes a dispersion parameter and better accounts for the observed overdispersion in the outcome variable (See supplementary file) than the Poisson regression model . This waModel 1 was used to examine risk levels of only socioeconomic characteristics before adjusting for control variables. Model 2 estimated the significance of the socioeconomic characteristics after adjusting for the demographic characteristics of respondents. Model 3, which is the full model, examined the significance of the socioeconomic factors after adjusting for both demographic characteristics and spatio-temporal factors such as place and region of residence and the survey year. The estimated population parameters were used to calculate risk ratios and 95 percent confidence intervals for the results. The risk ratios show how much the expected number of children is multiplied compared to the reference group. Owing to their pooled nature, the data were de-normalized by multiplying the standard weight by the ratio of estimated total females ages 15–49 in the country in 2003, 2008, and 2014  to the nThis section of the paper describes the sociodemographic characteristics of the respondents as presented in Respondents from rich households comprised 46.3 percent of the total sample while respondents from poor households comprised about one-third (33.8%). Further, about 59 percent of the respondents were married or living together as partners while 31.5 percent were never married or living together with any partner. Also, the majority of the respondents were in the labor force (74.4%) whereas a quarter was not working (25.6%). Regarding residence, about half of the respondents comprised those from urban settings (50.9%). The Ashanti (19.7%) and Greater Accra (18.5%) Regions had the highest representation of the total study sample while the Upper West Region (3.2%) had the least representation.Educational attainment was found to be negatively associated with cumulative fertility with women who had primary and secondary school or higher education having 0.94 and 0.80 times cumulative fertility risk, respectively, compared to those without formal education, after taking into consideration the control factors. Likewise, cumulative fertility was found to be negatively associated with the wealth status of the household. Women from the middle-status households had 0.93 times cumulative fertility risks while women from rich households had 0.76 times cumulative fertility risks compared to their counterparts from poor households. Unemployed women had significantly lower cumulative fertility risk (RR = 0.70) compared to employed women albeit the significance waned considerably after controlling for demographic characteristics and residential factors. Employer status of respondents–whether they are self-employed or employed by someone else–was also associated with cumulative fertility. Self-employed women had at least 1.11 times cumulative fertility risk compared to women who were employed by someone else. However, the regularity or seasonality of women’s employment was not associated with cumulative fertility, after considering the control factors. These two categories of women were found to have virtually the same cumulative fertility.Additionally, some demographic characteristics have been found to have an association with cumulative fertility as control factors. Expectedly, the current age of women was positively associated with cumulative fertility even after controlling for residential characteristics in model 3. Cumulative fertility risks were 2.67, 5.31, and 7.38 times among women aged 20–29, 30–39, and 40–49, respectively, compared to women aged less than 20. For age at first sex of respondents, the results show a significantly negative association with cumulative fertility. Women who had their first sexual intercourse at ages 20–29 had 0.78 times while those who had theirs at 30 or above had 0.25 times cumulative fertility risks compared to women who had their sexual debut before age 20. Marital status, as may be expected, had a significant association with cumulative fertility as well, with married women (RR = 3.76) and previously married women (RR = 3.53) having considerably higher cumulative fertility risks compared to their counterparts who had never married or lived together with a partner. Religious affiliation was associated with cumulative fertility whereby the risk was 1.12 times among women who identified as traditionalists or spiritualists compared to Christian women. Ethnicity was associated with cumulative fertility, with women of other ethnic groups  having significantly lower cumulative fertility than Akan women even though these effects attenuated after adjusting for time and residential factors.In estimating a temporal association with cumulative fertility, the results indicate that cumulative fertility reduced steadily over time. Cumulative fertility risk was 0.96 and 0.94 times for the years 2008 and 2014, respectively, compared to 2003. For the place of residence, women from rural settings had 1.05 times cumulative fertility risk compared to urban women. At the regional level, the results show some significant disparities in cumulative fertility among the regions. Compared to the Greater Accra region, women residing in the Western (RR = 1.13), Central (RR = 1.17), Eastern (RR = 1.12), and the Ashanti (RR = 1.18), as well as the Northern region (RR = 1.13) region, had significantly higher cumulative fertility.This study mainly examined the socioeconomic factors associated with cumulative fertility levels in Ghana. The descriptive findings show that while some fluctuations exist in the fertility composition of the study sample, these do not show any considerable changes over the study period. Furthermore, the findings of the multivariate analysis indicate that cumulative fertility is associated with most of the socioeconomic characteristics of women considered in this study. The study links higher cumulative fertility to women without formal education. Bongaarts  and Lee Moreover, cumulative fertility is found to have a significant negative relationship with household wealth status. The findings show pronounced reduced cumulative fertility among women from rich households, disparate from women from poor households who have significantly higher cumulative fertility. The association between wealth and fertility has been fairly debated in the literature. However, this appears to depend on the geographical context or development status of the place under consideration. For instance, studies in developing countries show a negative association between wealth and fertility –25, wherThe association between labor force participation and cumulative fertility as well appears to be significant, but only when demographic characteristics are not considered. In effect, employed women show significantly higher cumulative fertility than their unemployed counterparts. This finding supports the findings of previous research in sub-Saharan Africa . This isAdditionally, some demographic factors have shown some significant association with cumulative fertility. The current age of a woman is associated with cumulative fertility. Thus, the cumulative fertility risk increases as women’s age increases –44. ThisThe association between marriage and cumulative fertility has long been documented as a proximate determinant of fertility by Bongaarts  and his Furthermore, this study finds a religious association with cumulative fertility among the study sample. Women who identified with the traditional or spiritual religions are more likely to give birth than Christian women. The association with religious affiliation, and for that matter, the religious disparities in cumulative fertility levels differ substantially across developing countries ,48. For Regarding ethnic disparities in fertility, the cumulative fertility level is significantly higher among Akan women than women from all the other ethnic groups in the country, including other miscellaneous ethnic groups. It is, however, unclear why Akan women may have an unexpectedly higher cumulative fertility level. Analogously, considerable ethnic disparities in fertility levels have also been observed in Nigeria  and DR CThe study as well shows some temporal reductions in cumulative fertility levels for the study period. However, the pace of reductions in-between the survey periods has not been sharp enough to warrant a substantial decline in cumulative fertility levels over time. This appears to support the widely established evidence that even though cumulative fertility has consistently reduced over the past decades globally, the pace of decline has either slowed or stalled in sub-Saharan African countries ,4,12. ThThis study, however, has a few potential limitations that should be considered in the interpretation of the findings. First, the study data are based on retrospective information and, therefore, may be subject to recall bias or reporting errors. Second, since the data are cross-sectional, many of the independent variables provided a snapshot of the scenario whereas the outcome variable comprised lifetime fertility which is affected by the lifetime experiences of women. Consequently, there may be potential reverse relationships between the outcome variables and these independent variables. Despite these limitations, the study makes an invaluable contribution to the area of cumulative fertility in Ghana.Socioeconomic characteristics–educational attainment, household wealth, employment, and employer status–appear to be significantly associated with cumulative fertility levels in Ghana. Consequently, having no formal education, coming from a poor household, being employed, and self-employed are associated with higher cumulative fertility in Ghana. Similarly, rural residence, early sexual debut, and marriage are significantly linked to higher cumulative fertility. The study provides evidence that even though cumulative fertility has steadily reduced over time, the pace of decline appears to be slow and, therefore, not substantial enough to engender sharp reductions in cumulative fertility levels in the country.Ultimately, the cumulative fertility of women can be reduced significantly by alleviating socioeconomic inequalities at the individual and household levels. Thus, individual-level policies should prioritize improving women’s educational attainment and household economic wellbeing while placing serious attention on working women as well as self-employed women. Also, comprehensive sexual and reproductive health programs can focus on delaying sexual debut or promoting abstinence among adolescents. Women residing in regions with higher cumulative fertility levels can as well be targeted for specially tailored fertility regulation programs and interventions.S1 File(DOCX)Click here for additional data file.


