To summarize, categorize, and describe published research on how birthing room design influences maternal and neonate physical and emotional outcomes.The physical healthcare environment has significant effects on health and well-being. Research indicates that birthing environments can impact women during labor and birth. However, summaries of the effects of different environments around birth are scarce.We conducted a systematic review, searching 10 databases in 2016 and 2017 for published research from their inception dates, on how birthing room design influences maternal and neonate physical and emotional outcomes, using a protocol agreed a priori. The quality of selected studies was assessed, and data were extracted independently by pairs of authors and described in a narrative analysis.In total, 3,373 records were identified and screened by title and abstract; 2,063 were excluded and the full text of 278 assessed for analysis. Another 241 were excluded, leaving 15 articles presenting qualitative and quantitative data from six different countries on four continents. The results of the analysis reveal four prominent physical themes in birthing rooms that positively influence on maternal and neonate physical and emotional outcomes: (1) means of distraction, comfort, and relaxation; (2) raising the birthing room temperature; (3) features of familiarity; and (4) diminishing a technocratic environment.The evidence on how birthing environments affect outcomes of labor and birth is incomplete. There is a crucial need for more research in this field. To give birth is a central life event that women will remember and be affected by throughout their lives . It is aA birthing woman needs both high-quality care that minimize risks of complications and a familiar, calm, safe, and secure environment so her hormonal system can function optimally and facilitate a physiological and healthy labour and birth . The birA birthing woman needs both high-quality care that minimize risks of complications and a familiar, calm, safe, and secure environment so her hormonal system can function optimally and facilitate a physiological and healthy labour and birth.We know that the physical healthcare environment has significant effects on health and well-being. Physical aspects such as good ventilation, windows, conditions that promote orientation and distraction, a view of or access to nature, real or artificial, and ergonomic furniture can all have positive health effects . HoweverToday, most women give birth in hospitals with a mostly unfamiliar medically equipped environment, sometimes noisy, and brightly lit . Such enThe effect of healthcare environment on labor and birth has been only sparingly studied. A survey of 2,000 new mothers in Britain indicated that the physical environment could have both positive and negative effects on childbirth. Aspects of the birthing room identified as important were as follows: a nonclinical impression, a nicely decorated space, soundproofing so that people outside could not hear, possibility to control who had access to the room, possibility to control the temperature and the light, comfortable pillows and seating, rugs on the floors, and a private toilet, bath, and shower .We have only found one scoping review on whether the physical birthing environment impacts interventions during labor and how this might occur . The relThe effects of different models of care during labor and birth have been scientifically explored , demonstTo summarize, the physical healthcare environment has significant effects on patients’ health and well-being, but the influence of the physical environment on labor and birth has not been well-studied. There is only one review on how birthing environments may indirectly impact interventions during labor. The direct effects of physical birthing environments on health outcomes in women and neonates have not been systematically summarized. We wanted to explore the research field further, by doing a wide  and deep (limited to birthing room design) search on the basis of the following research question: What does the literature say regarding the influence that birthing room design has on maternal and neonate physical and emotional outcomes? We therefore conducted a systematic review to summarize, categorize, and describe published research on how birthing room design influences maternal and neonate physical and emotional outcomes. This review will include all studies that focus on aspects of the birthing room that have been explored and related to labor and birth outcomes in either women giving birth or their babies. Thus, the result of this study will partly overlap the result by For this systematic review, the following criteria were stated for inclusion and exclusion of literature. Criteria for inclusion were all types of peer-reviewed scientific papers which examined all health outcomes and experiences in women and their babies during labor and birth as well as shortly after birth. In December 2016, we searched the following electronic bibliographic databases because of their relevance to the topic of research, from their inception dates: Avery Index to Architectural Periodicals, CINAHL, the Cochrane Library, Compendex, the Design & Applied Arts Index, PsycINFO, ProQuest Dissertations & Theses Global, PubMed, Scopus, and Web of Science. No restrictions were applied to years searched, but language was limited to English, Swedish, Danish, Norwegian, and French publications only.Criteria for inclusion were all types of peer-reviewed scientific papers which examined all health outcomes and experiences in women and their babies during labor and birth as well as shortly after birth.Study protocols were excluded as having a different research focus. The birthing room was defined as the room where the birth takes place. Unintended or incidental birth