Aquaporin-4 (AQP4) is a predominant water channel expressed in astrocytes in the mammalian brain. AQP4 is crucial for the regulation of homeostatic water movement across the blood–brain barrier (BBB). Although the molecular mechanisms regulating AQP4 levels in the cerebral cortex under pathological conditions have been intensively investigated, those under normal physiological conditions are not fully understood. Here we demonstrate that AQP4 is selectively expressed in astrocytes in the mouse cerebral cortex during development. BMP signaling was preferentially activated in AQP4-positive astrocytes. Furthermore, activation of BMP signaling by in utero electroporation markedly increased AQP4 levels in the cerebral cortex, and inhibition of BMP signaling strongly suppressed them. These results indicate that BMP signaling alters AQP4 levels in the mouse cerebral cortex during development. In the cerebral cortex, AQP4 plays crucial roles in controlling water homeostasis. AQP4 regulates water movement across the blood–brain barrier (BBB), which restricts large polar substances and potentially neurotoxic compounds in circulation from passively diffusing into the brain5. Changes in AQP4 expression are often associated with various diseases such as epilepsy, edema and glioblastoma10. Thus, uncovering the molecular mechanisms regulating the expression of AQP4 is an important research topic. However, our understanding of the mechanisms of regulating AQP4 expression in the cerebral cortex is still rudimentary.Aquaporin-4 (AQP4) is a predominant water channel in the mammalian brain and is mainly expressed at the endfeet of astrocytes11. The induction of oxidative stress activates the p38 mitogen-activated protein kinase (MAPK) pathway, inducing transcriptional activation of AQP412. Activation of the cysteinyl leukotriene receptor 2 induces AQP4 upregulation through the p38 and ERK signaling pathways in astrocytes13. In contrast to the regulation of AQP4 expression under pathological conditions, the mechanisms regulating AQP4 expression under normal physiological conditions in the cerebral cortex are not fully understood.Previous reports have investigated the regulation of AQP4 expression in the context of inflammation in the mouse brain18. Here, we show that BMP signaling was activated in astrocytes in the cerebral cortex. Activation of BMP signaling increased AQP4 levels in astrocytes in the mouse cerebral cortex in vivo. Consistently, inhibition of BMP signaling decreased AQP4 levels in astrocytes. Taken together with a previous study showing that activation of BMP signaling increased AQP4 expression in cultured astrocytes19, these results clearly indicate that BMP signaling changes AQP4 levels in astrocytes under normal physiological conditions.Bone morphogenetic proteins (BMPs), members of the TGF-β family of signaling molecules, have numerous functions in the brain20, vascular structures were labeled with AQP4 immunostaining  , the neuron marker NeuN or the oligodendrocyte marker CC1  using cortical sections of mice at P16. Consistent with a previous reportAQP4 and pSmad were predominantly distributed in the WM, we hypothesized that BMP signaling affects AQP4 levels. To test this, we performed immunohistochemistry for pSmad and in situ hybridization for AQP4 and found that most pSmad-positive cells in the WM expressed AQP4 (92 ± 1%)    have shown that BMP family members such as BMP3 and BMP7 are expressed in the cerebral cortex during development24. Because it was reported that BMP3 does not have the ability to activate Smad1/5/8 or to induce bone and cartilage tissues in vivo, and because most BMP ligands share Smad1/5/8 as downstream mediators25, here we utilized BMP7 to activate BMP signaling. In situ hybridization for BMP7 confirmed that BMP7 mRNA was indeed produced as a result of introducing BMP7-expressing plasmids into the mouse cerebral cortex  at embryonic day 14 (E14) and examined AQP4 expression at P16. Previous studies and the Allen Brain Atlas  which lacks both transmembrane and serine/threonine kinase domains. Consistently, inhibition of BMP signaling decreased AQP4 levels in astrocytes in the cerebral cortex. Our findings indicate that BMP signaling increases AQP4 levels in the mouse cerebral cortex during development.Our experiments demonstrated that BMP signaling was activated in astrocytes which expressed AQP4. Activation of BMP signaling increased AQP4 levels in astrocytes11. For example, the exposure of astrocytes to ammonium chloride, which is an in vitro model of hepatic encephalopathy, activates the p38 MAP kinase pathway, and as a result, AQP4 expression is upregulated12. Oxygen–glucose deprivation induces AQP4 upregulation in astrocytes through the p38 MAP kinase and ERK signaling pathways13. However, little was known about the regulatory mechanisms of AQP4 levels under normal physiological conditions. A previous pioneering study using cultured astrocytes reported that BMP activation increased the expression levels of AQP4 protein in astrocytes in vitro19. Our gain- and loss-of-function experiments clearly demonstrated that endogenous BMP signaling increases AQP4 levels in the mouse cerebral cortex in vivo during development. Because a previous study reported that BMP signaling promoted astrocyte maturation, increased AQP4 expression and downregulated EGF receptor expression19, it seems possible that activation of BMP signaling induced astrocyte maturation and, as a result, AQP4 expression was increased in the cerebral cortex.Although previous studies have examined the regulatory mechanisms of AQP4 levels, most of the studies investigated these mechanisms under pathological conditions such as inflammatory responses, hepatic encephalopathy, epilepsy, brain edema and glioblastomaBMP can activate both canonical and non-canonical intracellular signaling pathways. In the canonical BMP signaling pathway, activation of BMP receptors phosphorylates Smad1/5/8, which makes a complex with Smad4. Then, the complex translocates into the nucleus and regulates gene expression. In the non-canonical BMP signaling pathway, BMP activates several molecules, which are distinct from Smad, such as p38 MAP kinase, JNK, ERK, PI3 kinase and Cdc42. Because previous studies demonstrated that p38 MAP kinase regulates AQP4 expression downstream of ammonium chloride and oxygen–glucose deprivation, it seems plausible that p38 MAP kinase mediates the effect of BMP on AQP4 levels in the cerebral cortex. In addition, because our experiments showed that Smad1/5/8 was activated in AQP4-positive astrocytes, it remains possible that the canonical BMP signaling pathway is involved in AQP4 expression. Further investigation would be necessary in order to uncover the entire picture of the mechanisms underlying the physiological regulation of AQP4 levels.28. In contrast, our experiments using BMP and noggin did not show obvious effects on the cortical layers at P16  sleep31. It seems plausible that the regulation of AQP4 expression by BMP signaling, which we uncovered in this study, is involved in the regulation of water and waste clearance and sleep quality. It would be intriguing to examine whether BMP signaling alters water and solute clearance and sleep quality through regulating AQP4 expression.The importance of AQP4 in the brain has been investigated intensively. It was reported that AQP4 has an important role in water and waste clearance in the brain. The deletion of AQP4 expression resulted in the reduction of interstitial solute clearance, and in the accumulation of amyloid β, a peptide thought to be pathogenic in Alzheimer’s disease5. Therefore, it is important to elucidate the mechanism underlying the structural and functional development of the BBB. AQP4 is one of the major functional components of the BBB and is crucial for water transport at the blood–brain interface5. Our data showed that BMP signaling is necessary and sufficient for the expression of AQP4 in vivo, raising the possibility that BMP signaling regulates the function of the BBB by modulating AQP4 expression. Interestingly, it was reported that the structure of the BBB is also regulated by BMP signaling32. Taken together, BMP signaling may regulate the BBB not only structurally but also functionally through regulating AQP4 expression.The BBB is important for brain homeostasis because the BBB restricts large polar substances and potentially neurotoxic compounds in the circulation from passively diffusing into the brainICR mice were purchased from SLC  and reared on a normal 12 h light/dark schedule. The day of conception and that of birth were counted as embryonic day 0 (E0) and postnatal day 0 (P0), respectively. All procedures were performed in accordance with protocols approved by the Animal Care Committee of Kanazawa University and with the ARRIVE guidelines.34. Briefly, pregnant ICR mice were anesthetized, and the uterine horns were exposed. Approximately 1–2 μl of DNA solution was injected into the lateral ventricle of embryos using a pulled glass micropipette. Each embryo within its uterus was placed between a tweezer-type electrode . Square electric pulses  were passed 5 times at 1 s intervals using an electroporator . Care was taken to quickly place embryos back into the abdominal cavity to avoid temperature loss. The wall and skin of the abdominal cavity were sutured, and embryos were allowed to develop normally.IUE using mice was performed as described previously with slight modifications35. Plasmids were purified using the EndoFree Plasmid Maxi Kit (QIAGEN). To activate BMP signaling, a mixture of pCAG-EGFP (0.5 mg/ml) plus either pCAG-BMP7 or pCAG control plasmid (5 mg/ml) in PBS was used. To inhibit BMP signaling, a mixture of pCAG-EGFP (0.5 mg/ml) plus either pCAG-noggin, pCAG-sBMPR or pCAG control plasmid (5 mg/ml) was used. Fast Green solution was added at a final concentration of 0.3% to monitor the injection.Noggin was kindly provided by Dr. Daisuke Saito (Kyushu University) and was inserted into pCAG vector, yielding pCAG-noggin. sBMPR was kindly provided by Dr. Naoto Ueno  and was inserted into pCAG vector, yielding pCAG-sBMPR. The cDNA for mouse BMP7 was amplified using PCR with the following primers: 5′-ATTCTCGAGGCCACCATGCACGTGCGCTCGCTGC-3′ and 5′-AAGCGGCCGCCTAGTGGCAGCCACAGG-3′. The amplified fragment was digested with XhoI and NotI, and then subcloned into pCAG vector. pCAG-EGFP was described previously37. Mice were deeply anesthetized and transcardially perfused with 4% paraformaldehyde (PFA)/PBS. Brains were dissected and post-fixed with an overnight immersion in 4% PFA/PBS, cryoprotected with 2-day immersion in 30% sucrose/PBS, and embedded in OCT compound. Coronal sections of 20–30 μm or 50 μm thickness were made using a cryostat.Tissue sections were prepared as described previously39. Briefly, free-floating sections were permeabilized with 0.3% Triton X-100/PBS, incubated overnight with primary antibodies in 2% bovine serum albumin (BSA)/PBS, and then washed with PBS 3 times for 10 min each. The sections were incubated with secondary antibodies and Hoechst 33342 in 2% BSA/PBS for 2 h, and then washed with PBS 3 times for 10 min each. The sections were mounted on slides with Mowiol (Sigma-Aldrich). Antibodies used were as follows: rabbit anti-aquaporin-4 (AQP4) antibody (Millipore), rabbit anti-glutamine synthetase (GS) antibody (Sigma-Aldrich), mouse anti-GS antibody (Roche), rabbit anti-NeuN antibody , mouse CC1 antibody , rabbit anti-phospho-Smad1/5/8 antibody , rabbit anti-Cux1 antibody (Santa Cruz), rat anti-Ctip2 antibody (Abcam), donkey secondary antibodies conjugated with Cy3 (Jackson ImmunoResearch), donkey secondary antibodies conjugated with Alexa Fluor 647 (Molecular Probes) and donkey secondary antibodies conjugated with Alexa Fluor 488 (Molecular Probes).Immunohistochemistry was performed as described previously with slight modifications41. Briefly, sections of 20–30 μm thickness were incubated overnight with digoxigenin-labeled RNA probes in hybridization buffer . The sections were then incubated with an alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche) and were visualized using NBT/BCIP as substrates. In some experiments, the sections were then subjected to immunohistochemistry and Hoechst 33342 staining.In situ hybridization was performed as described previously with slight modificationsAQP4, the cDNA fragment for mouse AQP4 was amplified with RT-PCR using the following primers: 5'-AAAGAATTCTTTTGCTCGGTGTGGGAT-3' and 5'-AAAGAATTCGAAGCAAGAAACCCGCAA-3'. The amplified fragment was digested with EcoRI and then subcloned into pCRII-TOPO vector, and digoxigenin-labeled RNA probes were made. To make an RNA probe for mouse BMP7, digoxigenin-labeled RNA probes were made using pCAG-BMP7 plasmid.To make an RNA probe for mouse Epifluorescence microscopy was performed with BZ-9000 and BZ-X710 microscopes (KEYENCE).AQP4. Images captured using a BZ-9000 microscope were used for quantification. The background signal was removed by subtracting the average signal intensity of negative cells.Coronal sections were stained with Hoechst 33342 plus either anti-GS antibody, anti-NeuN antibody, CC1 antibody, anti-AQP4 antibody or anti-phospho-Smad1/5/8 antibody. In some experiments, the sections were subjected to in situ hybridization for For cell counting, the numbers of immunopositive cells in columns with 200 μm width were manually counted using the “Cell Counter” tool of Image J software.To measure the average intensities of AQP4 immunoreactivity, the regions in columns with 200 μm width corresponding to the GM or the WM were selected using the “Rectangle Selection” function of ImageJ. Average signal intensities in the regions were measured using the “ROI Manager” tool of ImageJ. To minimize variation in signal intensity depending on the position of each section, the average intensity of AQP4 on the electroporated side and that on the contralateral non-electroporated side of the cortex in the same brain section were measured, and the former was divided by the latter. Values in graphs are mean ± SD. An unpaired two-tailed Student's t-test was used to determine statistical significance. P values<0.05 were considered statistically significant.Supplementary Information 1.


Major depressive disorder (MDD) is a common and severe mental disorder that is usually recurrent and has a high risk of suicide. This disorder manifests not only with psychological symptoms but also multiple changes throughout the body, including increased risks of obesity, diabetes, and cardiovascular disease. Peripheral markers of oxidative stress and inflammation are elevated. MDD is therefore best described as a multisystem whole-body disease. Pharmacological treatment with antidepressants usually requires several weeks before the desired effects manifest. Previous theories of depression, such as the monoamine or neurogenesis hypotheses, do not explain these characteristics well. In recent years, new mechanisms of action have been discovered for long-standing antidepressants that also shed new light on depression, including the sphingolipid system and the receptor for brain-derived neurotrophic factor (BDNF). For many clinically successful drugs, new and relevant mechanisms of action have been discovered over time. For example, we have demonstrated relevant antagonistic effects of memantine, amantadine and budipine, which until then had been considered dopaminomimetic substances, on N-methyl-D-aspartate (NMDA) receptors ,2,3. A nA receptor-positive allosteric modulatory activity, shows a rapid antidepressant effect that lasts more than a week in postpartum depression [In recent years, a number of new antidepressants with novel mechanisms of action have been developed and clinically evaluated. The inhibition of NMDA receptors by esketamine shows a rapid antidepressant effect . Howeverpression ,9. Zuranpression  and postpression . In addipression .In parallel with the development of new compounds and the exploitation of new molecular targets, important cellular and systemic mechanisms of action of antidepressants have been discovered, such as neurogenesis , neuroplThe focus of this review is relatively narrow. We discuss only novel molecular targets other than the previously described targets for the groups of licensed antidepressants , and we only consider developments during the last 10 years. The mechanisms should apply not only to individual substances but also to many antidepressants, even structurally different ones. We discuss in detail the effect of antidepressants on the sphingolipid system and the tyrosine kinase receptor 2 (TRKB) receptor for BDNF.MDD is a common mental disorder with a lifetime prevalence of approximately 10%. Patients suffer a high overall burden of disease due to severe impairment during each episode of illness, long individual episodes of illness, an often early age of onset, and an often recurrent course . MDD is The diagnostic criteria for major depression according to the DSM-5  include The group of antidepressants defined in the ATC system (N06A) includes substances with very different structures and mechanisms of action. According to the current view, antidepressants can act, for example, by modulating central monoaminergic transmission and NMDA receptors or via the melatonergic system. Antidepressants are more effective than placebos in treating major depression, and amitriptyline has the highest efficacy . AntidepThere are a number of unmet needs in the pharmacological treatment of MDD. The potential for optimization exists with regard to low remission rates and high treatment resistance, therapeutic latency, side effects and drug-drug interactions. Currently, there are limited approaches to individualizing pharmacologic therapy. The molecular mechanisms leading to depression are not sufficiently understood. As a result, important characteristics, such as the multisystem and whole-body nature of MDD, remain unexplained and are therefore not treatable in a targeted manner.MDD is treated with pharmacologic and nonpharmacologic methods; these treatments include antidepressants, sleep deprivation, physical activity, and ECT. The monoamine hypothesis of depression , which wDecreased hippocampal neurogenesis is thought to be a key element in the pathophysiology of depression ,31,32,33Sphingomyelin is cleaved by acid sphingomyelinase (ASM) to form phosphorylcholine and ceramide. ASM is found in the lysosomes of all cells; since lysosomes constantly fuse with the plasma membrane, ASM is also found on the cell surface ,40. SphiIncreased ASM activity increases the ceramide level in the cell membrane. Ceramide is very hydrophobic and aggregates spontaneously in the cell membrane into large, hydrophobic rafts , therebyIn addition to influencing the biophysical properties of the cell membrane and forming ceramide-rich rafts, sphingolipids can also interact directly with the transmembrane structures of proteins ,51. BothIntracellular ceramide levels can be increased by various stimuli that activate the de novo synthesis pathway, the ASM pathway, or both. One of the major activators of ceramide formation is oxidative stress induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS) ,53. In tThe ASM/ceramide system has been intimately linked to the generation of ROS. In particular, in macrophages and endothelial cells, it was shown that ceramide-enriched membrane domains cluster components of NADPH oxidase; this clustering is a prerequisite for the correct assembly and function of NADPH oxidase and, thus, the release of ROS ,56. CeraSince the 1970s, weak organic bases such as desipramine have been shown to inhibit ASM activity ,60,61,62The functional inhibition of ASM requires few structural conditions; molecules must contain a lipophilic part that integrates into the inner lysosomal membrane, a short spacer, and a charged tertiary amine group that displaces ASM from the inner lysosomal membrane, leading to proteolytic degradation of the enzyme in the lysosomal lumen ,65. Thuswww.drugbank.ca; accessed on 1 August 2021) and high tissue concentrations in all organs [The accumulation in lipophilic membranes and acidic intracellular compartments explains the high volume of distribution of FIASMAs. The FIASMA amitriptyline will be described in more detail as an example. Due to its high lipophilicity and weak basicity, amitriptyline highly accumulates in tissue compartments, such as lysosomes , resultil organs ,67,68.FIASMAs are cationic amphiphilic drugs (CADs) that act within acidic intracellular compartments to affect not only ASM but also the catalytic activities of other lysosomal enzymes. This can lead to the side effect of phospholipidosis (PLD), which is a deleterious increase in phospholipids in lysosomes. FIASMAs differ only slightly in their physicochemical properties, but their effects on ASM activity and the induction of lysosomal phospholipid content vary significantly. Systematic chemical modifications to the model FIASMAs imipramine, desipramine, and fluoxetine may lead to other more suitable compounds with more pronounced inhibition of ASM and less induction of PLD. Small chemical changes to FIASMAs may therefore result in compounds with better antidepressant properties and fewer side effects than the parent compounds .By inhibiting ASM, FIASMAs result in lower cellular concentrations of ceramides. Human studies have shown reduced ceramide concentrations in lung endothelial and nasal epithelial cells after treatment with 25–75 mg/day amitriptyline for 2–4 weeks ,71.There are several a priori arguments for the involvement of the ASM/ceramide system in the pathophysiology of MDD. (1) It has long been known that tricyclic antidepressant drugs such as desipramine functionally inhibit ASM. (2) This system gathers and amplifies a wide range of stress signals and thereby regulates important cellular functions . (3) TheThe systematic review by Dinoff et al. on the relationship between ceramides and depression summarizes the data through 2017. Preclinical and clinical studies have suggested the involvement of ceramides in the pathophysiology of depression, with a particularly strong association of the ceramides C18:0 and C20:0 with depression in humans . Since tThe hippocampus is one of the most interesting targets of antidepressant drugs. The hippocampus is important not only for learning and memory but also for emotional regulation. Patients suffering from depression and anxiety often show hippocampal atrophy , which iWe have shown a central role of hippocampal ceramide in depression development. Lowering hippocampal ceramide by therapeutic concentrations of the antidepressants amitriptyline and fluoxetine normalizes hippocampal neurogenesis and behavior in mouse models of stress-induced depression. Genetic ASM deficiency abrogates these effects of antidepressants. In contrast, increasing ceramide levels such as by overexpressing ASM, blocking ceramide degradation, or directly injecting C16 ceramide into the hippocampus results in reduced neurogenesis and depression-like behavior, even in the absence of stress. The antidepressant-mediated reduction in ceramide concentrations via the inhibition of ASM normalizes these effects. Thus, lowering the ceramide concentration may be a key strategy in future antidepressant development . The abiBased on the crystal structure of ASM ,86,87, dAntidepressants such as amitriptyline or fluoxetine induce hippocampal autophagy . FunctioThese results argue for a central role of autophagy in the