places, such as in an ambulance during transport, were excluded as the features of these spaces are too diverse to inform meaningful conclusions. Excluded were studies exploring all other persons other than the birthing mother and baby present in the birthing room; that is, partners, staff , and doulas/birth companions.A search strategy was developed by the librarian authors  in collaboration with the other authors. The search strategy was reviewed for accuracy using the Peer Review of Electronic Search Strategies criteria . DetailsReview team members working in pairs evaluated the quality of each article chosen for inclusion. The The quality of the qualitative studies was assessed using the In total, 3,373 records were identified. The initial database searches were performed on December 1, 2016, and resulted in 3,192 records. An update of the searches was performed on August 31, 2017, resulting in an additional 181 records. After removing duplicates, 2,341 unique records were screened by title and abstract and 2,063 records were excluded. The full texts of the remaining 278 articles were read, and 241 were excluded due to either study design or outcome not related to women in birthing rooms , leaving 37 for quality assessment  to analyze findings from the different articles. Five characteristics of birthing room design were derived: Snoezelen room, room temperature, place for partner, sensory stimulation, and familiarity, noninstitutionalized, institutionalized environment. The concept of Snoezelen, which was specifically explored in two of the studies, refers to the means whereby an indoor environment is created to provide comfort using controllable stimuli .Despite the limited number of studies showing which specific aspects of birthing room design have an influence on maternal and neonate physical and emotional outcomes, four physical themes were more prominent and repeated than others: means of distraction, comfort, and relaxation; raising the birthing room temperature; features of familiarity; and diminishing a technocratic environment. These themes are presented individually below, but it should be noted that several of them seem to be closely related and interact with each other.Some of the studies stressed the importance of incorporating means of distraction, comfort, and relaxation in the design of the hospital environment, showing the resulting positive effects on labor and pain , the womThis was shown in controlled studies to have a significant impact on both the birthing woman and the neonate, measured as less postpartum shivering in women  and lessThese were shown to shorten the length of labor  and to dThis is important in order to support the woman’s freedom of movement and self-expression, to decrease unnecessary suffering due to fear of childbirth and to enhance the possibility for positive encounters between the women, the midwives, and other staff ; to enabThe result of this literature review on how birthing room design influences maternal and neonate physical and emotional outcomes revealed four prominent physical themes that affect women and their babies: means of distraction, comfort, and relaxation; raising the birthing room temperature; features of familiarity; and diminishing a technocratic environment.The findings of this review also show that research into the birthing environment is far too limited, and there is a need for high-quality scientific studies on the effect of the birthing room itself. Existing research is fragmented, describing only some parts of the effect of environments surrounding labor and birth. The results indicate knowledge gaps in several different dimensions of birthing room design: for example, the role of distraction, how and when is it beneficial to women , and whe…research into the birthing environment is far too limited, and there is a need for high-quality scientific studies on the effect of the birthing room.Moreover, the findings also point to a favoring of a “birth territory ideology”; a theory in which the birthing environment is described as a safe space, where women’s power to give birth physiologically is promoted and their privacy is protected . This thmeans of distraction, comfort, and relaxation  means of distraction, comfort, and relaxation; (2) raising the birthing room temperature; (3) features of familiarity; and (4) diminishing a technocratic environment.Given that many women are expected to give birth at labor wards in hospitals, it is surprising that the evidence on how such environments affect the health of women and their babies is insufficient.The results indicate knowledge gaps in several different dimensions of birthing room design. The evidence on how birthing environments affect outcomes of labor and birth in women and neonates is all too limited. There is crucial need for more research in this field.Birthing room environments including means of distraction, comfort, and relaxation; raising the birthing room temperature; and features of familiarity may have positive effects on outcomes of labor and birthDiminishing a technocratic birthing environment can positively affect outcomes of labor and birthThe results of this review can be used to guide the design of birthing room environments in order to improve patient safety and well-beingClick here for additional data file.Supplemental Material, sj-docx-1-her-10.1177_1937586720903689 for Effects of Birthing Room Design on Maternal and Neonate Outcomes: A Systematic Review by Christina Nilsson, Helle Wijk, Lina Höglund, Helen Sjöblom, Eva Hessman and Marie Berg in HERD: Health Environments Research & Design JournalClick here for additional data file.Supplemental Material, sj-docx-2-her-10.1177_1937586720903689 for Effects of Birthing Room Design on Maternal and Neonate Outcomes: A Systematic Review by Christina Nilsson, Helle Wijk, Lina Höglund, Helen Sjöblom, Eva Hessman and Marie Berg in HERD: Health Environments Research & Design Journal