pathophysiology of MDD and the effect of antidepressants. Environmental stress and corticosterones induce autophagy; antidepressants such as amitriptyline or fluoxetine attenuate autophagy. A direct increase in ceramide levels in the ER induces autophagy much more rapidly than amitriptyline or fluoxetine. Drugs, such as D609 or derivatives thereof, that cause the rapid induction of autophagy could be a new class of rapidly acting antidepressants. D609 has also been shown to induce ceramide synthesis , which whttp://mousebrain.org/genesearch.html; accessed on 1 August 2021). This brain regional distribution suggests an antidepressant effect for both enzymes and perhaps even an optimal antidepressant effect through the simultaneous inhibition of both enzymes.The substance D609 used in previous investigations inhibits both SMS1 and SMS2 ,97, whicCeramide generated in mitochondrial membranes has also been shown to bind microtubule-associated protein 1 light chain 3 β-lipidation, forming LC3B-II and thereby promoting mitophagy . The dysCaenorhabditis elegans and Drosophila, and autophagy-stimulating drugs, such as rapamycin, extend lifespan in rodents [The results regarding autophagy are consistent with important clinical observations. First, the typical symptomatology of MDD is compatible with a reduction in autophagy. Adult hippocampal neurogenesis is reduced under stress and in animal models of depression ,32,33,88 rodents ,107.Second, predisposing factors for MDD are also consistent with a reduction in autophagy. Genetic predisposition to MDD may be partially explained by an effect on autophagy . Older iThird, the effects of antidepressant therapies are also consistent with an autophagy-enhancing effect. Stimuli that promote autophagy counteract MDD. Many publications report an increase in autophagy with the application of antidepressants ,116. LitFourth, the therapeutic latency of antidepressants such as amitriptyline or fluoxetine is explained through the backward effects after inhibiting ASM by the slow accumulation of the precursor substance sphingomyelin in the lysosome and ceramides in the ER. Even in the case of genetic ASM deficiency, sphingomyelin accumulates only slowly in lysosomes ,135. AppIn summary, the associations between important clinical characteristics of depression and autophagy compiled here support an important role of autophagy in the development and treatment of depression.Sphingolipid metabolism offers multiple opportunities for pharmacological interventions. The limitation is that intervention at a single node in this highly regulated metabolic system can affect the entire metabolic network in potentially unexpected ways. Whenever we make even small changes within a complex system, we risk dramatic unintended consequences. In addition to the expected “forward effects”, “backward effects” must also be considered. For example, FIASMAs inhibit ASM in acidic intracellular domains and thus reduce the product ceramide as a forward effect. However, the inhibition of ASM also leads to the slow accumulation of the precursor sphingomyelin in lysosomes and Golgi membranes and of ceramide in the ER, which in turn activates an autophagy cascade . These cBDNF stimulates neuronal plasticity and induces antidepressant effects . The recAntidepressants have been used very successfully worldwide for over 60 years as safe drugs for treating depression. These drugs form a structurally diverse group of small molecular weight compounds, all of which are active in the CNS. A common feature of many antidepressants is their actions on monoaminergic transporters. However, important clinical characteristics of depression are not explained by the monoamine hypothesis, such as the latent onset of antidepressant effects, the typical diurnal variations with morning lows, the higher prevalence in women than in men, and the often observed increase in symptomatology in older individuals. Most importantly, the monoamine hypothesis does not explain the multisystem whole-body character of the disorder. In the last 10 years, new molecular targets have been discovered for long-established antidepressants. These targets are mostly associated with the effects of antidepressants on the sphingolipid system. Antidepressants accumulate as weak, lipophilic bases in acidic intracellular compartments and can displace ASM localized there from the inner lysosomal membrane. ASM then undergoes intralysosomal proteolytic digestion; thus, many antidepressants act as FIASMAs. ASM cleaves sphingomyelin to form ceramide. Therefore, the inhibition of this enzyme leads to a reduction in ceramide that has a positive effect on neurogenesis and behavior. The “backward effect” of ASM inhibition results in the slow accumulation of sphingomyelin in the lysosome and subsequently an increase in the abundance of ceramide in the ER. This effect induces autophagy, which reduces depression. Investigations of this signaling pathway have provided hints for a new group of rapidly acting antidepressants through the inhibition of SMSs. Both types of effects of antidepressants on ASM—the forward effect on neurogenesis and the backward effect on autophagy—explain important clinical characteristics of depression. A second important molecular target for antidepressants has recently been described: interference with TRKB, the receptor for BDNF. This mechanism also explains important characteristics of the disease.Of the aforementioned unmet needs in the pharmacological treatment of depression see , recent


Melanin has been a widely researched pigment by scientists for decades as it is undoubtedly the most ubiquitous and ancient pigment found in nature. Melanin plays very significant roles in structural plumage colors in birds: it has visible light-absorbing capabilities, and nanoscale structures can be formed by self-assembling melanin granules. Herein, we review recent progress on melanin-based structural coloration research. We hope that this review will provide current understanding of melanin’s structural and optical properties, natural coloration mechanisms, and biomimetic methods to implement artificial melanin-based structural colors. Most animals survive by obtaining information from colors. Colors can be used for communication between conspecifics, and colors can also be used to convey subordination signals, nutritional conditions, health quality, and even genetic conditions. Particularly, birds are representative animals that display vivid, bright, and colorful colors. Over 10,000 species of birds possess and utilize an amazing diversity of colors in their feathers   surrounding melanosomes also affect the structural colors because the effective refractive index changes with keratin and air having different refractive indices , an artificial melanin, is the most widely used material for artificially implementing structural colors. Similar to natural melanin in birds, artificial melanin’s broadband absorption of UV to visible light helps to increase color saturation of structural colors by suppressing incoherent scattering. Biomimetic structural colors have often displayed low-saturation colors due to the incoherent scattering of light. To suppress the incoherent scattering, absorbing materials such as carbon black have been used conventionally. However, artificial melanin-based structural colors do not need to add extra absorbing materials, because artificial melanin can absorb broadband light and form nanostructures. As PDA can be synthesized in various forms from thin films to core-shell particles, many researchers have used PDA to produce structural colors Kohri . Self-asAs described earlier, PDA has been synthesized in the form of thin films to core-shell particles. The most representative study in thin film-based structural coloration is shown in Fig. In this review, we broadly summarized melanin’s structural and optical properties, current understanding of melanin-based structural coloration, and fabrication methods to implement structural colors using synthetic melanin. Information on chemical structures of pheomelanin is less understood than eumelanin. Melanosomes’ shapes vary from spherical, rod, and hollow rod to hollow platelets. Melanosome morphologies may depend on the chemical structures of melanin. We also discussed the optical properties of melanin. Eumelanin and pheomelanin absorb visible light, and eumelanin absorbs UV to blue light more than pheomelanin. Eumelanin and pheomelanin’s absorption spectra do not have peaks. Their monotonic absorption spectra are unusual compared to other biological pigments. Not only melanin acts in pigment as an absorbing material of broadband visible wavelengths for coloration, but melanin granules also produce iridescent structural colors when they are arranged with high crystallinity. We introduced a few studies on the optical advantages of hollow melanosomes, and briefly addressed the need for future research on the biosynthetic mechanisms of hollow melanosomes. Although many optical applications have been implemented based on the understanding of natural melanin granules, continuous research on the natural coloration mechanisms and biological synthetic pathways will be required to open up new possibilities for more energy-efficient biomimetic applications.


Deficits in neuronal structure are consistently associated with neurodevelopmental illnesses such as autism and schizophrenia. Nonetheless, the inability to access neurons from clinical patients has limited the study of early neurostructural changes directly in patients’ cells. This obstacle has been circumvented by differentiating stem cells into neurons, although the most used methodologies are time consuming. Therefore, we recently developed a relatively rapid (~20 days) protocol for transdifferentiating human circulating monocytes into neuronal-like cells. These monocyte-derived-neuronal-like cells (MDNCs) express several genes and proteins considered neuronal markers, such as MAP-2 and PSD-95. In addition, these cells conduct electrical activity. We have also previously shown that the structure of MDNCs is comparable with that of human developing neurons (HDNs) after 5 days in culture. Moreover, the neurostructure of MDNCs responds similarly to that of HDNs when exposed to colchicine and dopamine. In this manuscript, we expanded our characterization of MDNCs to include the expression of 12 neuronal genes, including tau. Following, we compared three different tracing approaches (two semi-automated and one automated) that enable tracing using photographs of live cells. This comparison is imperative for determining which neurite tracing method is more efficient in extracting neurostructural data from MDNCs and thus allowing researchers to take advantage of the faster yield provided by these neuronal-like cells. Surprisingly, it was one of the semi-automated methods that was the fastest, consisting of tracing only the longest primary and the longest secondary neurite. This tracing technique also detected more structural deficits. The only automated method tested, Volocity, detected MDNCs but failed to trace the entire neuritic length. Other advantages and disadvantages of the three tracing approaches are also presented and discussed. Neurodevelopmental disorders such as autism and schizophrenia are relatively common alignments ,2 causedOne of the many challenges researchers face when studying neurodevelopmental disorders is that they are diagnosed once most neurodevelopmental stages have been completed. For instance, schizophrenia is diagnosed in late adolescence or early adulthood. Autism is often recognized earlier in life but still too late to study neuronal processes such as neurite formation, neuronal polarization and pruning of neuronal extensions. These neurodevelopmental processes are of particular importance, as the neuronal structure has been consistently associated with the pathophysiology of schizophrenia and autism ,11,12,13An additional challenge when ascertaining neurodevelopmental disorders is the accessibility of neurons coming directly from clinical patients. This obstacle has been circumvented by several different methods. The collection of olfactory neuroepithelial cells (ONCs) is the only approach presently available that provides access to mature neurons . It alsoWe have recently developed a methodology for transdifferentiating human circulating monocytes into neuronal-like cells in only 20 days . These mIn a prior publication, we directly compared the structure of MDNCs with that of human developing neurons (after 5 days in culture) as well as with that of differentiated human neuroblastoma cells . The strNeurite outgrowth is a key neuronal feature, and therefore characterizing neurites is important for understanding MDNCs’ neuronal properties and their application in disease and pharmacology research. Neurites are numerous, and accurate measurements through individual neurite tracing are labor-intensive. Therefore, in order to take advantage of this faster yield of neuronal-like cells, an efficient neurite tracing method is critical for extracting neurostructural data from MDNCs. The current available options can be divided into two general tracing methodologies: automated and semi-automated. Most automated alternatives are similar. They rely on software capable of detecting neurons stained with a fluorochrome. Tagged cells are automatically traced. The output of such softwares is faster than semi-automated methods, as these latter options require the researcher to select the cell to be traced and then identify the beginning and end of the neurite of interest. One of the advantages of semi-automated methods is that researchers have more flexibility when deciding which cells to trace, as they are not bound by the expression of a specific marker. Another advantage is that immunofluorescence or the expression of a fluorescent marker such as green fluorescent protein (GFP) can be avoided. These techniques are not always desirable, as both can lead to cellular damage ,35.Postmortem studies indicate that defects in the neuronal structure of patients with neurodevelopmental disorders are subtle ,11,12,13In order to avoid the use of fluorochromes and to utilize each neuronal-like cell as its own control, we decided to test three different neurite tracing approaches. First, we traced each MDNC in its entirety using a semi-automated method. Since this approach is lengthy, we also tested a simplified version in which only the longest primary neurite and longest secondary neurite were traced. Finally, the third and most intriguing alternative was to use an automated method without the addition of a fluorochrome. Automated software are triggered by brightness. Neuronal-like cells appear significantly brighter than the background when pictures are taken using light microscopy, and thus, cell recognition is expected.The first goal of this study was to compare the expression of several previously unreported neuronal markers between MDNCs, human neuroblastoma cells and THP-1 cells (a human monocytic cell line) to further validate MDNCs as neuronal-like cells. The second and main objective was to determine which of the three neurite tracing methods was faster. We hypothesized that an automated method would be faster than the other two semi-automated approaches. The third and final goal was to establish whether any of the three techniques was better at exposing neurostructural defects, with the expectation that whole-cell tracing of MDNCs would reveal the highest number of structural deficiencies, considering the thoroughness of this approach.2 flasks (13.5 million PBMCs per flask). The remaining PBMCs were used for isolation of CD14+ cells (monocytes) by positive immunomagnetic selection based on the manufacturer protocol . CD14+ cells were cultured on fibronectin-coated wells at a concentration of 180,000 cells per cm2. Plastic plates and flasks came from BD Falcon, Glendale, AZ, USA . Human fibronectin from plasma  was used at a concentration of 20 µg/mL, and coating was carried out overnight at 4 °C. Macrophage colony-stimulating factor (MCSF) from AbCys, Paris, France (300-25) was added to monocytes right before culturing at a final concentration of 50 ng/mL  at Penn State University (Study #00006911). Fresh blood was obtained from healthy individuals. We then followed our transdifferentiation protocol, as previously described . Briefly50 ng/mL A. All cePictures of cells were taken using a Nikon  Eclipse Ti-S/L 100 inverted microscope equipped with a CoolSNAP Myo, 20 MHz, 2.8 Megapixel, 4.54 × 4.54 µm pixels camera  and with a Nikon CFI Super flour 20X DIC prism objective . Pictures were taken immediately after monocyte extraction and on days 20–21 when transdifferentiation was completed B. PicturImmunofluorescence was performed as previously described  (f. BrieTM Single-Cell Autoprep System (C1) to explore genome-wide gene expression in 17 cells exposed to our transdifferentiation protocol and for THP-1 cells. We followed the manufacturer’s protocol, as previously described [Rapid mode of Illumina HiSeq 2500 was used to generate sequencing reads of sufficient depth (about 3 million of sequencing reads) per each cell. De-multiplexed sequencing reads passed the default quality filtering of the Illumina CASAVA pipeline  and were then exposed to further quality trimming/filtering using FASTX-Toolkit . The filtered reads were aligned to the most recent reference genome (hg38) using Tophat  [We utilized microfluidic single-cell capture and single-cell mRNA sequencing technologies via Fluidigm’s C1escribed . In shorMD, USA)  by allowMD, USA) . Some reMD, USA) , but theGene expression for human neuroblastoma cells was obtained from a public database generated by Li et al. .p values lower or equal to 0.05 were considered significant. The non-parametric Mann–Whitney test was used to make pairwise comparisons between MDNCs treated with colchicine versus MDNCs under control conditions. A one-way ANOVA followed by Bonferroni correction was used to make comparisons between the time it took to trace MDNCs using each of the three tracing methods tested. We compared the expression of 12 neuronal markers between (a) MDNCs; (b) SH-SY5Y cells, a human neuroblastoma cell line commonly used to study neuronal processes; and (c) THP-1 cells, a human monocytic cell line (to serve as negative control). Of the 12 neuronal markers, 7 are involved in synaptic functions, 4 are part of the neuronal structure and one is a gamma-aminobutyric acid (GABA) type A receptor . The expAfter 20 days in culture following our protocol , transdiThe principal investigator (PI), who has ample experience tracing cells, traced these four sets of MDNCs, meaning cells that were cultured under control conditions at T0 h and T1 h, as well as cells treated with colchicine at T0 h and T1 h. Since the entire neuropil of each MDNC was traced, we can report differences in the longest primary neurite (LPN), longest secondary neurite (LSN), number of primary neurites, number of secondary neurites, number of tertiary neurites and total number of neurites. MDNCs were traced using a semi-automated software called FIJI , which is a plugin for ImageJ, an open source image processing program provided by the National Institutes of Health (NIH). p = 0.0006), while there were no differences in LSN  (p = 0.48), secondary , tertiary  or total neurites pruned   for MDNCs cultured under control conditions, as well as cells treated with colchicine 0.5 µM. Then, non-parametric statistical analyses were conducted to establish whether there were differences between control (CTL) and colchicine (Colchi). Whole-cell tracing by the PI evidenced a statistically significant reduction in the percentage of LPN after treatment with colchicine 0.5 µM  B. To est = 0.27) B.Three neurite tracing approaches were tested by four individuals with research experience . One of the medical students had performed cell tracing before participating in this study, whereas all other participants had no experience in tracing. The tracers were blinded to the treatment condition each of the two groups of MDNCs had received (either CTL or Colchi), and they were unaware of that we were expecting pruning of neuronal extensions with Colchi. Participants were trained on how to use FIJI , a semi-automated software, and Volocity , an automated software. After training was completed, participants were told to use FIJI for whole-cell tracing, and they were instructed to trace all neurites present in MDNCs for this first tracing method. For the second tracing method, they were again instructed to use FIJI, but this time to trace only the longest primary and longest secondary neurite in each of the MDNCs. This second approach was named the longest neurite method (LN). For the third method, participants processed the photographs of MDNCs for each of the two treatment conditions through Volocity and then confirmed that the MDNCs had been traced. Finally, participants were asked to record the time it took them to trace MDNCs with each of the three tracing methods.p = 0.35; P2, CTL, 100 ± 5%; Colchi, 100 ± 2%; p = 0.86; P3, CTL, 100 ± 2%; Colchi, 99 ± 2%, p = 0.44; P4, CTL, 99.9 ± 2.8%; Colchi, 100 ± 4.6%; p = 0.45) (p = 0.004), while all other participants observed no differences  , CTL, 95 ± 2%; Colchi, 100 ± 3%;  = 0.45) A. Partic = 0.71) A.p = 0.04) . The same participant who found a significant retraction of LSN while tracing the entire cell again encountered retraction elicited by colchicine while tracing only longest neurites  , and one participant did not trace LSN  B. All ot= 0.001) B. Two otrace LSN B.p = 0.14; P2, CTL, 94.6 ± 9.6%; Colchi, 120.7 ± 17.4%; p = 0.18; P3, CTL, 95 ± 6.4%; Colchi, 91.7 ± 5.7%, p = 0.68; P4, CTL, 116.6 ± 18.2%; Colchi, 92.2 ± 11.8%; p = 0.27) (p = 0.05)   C. Howeve = 0.05) C. None o = 0.31) C.p < 0.00001) (p = 0.001), while tracing the entire cell was more efficient than using Volocity  (p = 0.00001).A one-way ANOVA revealed that the amount of time necessary to complete all tracings was significantly different between each of the three approaches  = 25.74, 0.00001) D. Bonfer= 0.001) D. Since We have previously shown that MDNCs conduct electrical activity and express a wide variety of neuronal markers . Here weSeveral lines of evidence strongly indicate that deficits in the neuronal structure are implicated in the pathophysiology of autism and schizophrenia ,66,67,68MDNCs’ ability to reproduce characteristics of the structure of human neurons opens the opportunity for studying these aspects of neurodevelopmental illnesses directly in living patients’ cells. This means that MDNCs provide a window into early neurodevelopmental processes in vitro, even when patients are already adults. Nonetheless, in order to maximize the delivery of neurostructural results, it is imperative to determine which neurite tracing method is more efficient in extracting data from MDNCs.Unfortunately, there is no universal tracing method that can efficiently extract neurostructural data under all research conditions. Instead, experts recommend testing several tracing approaches to determine which is the best suited for each laboratory ,70. CurrUsing the more thorough tracing approach—namely, whole-cell tracing—the PI found that colchicine elicited, as expected, a statistically significant retraction of LPN B. None oThe most surprising finding was that Volocity, the automated tracing method, was the slowest in delivering structural results D. This dThe fastest tracing approach was tracing only the longest primary and longest secondary neurites D. This aThere are other factors that need to be considered when selecting a tracing approach. One is that tracing only the longest neurites neglects other structural parameters. Additionally, Volocity is just one of the many commercially available automated software products. It is possible that other automated applications would recognize the entire structure of MDNCs, even when analyzing photographs of live cells using light microscopy. However, if automated software products become a viable alternative, cost will have to be factored in, as semi-automated methods usually do not bear any cost to the researcher, while most automated software have to be purchased ,70. In summary, selecting a tracing method is a complicated process that depends on the specific research conditions to be tested ,70. For MDNCs express a wide variety of neuronal markers that have been associated with the pathophysiology of autism and schizophrenia. Since MDNCs originate from a blood sample taken directly from patients, these cells carry the genetic susceptibility to the neurodevelopmental illness that the patients are afflicted with. In contrast with rodent neurons in culture or neuronal cell lines such as neuroblastoma cells, MDNCs allow researchers to study directly in patients’ cells early neurodevelopmental processes involving changes in neuronal structure. In order to maximize efficiency in studying MDNCs’ structure, the best approach is to only trace the longest primary neurite and the longest secondary neurite using FIJI, a semi-automated software made available by the NIH.


The aim of study was to evaluate the single oral dose and 28 day repeated oral administration toxicity profile of the synthetic compound Gαq-RGS2 signaling inhibitor, (1-(5-chloro-2-hydroxyphenyl)-3-(4-(trifluoromethyl)phenyl)-1 H-1,2,4-triazol-5(4 H)-one) as per OECD guideline 425 (2008a) and 407 (2008b), respectively.In acute toxicity study, a single oral dose administration of Gαq-RGS2 signaling inhibitor did not show any mortality at doses of 5, 50, 300 and 2000 mg/kg within 24 h and 14 days. The treatment of Gαq-RGS2 signaling inhibitor at dose 10 and 100 mg/kg for 28 days did not show any mortality, significant changes in the increase of body weight, various organ damage markers, hematological parameters, relative organ/body weight ratio and microscopic anatomical texture of essential organs as compared to vehicle and normal control.A single oral administration of Gαq-RGS2 signaling inhibitor up to dose of 2000 mg/kg in mice and repeated administration of Gαq-RGS2 signaling inhibitor at higher dose 100 mg/kg for 28 days in the rats is safe.The online version contains supplementary material available at 10.1186/s42826-021-00093-1. The major barrier for drug failure in the late stage of drug development process is toxicity and adverse effects. A troglitazone was withdrawn from the market due to identification of the unexpected liver toxicity as adverse effect in the later phase of drug surveillance phase . Therefoin-vitro and in-vivo preclinical studies [The synthetic compound Gαq-RGS2 signaling inhibitor, (1-(5-chloro-2-hydroxyphenyl)-3-(4-(trifluoromethyl)phenyl)-1 H-1,2,4-triazol-5(4 H)-one) is a triazolone ring containing compound [ studies . It has  studies . An incr studies . Thus, RA single administration of Gαq-RGS2 signaling inhibitor did not show the any mortality up to dose of 2000 mg/kg within 24 h and 14 days. None of the animals showed the alteration in the muscle activity, reflex activity and secretory during the observation period (14 days) after administration of Gαq-RGS2 signaling inhibitor .The repeated administration of vehicle (1 % v/v DMSO) and Gαq-RGS2 signaling inhibitor at doses of 10 and 100 mg/kg/d for 28 days did not show the any mortality and clinical abnormalities as compared to normal control. The increase in the body weight of rats treated with vehicle and Gαq-RGS2 signaling inhibitor did not show the significant difference as compared to normal control at weekly interval on 7th, 14th, 21st and 28th day  and Gαq-RGS2 signaling inhibitor at doses of 10 and 100 mg/kg/d for 28 days did not show significant changes in the hematological parameters such as Red blood cells (RBC) count, total and differential white blood cells (WBC) count and hemoglobin (Hb) content as compared to normal control Table , liver dThe current study showed the acute and subacute toxicity profile of the Gαq-RGS2 signaling inhibitor in the rodents. In acute oral dose toxicity study, a single oral administration of Gαq-RGS2 signaling inhibitor at the dose up to 2000 mg/kg in the female mice did not show the mortality and adverse symptoms. As per Hodge and Sterner Scale, Gαq-RGS2 signaling inhibitor is fall under the slightly toxic category .The OECD guideline 407 was followed for 28 days repeated oral dose toxicity study in the rats. The two doses 10 and 100 mg/kg of Gαq-RGS2 signaling inhibitor were used in the subacute toxicity study. These doses in subacute toxicity were selected from the effective dose (10 mg/kg) of Gαq-RGS2 signaling inhibitor against the cardiac arrhythmia in the rats in our previous study . TherefoThe Gαq-RGS2 signaling inhibitor did not produce any mortality on single oral administration up to dose of 2000 mg/kg in the mice. The treatment of Gαq-RGS2 signaling inhibitor at doses of 10 and 100 mg/kg in the rats for 28 days did not produce any lethality and abnormalities on metabolic and hematological parameters, various organ damage markers and essential organs.A permission to conduct the experiment  was taken from Institutional Animal Ethics Committee (IAEC). A guideline of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) was followed throughout the experiment. Albino mice (20–25 g) and Sprague-Dawley rats (200–250 g) were obtained at 1 week prior the starting of study from Zydus research center (ZRC), Ahmedabad, India. Animals were free access to standard pellet diet and filtered tap water. They were maintained at 22 ± 1 °C, 55 ± 5 % relative humidity and 12-hr light-dark cycle in the animal house facility of institute.Gαq-RGS2 signaling inhibitor (1-(5-chloro-2-hydroxyphenyl)-3-(4-(trifluoromethyl)phenyl)-1 H-1,2,4-triazol-5(4 H)-one) was synthesized and purified in our laboratory according to reported data [ad libitum) prior to dosing and food was restricted further 4 h after dosing. The mortality and clinical observations such as muscle activity , reflex activity  and secretory activity  were observed at 1 h, 2 h, 4 h, and 24 h after dosing and subsequentialy once daily for 14 days [A single oral administration of Gαq-RGS2 signaling inhibitor was performed in the albino mice as per the OECD guideline 425 . The Gαq-RGS2 signaling inhibitor was dissolved in the absolute DMSO and desired concentration of drug and DMSO (1 % v/v) was achieved with distilled water. In the control group, mice were treated with vehicle . The Gαq-RGS2 signaling inhibitor was administered subsequentially in the ascending order of dose 5, 50, 300 and 2000 mg/kg in the respective group. The Gαq-RGS2 signaling inhibitor was administered in the next group after 48 h survival of animals. The mice were fasted overnight . The rats were randomly divided into four groups; normal control, vehicle control and two treatment groups (10 and 100 mg/kg treated). Normal control group was treated with saline . Vehicle control group was treated with DMSO . The Gαq-RGS2 signaling inhibitor was administered at the doses of 10 and 100 mg/kg/d, for 28 days in the individual treatment group, respectively. These two doses were selected on basis of our All the animals were examined once daily for clinical signs, mortality and morbidity throughout the study. Body weights of rats were measured on day 1 before dosing followed by once a week during study. At the end of study, various organ damage markers, hematological parameters, relative organ/body weight ratio and histological examination of essential organs were performed .Blood was collected from retro-orbital plexus of anesthetized rats. It was kept at room temperature for 30 min, centrifuged at 1000 g for 15 min, and serum was isolated by aspiration. Various biochemical parameters such as liver damage markers  and metabolic abnormalities , cardiac damage markers , kidney damage markers  were determined using spectrophotometer based kits  .The blood was collected through heart puncture under ketamine (100 mg/ml)/xylazine (20 mg/ml) anesthesia in the EDTA coated tubes. The hematological parameters including hemoglobin concentration, erythrocytes, total and differential WBC  and thrombocytes count were measured in the collected blood using Auto cell analyzer  .Rats were sacrificed and essential organs heart, liver, kidney, brain and lungs were isolated. A weight of each organ was measured using weighing balance. An organ-to-final-body-weight ratio was calculated for each organ . All theP < 0.05 was considered as statistically significant.Data were expressed as Mean ± SEM. Statistical analysis was performed by one way analysis of variance (ANOVA), followed by Bonferoni’s multiple comparison test using Graph pad prism 5.0 software. Additional file 1:Table 1. Effect of single oral dose treatment of Gαq-RGS2 signaling inhibitor on mortality. Table 2.Effect of single oral dose treatment of Gαq-RGS2 signaling inhibitor on locomotion. Table 3. Effect of single oral dose treatment of Gαq-RGS2 signaling inhibitor on muscle co-ordination. Table 4. Effect of single oral dose treatment of Gαq-RGS2 signaling inhibitor on catatonia. Table 5. Effect of single oral dose treatment of Gαq-RGS2 signaling inhibitor on convulsive episode. Table 6. Effect of single oral dose treatment of Gαq-RGS2 signaling inhibitor on visual place response. Table 7. Effect of single oral dose treatment of Gαq-RGS2 signaling inhibitor on writhing response. Table 8. Effect of single oral dose treatment of Gαq-RGS2 signaling inhibitor on tail pinch response. Table 9. Effect of single oral dose treatment of Gαq-RGS2 signaling inhibitor on piloerection. Table 10. Effect of single oral dose treatment of Gαq-RGS2 signaling inhibitor on lacrimation. Table 11. Effect of single oral dose treatment of Gαq-RGS2 signaling inhibitor on salivation. Table 12. Effect of single oral dose treatment of Gαq-RGS2 signaling inhibitor on sniffing. Table 13. Effect of single oral dose treatment of Gαq-RGS2 signaling inhibitor on defecation.


Palmoplantar hyperkeratosis is a cutaneous manifestation that had not been clearly associated with infection by the human T-cell lymphotropic virus, which is a retrovirus that in most cases does not develop clinical pathologies and its symptoms may be undetected. The skin is one of the most affected organs, however until now only seborrheic dermatitis, xerosis/ichthyosis and infective dermatitis associated with HTLV-1 have been described as cutaneous clinical manifestations of this disease.Strongyloides stercoralis, and in whom a physical examination revealed an association with generalized xerosis and palmoplantar keratoderma confirmed by skin biopsy. Other infectious etiologies and malignancy were ruled out. This clinical manifestation was managed with dermal hydration, and skin care which improved the thickened skin and make it less noticeable.We present the case of a 36-year-old male patient with serologically documented HTLV-1 infection, who presented symptoms of diarrhea, malabsorption due to According to our experience, this is the first reported case of palmoplantar keratoderma associated with a human lymphotropic virus infection. This is a skin manifestation that has not been confirmed in conjunction with HTLV-I before. This implies that palmoplantar keratoderma is a new clinical manifestation of this infection, that should be considered in the initial approach of patients in endemic areas with these dermatological characteristics. Human T-cell Lymphotropic Virus (HTLV) is a retrovirus first isolated in 1980 from a T lymphocyte in a patient with cutaneous lymphoma (HTLV-1), after that, HTLV-2 was identified in cells from a patient with hairy cell leukemia –6. HTLV-HTLV-1 infection is endemic in southern Japan, the Caribbean, Central and South America, some regions of Africa, also Australia and the Pacific . The devPalmoplantar keratodermas (PPK) are a heterogeneous group of keratinization disorders with hyperkeratotic thickening of palms and soles. The etiology can be hereditary, caused by mutations in genes that encode proteins that are components of the intracellular cytoskeleton such as keratin or that participate in intercellular adhesion, cell-to-cell communication  and cell signaling. The other group of PPK are acquiredes which are secondary to toxic, infectious diseases and malignant neoplasms including esophageal cancer, Hodgkin’s disease, prolymphocytic leukemia, renal, mammary, pancreatic, colonic adenocarcinomas and malignant melanoma .Strongyloides stercoralis infection.We present the case of a patient with serologically documented HTLV infection associated with acquired palmoplantar keratoderma transgrediens, a cutaneous manifestation of HTLV not clearly described in the literature, also in the context of an adult patient with malabsorptive diarrhea and A 36-year-old male patient, from Valle del Cauca, Colombia, incarcerated in a penitentiary center, without medical or family history, who was admitted to the emergency department due to a 3-month clinical course consisting of liquid stool, approximately 7 episodes daily, associated with multiple emetic episodes of food content and weight loss. In addition, patient reported an 8-month history of palmoplantar hyperkeratosis with onychrygriphosis and generalized cutaneous xerosis Fig. , withoutStrongyloides stercoralis, so treatment with ivermectin was started (200 mcg/kg/dose), with the consequent decrease in the frequency and volume of bowel movements.Among the laboratory studies carried out, the presence of positive serology for HTLV-1 and other negative infectious studies stands out. To rule out malignancy, bone marrow studies were performed, including biopsy, which had no evidence of malignancy  but never with HTLV . ParticuStrongyloides stercolaris is known. The available evidence suggest HTLV-1 infection is more frequently associated (up to 2.4 times more) with Strongyloides stercolaris infection [Strongyloides hyperinfection syndrome, is a severe form of strongyloidiasis, has been described in patients with drug immunosuppression or secondary to malignancy, but also in cases of HTLV-1 infection [Furthermore, the association between HTLV-1 infection and nfection , 22. Simnfection , a view nfection . AlthougAccording to that, the evaluation of this coinfection and this cutaneous manifestation not previously described in relation to HTLV-1 infection, is important in order to carry out an adequate evaluation and follow-up, monitoring the possible progression of the disease and the development of complications to short and long term in an endemic region.


C-X-C motif chemokine ligand 1 (CXCL1) was upregulated in renal tubules from mice subjected to uric acid (UA)-induced nephropathy. Given that CXCL1 is a master chemokine responsible for the recruitment of macrophage by binding with its receptor C-X-C motif chemokine receptor 2 (CXCR2), we thus hypothesized that UA-induced cardiac injury is via promoting the recruitment of CXCR2 + macrophages into the heart, which enhances cardiac inflammation. Within a mouse model of UA injection , we measured the level of cardiac CXCL1. We also tested the efficacy of the CXCR2 antagonist on UA-induced cardiac inflammation and remodeling. We found a high plasma level of UA-induced upregulation of CXCL1 in heart tissues. CXCR2 antagonist relieved UA-induced cardiac hypertrophy and suppressed cardiac inflammation and fibrosis. The silencing of CXCR2 in human monocytes abolished the migration of UA-induced monocyte. Thus, the interventions against CXCL1/CXCR2 may be effective for the prevention and treatment of UA-induced cardiac hypertrophy and inflammatory responses.Hyperuricemia-induced cardiac remodeling is at least in part  Uric acid (UA) is the end product of purine metabolism and contributes to the formation of gout . Patientvia binding with surface receptors on inflammatory cells, which are designated as C-C motif chemokine receptor (CCR), C-X-C motif chemokine receptor (CXCR), C-X3-C motif chemokine receptor (CX3CR), and XCR chemokine receptor (XCR). CXCL1 is a master factor responsible for the recruitment of macrophages  guidelines and carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals . Mice were treated with the CXCR2 antagonist , which was administered intraperitoneally 1 day before UA injection for 14 days.Hyperuricemia was induced by UA injection in Kunming (KM)-background mice . UA  was injected intraperitoneally twice daily for 14 days. The control group only received 0.5% carboxymethyl cellulose sodium (CMC-Na)  injection for 14 days . ExperimEchocardiography was performed on mice with a 30 MHz probe . Left ventricular (LV) dimensions at end-diastole and end-systole (LVEDd and LVEDs) and anterior wall (Awd/Aws) and posterior wall (Pwd/Pws) thicknesses of end-diastole and end-systole were analyzed. Ejection fraction (EF%) and fractional shortening (FS%) were calculated. Measurements were taken from five consecutive cardiac cycles, and the average was used.Blood pressure was measured by the tail-cuff method every 3 days after UA injection.Mice were anesthetized by 4% chloral hydrate, and blood samples were collected from orbital veins at different time points after UA or vehicle injection. Plasma levels of UA were measured by the Uric Acid Assay Kit  according to the instructions of the manufacturer.Immunohistochemistry (IHC) was used to determine the contents of CXCL1 or CXCR2 in the LV sections. Briefly, antigen retrieval was conducted by immerging slides in the citrate-ethylenediaminetetraacetic acid (EDTA) buffer and heated in a microwave oven for 5 min. About 10% goat serum was used to block non-specific staining. After blocking, 50 μl of diluted primary antibodies  was applied onto each section at 4°c overnight. After incubation with biotinylated goat-anti-rabbit secondary antibody  for 1 h, sections were incubated with diaminobenzidine (DAB) for 3 min. Sections were counterstained with Mayer’s hematoxylin for 2 min, then dehydrated, then and mounted with DePex.Briefly, LVs were minced into multiple small pieces, digested in an enzyme mixture (0.04% collagenase type I and 0.25% trypsin) for 10 min at 37°C, and then stopped by adding 2% fetal bovine serum (FBS). The single cell suspension was resuspended in 100 μl phosphate-buffered saline (PBS) and then incubated with peridinin chlorophyll protein complex (PerCP)-conjugated anti-mouse CD45 antibody , BV421-conjugated anti-mouse CXCR2 antibody , phycoerythrin (PE)-conjugated anti-mouse CD163 antibody , PE-Cy7-conjugated anti-mouse CD86 antibody , fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD68 antibody , and allophycocyanin (APC)-conjugated anti-mouse CD11b  for 30 min in the dark. After staining, samples were analyzed with the FACSCanto II flow cytometer.Alexa Fluor 488 conjugated with wheat germ agglutinin (WGA)  was used to identify individual cardiomyocytes. After 2-h incubation at room temperature, images of five visual fields for each section were acquired across the whole LV with fluorescence microscopy . The cross-sectional area of cardiomyocytes was measured.Hearts were collected for gene expression of inflammatory mediators by real-time PCR. The primers used were MCP-1: 5′-CAC TCA CCT GCT GCT ACT CA-3′, 5′-GCT TGG TGA CAA AAA CTA CAG C-3′; IL-8: 5′-CAA GGC TGG TCC ATG CTC C-3′, 5′-TGC TAT CAC TTC CTT TCT GTT GC-3′; CXCL1: 5′-ACT GCA CCC AAA CCG AAG TC-3′, 5′-TGG GGA CAC CTT TTA GCA TCT T-3′; ANF: 5′-CGT CTT GGC CTT TTG GCT TC-3′, 5′-GGT GGT CTA GCA GGT TCT TGA AA-3′; BNP: 5′-GCC ATG TGA GAG TCA GCA AAC-3′, 5′-GTG AGG CCT TGG TCC TTC AA-3′; MHC: 5′-CCT GCG GAA GTC TGA GAA GG-3′, 5′-CTC GGG ACA CGA TCT TGG C-3′; α-SMA: 5′-CCC AGA CAT CAG GGA GTA ATG G-3′, 5′-TCT ATC GGA TAC TTC AGC GTC A-3′; Collagen I: 5′-GCT CCT CTT AGG GGC CAC T-3′, 5′-ATT GGG GAC CCT TAG GCC AT-3′; Collagen III: 5′-TGA CTG TCC CAC GTA AGC AC-3′, 5′-GAG GGC CAT AGC TGA ACT GA-3′; GAPDH: 5′-CAG GAG AGT GTT TCC TCG TCC-3′, 5′-TTT GCC GTG AGT GGA GTC AT-3′. The cycling conditions consisted an initial, single cycle of 5 min at 95°C, followed by 30 cycles of 30 s at 95°C, 30 s at 54°C, and 15 s at 72°C. The gene expression levels were quantified relative to the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).Hearts were collected from anesthetized mice and then fixed in phosphate-buffered 4% formalin for 24 h, followed by 30% of sucrose for another 24 h. Tissues were embedded in paraffin. Heart histology and fibrosis were stained by H&E and Masson’s trichrome as described earlier. Images were viewed and captured using the microscope .Hearts from neonatal rats (2–3 days) were isolated and cut into small pieces and then digested in trypsin (Gibco) for 30 min at 37°C. The cell suspension was centrifuged at 1,000 rpm for 5 min and then cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS. After the attachment of fibroblasts (about 2 h), the remaining unattached cells (cardiomyocytes) were collected and then centrifuged at 1,000 rpm for 5 min. The cell pellet was resuspended in DMEM supplemented with 10% FBS.7/ml) were resuspended in Roswell Park Memorial Institute Medium (RPMI 1640) supplemented with 10% FBS (Gibco), penicillin 100 IU/ml, and streptomycin 100 g/ml at 37°C with 5% carbon dioxide (CO2). The adenovirus-based small interfering RNA (siRNA) vectors were used for silencing of CXCR2 in monocytes. Neonatal cardiomyocytes were seeded to the lower chamber for 24 h. Cardiomyocytes were stimulated with UA (9 mg/ml) or CMC-Na-saline for another 24 h. About 5 × 104 cells, with or without CXCR2 knockdown, in serum-free RPMI solution (100 μl) were added to the upper chamber of a transwell chamber (Corning). After 12 h, transwell membranes were stained by 4′,6-diamidino-2-phenylindole (DAPI) to detect nuclei. Monocytes accumulating on the lower side of the transwell membrane were acquired by microscopy.Blood samples (30 ml) were collected from healthy volunteers. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque Plus  according to the instructions of the manufacturer. Monocytes were further sorted by Aria II flow cytometer. Monocytes  staining, and expression level of α-SMA, collagen I, and collagen III, was attenuated by the CXCR2 antagonist . H&E starophages . These erophages . Althougmulation , the folTo test whether UA could directly trigger monocyte migration by activating CXCR2, neonatal cardiomyocytes were cultured in the lower chamber of the transwell and then were stimulated by vehicle or UA for 24 h. Monocytes with or without CXCR2 knockdown were loaded to the upper chamber of the transwell. Compared with the vehicle control group, cardiomyocytes with UA stimulation augmented monocyte migration . Such efIn this study, several novel findings have been made. (1) Mice with UA injection upregulated CXCL1 protein levels in hearts; (2) CXCR2 antagonist preserved UA-induced cardiac hypertrophy and suppressed cardiac inflammation and fibrosis; (3) silencing of CXCR2 in human monocytes abolished UA-induced monocyte migration. Thus, the interventions against CXCL1/CXCR2 may be effective for the prevention and treatment of UA-induced cardiac hypertrophy and inflammatory responses.via a pressure-independent pathway.Clinical and experimental studies reported that high plasma levels of UA induced hypertension and further led to endothelial dysfunction . Clinica+ monocytes to the local sites  mic+ macrophages, leading to ventricular hypertrophy. Therefore, inhibition of CXCL1 or CXCR2 would be a potential therapeutic approach against UA-induced heart injury.A high level of UA is able to induce upregulation of chemokine CXCL1 in the heart. Elevated CXCL1 is responsible for attracting CXCR2The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Dalian Medical University Human Research Ethics Committee. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by The Animal Care and Use Committee of Dalian Medical University.HW and XK designed the study and wrote the manuscript. MX, XZ, DW, XF, YX, and YL performed the experiments and data analysis. All authors contributed to the revision of the manuscript and approved the final version.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.


Increased demand for in vitro fertilization (IVF) due to socio-demographic trends, and supply facilitated by new technologies, converged to transform the way a substantial proportion of humans reproduce. The purpose of this article is to describe the societal and demographic trends driving increased worldwide demand for IVF, as well as to provide an overview of emerging technologies that promise to greatly expand IVF utilization and lower its cost. Since its clinical introduction in 1978, in vitro fertilization (IVF) has redefined the ability of the human species to procreate. Initially developed to aid the infertile couple, clinical indications for IVF have since rapidly expanded to include medical and genetic conditions, as well as fertility preservation. While IVF access and utilization vary widely globally, the practice now accounts for the conception of over 5% of all newborns in some European countries where IVF is more affordable and/or is covered by insurance , 3. InfeChanging demographics and societal norms are driving increased IVF utilization. Improved access of women to educational and career opportunities, as well as effective contraception has contributed to progressively delayed childbearing and overall lower fertility rates worldwide. In many countries and in virtually all US states, fertility rates are now substantially below population replacement levels of 2100 births per 1000 women. In a growing number of metropolitan areas as well as in entire highly developed countries, the average age at first birth now exceeds 30 years, that is, well beyond peak fertility which occurs in the mid 20s. Inadvertently, a growing proportion of women is delaying childbearing to a point where age-related fertility decline contributes to the prevalence of infertility and to increased demand for fertility treatments including IVF and oocyte cryopreservation. These trends will likely accelerate due to the socio-economic impact of the COVID-19 pandemic, which has forestalled new family formation. Indeed, preliminary data from Chinese cities indicate that birth rates declined between 9 and 32.6% in the second half of 2020 compared with 2019, reflecting effects of the COVID-19 lockdowns . Declinifertility is a basic human right is just starting to gain traction and is sure to accelerate wider adoption of such policies [The utilization of IVF is closely linked to its affordability and accessibility . Indeed,policies . AnotherThe distribution of established fertility clinics thus closely corresponds to affluent metropolitan areas with the lowest fertility rates and the most advanced maternal ages at birth. Conversely, less densely populated and less affluent areas are characterized by relatively poor IVF access. Moreover, racial and ethnic disparities in the utilization of IVF, largely due to socio-economic factors, are inversely correlated with fertility rates . An addiThe IVF process is complex and stressful, it consists of multiple steps which can take up to several months to complete. The main reasons patient prematurely drop-out of IVF prior to achieving a pregnancy are the financial, physical, and psychological burdens of the treatment regimen . Here, wControlled ovarian hyperstimulation (COH) is performed to increase the number of oocytes available for IVF. COH involves multiple injections of gonadotropins and serial visits to the fertility clinic for the conduct of transvaginal ultrasound evaluations and the measurement of circulating hormone levels. It follows that COH is complex, time sensitive, and intensive. Various strategies intent on reducing the number of injections by utilizing long-acting gonadotropins or oral medications are already available and are gaining increased acceptance in the field for the treatment of select patient populations , 11. Simidentification and separation of sperm and oocytesfertilizationembryo cultureembryo selection for transfercryopreservation of surplus embryos and gametesPerhaps the most promising technological development that might democratize IVF access in the near-term is the automation and miniaturization of the IVF laboratory. Building, staffing, and manually operating an IVF laboratory account for much of the high cost, maldistribution in access, and variability of outcomes. The basic steps in the IVF laboratory include:Great progress has already been made towards the automation of these individual steps by way of new technologies. Still, the IVF process in its entirety remains highly manual. The altogether novel IVF lab-on-a-chip concept has the potential to revolutionize IVF by enabling the automation of virtually all of the steps involved in a single system –18.Microfluidics is defined as a multidisciplinary field of study and design whereby fluid behaviors are accurately controlled and manipulated with small scale geometric constraints that yields dominance of surface forces over volumetric counterparts. Past procedures in the IVF laboratory, though successful, apply a macroscale approaches to microscale cellular biological events . IntegraMicrofluidic sperm-sorting devices –21 and aMicrofluidic in vitro insemination has been demonstrated , whereasEmbryo culture has already been fully automated with use of time-lapse incubators which allow continuous monitoring of embryo development. Data generated from time-lapse incubators can be analyzed with machine learning to aid in the selection of embryos with the highest pregnancy potential –38. AddiFinally, cryopreservation of sperm, oocytes, and embryos has become the standard of care. Vitrification has become the dominant method for oocyte and embryo cryopreservation. While semi-automated/automated systems for oocyte/embryo vitrification have been reported and are now in early stages of clinical adoption –56, thesFigure Fertility preservation research has steadily increased our understanding of the mechanisms that govern folliculogenesis . The devPerhaps the most revolutionary concept in modern reproductive science is that of in vitro gametogenesis (IVG). IVG comprises various approaches, including organ culture systems, embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), and spermatogonial stem cells (SSCs). Several of these approaches led to the creation of functional gametes in rodent models . JapanesThe existence of human oogonial stem cells (OSCs) capable of giving rise to new oocytes has been an area of some controversy for nearly a decade. Reports to the effect that cells isolated from human ovarian tissue using fluorescence-activated cell sorting and an antibody against the DDX4 protein constituted OSCs challenged the long-standing dogma that the ovarian reserve is finite , 72. MulSSCs constitute the progenitor cells in the process of spermatogenesis. As such, these cells are the focus of in vitro spermatogenesis (IVS) and in vivo restoration of male fertility. While IVS has been achieved in rodent models, it has proven far more difficult to realize in primate counterparts . One recThe convergence of IVF with reproductive genetics has been at the forefront of the field for the past few decades. The development of next generation sequencing has expedited the adoption of PGT of embryos with an eye toward detecting the presence of chromosomal abnormalities. Moreover, increased use of carrier screening of infertile couples has increased the use of PGT for monogenic diseases. As cost of carrier screening decreases and the number of detected mutations expands, a substantial new population of patients identified as carriers may pursue IVF with PGT to build their families. Indeed, population genomic screening of young adults may offer significant healthcare savings through the prevention of rare disorders and cancers . Future The growing utilization of IVF will transform the way a substantial proportion of the human species procreates. It is likely that in the near future, as many as 10% of all children will be conceived through IVF in many parts of the world. Given the rapid scientific and technological evolution of IVG and of reproductive genetics, it is imperative that both the public and regulatory bodies be engaged in establishing a framework for the ethical evaluation of emerging technologies –84. Such