Impaired cognitive functioning is a core feature of schizophrenia, and is hypothesized to be due to myelination as well as interneuron defects during adolescent prefrontal cortex (PFC) development. Here we report that in the apomorphine-susceptible (APO-SUS) rat model, which has schizophrenia-like features, a myelination defect occurred specifically in parvalbumin interneurons. The adult rats displayed medial PFC (mPFC)-dependent cognitive inflexibility, and a reduced number of mature oligodendrocytes and myelinated parvalbumin inhibitory axons in the mPFC. In the developing mPFC, we observed decreased myelin-related gene expression that persisted into adulthood. Environmental enrichment applied during adolescence restored parvalbumin interneuron hypomyelination as well as cognitive inflexibility. Collectively, these findings highlight that impairment of parvalbumin interneuron myelination is related to schizophrenia-relevant cognitive deficits. Dysfunction of GABAergic neurons in the prefrontal cortex has been reported in schizophrenia. Here, the authors use the apomorphine-susceptible rat, which displays some schizophrenia-like behaviors, and show that interneurons in the medial prefrontal cortex are hypomyelinated, which may contribute to this behavioral phenotype. Interneurons in the PFC microcircuit as well as proper PFC myelination are known to be crucial for proper cognitive functioning3. Cognitive impairment is a core feature of schizophrenia (SZ), a complex neuropsychiatric disorder affecting 1% of the world population and with a large economic burden4. Human genetic and postmortem, as well as animal model studies have suggested an intrinsic defect of interneurons in SZ (refs. 10). GABAergic interneurons play an important regulatory role in cortical microcircuits and encompass ~20% of all myelinated axons in the neocortex12. The interneuron defect in SZ is characterized by a reduced PFC expression of GABA-related mRNAs, while the number and dendritic arborization of PFC interneurons is not affected15. In addition, in the PFC of SZ patients lower myelin levels are found16 and postmortem SZ brain tissue shows reduced myelin- and oligodendrocyte (OL)-related gene expression levels18. Rodent models of SZ are also characterized by myelination defects20, whereas conversely animal models of demyelination display SZ-related behavioral characteristics21. It is further noteworthy that the onset of SZ coincides with both interneuron maturation and PFC myelination during late adolescence. On the basis of these findings, defective myelination of PFC interneurons during adolescence has been hypothesized to underlie cognitive impairment in SZ (refs. 22). However, no studies have assessed the development and characteristics of PFC interneuron myelination in this disorder14.Higher cognitive functions, such as working memory and attentional flexibility develop during adolescence, are optimal in early adulthood and are dependent on correct maturation of the prefrontal cortex (PFC)23. One such model is the apomorphine-susceptible (APO-SUS) rat that, together with its phenotypic counterpart the apomorphine-unsusceptible (APO-UNSUS) rat, has been extensively studied as an idiopathic model of SZ (refs. 25). Without requiring genetic or pharmacological manipulation, APO-SUS rats show SZ-relevant behavioral traits in the positive 28, negative 30, and cognitive 31 domains, as well as neurobiological similarities with SZ patients, such as a hyperactive HPA axis and elevated dopamine D2-receptor binding32. In particular, APO-SUS rats have a defective PFC microcircuit due to a reduced excitability of GABAergic interneurons, reminiscent of the interneuron abnormalities described in SZ patients15. We use the APO-SUS and APO-UNSUS rats as a model to examine the role of  interneuron myelination in PFC-dependent cognitive impairment. Here, we report that APO-SUS rats display cognitive inflexibility, that interneurons are hypomyelinated during PFC development, and that environmental enrichment during adolescence restores interneuron hypomyelination and rescues cognitive impairment.Understanding the causal pathophysiology of SZ will allow the development of new treatment strategies. However, because of its highly complex nature SZ is mechanistically enigmatic and not easy to mimic in animals. Yet, animal studies are pivotal for mechanistic insight. A number of animal models exist, in which a combination of genetic and environmental factors generates a SZ-related phenotypet-tests showed no difference in performance per session at the 10-s delay , but confirmed a significant decrease in the 60-s delay performance per session  in APO-SUS versus APO-UNSUS rats. These findings indicate that mPFC-linked spatial working memory is impaired in APO-SUS rats.To explore cognitive functioning of the APO-SUS rats relative to that of the APO-UNSUS rats, we performed SZ-relevant cognitive behavioral tests in adulthood 90). The first test was to examine spatial working memory using the continuous delayed alternation paradigm with 10-s or 60-s delays between trials  until they reached a criterion of ten correct trials in a row. There were no differences in the number of errors made during visual cue discrimination in APO-SUS and APO-UNSUS rats , suggesting reduced mPFC-dependent cognitive flexibility in APO-SUS rats. The increased number of errors made by APO-SUS rats represented perseverative errors, indicating a lack of inhibition of previously learned behavior .To confirm mPFC dysfunction, we next performed an extra-dimensional operant set-shifting test35. In the T-maze, rats were trained to retrieve a reward from one arm until they reached a criterion of 70% correct trials per session . We confirmed this result in the operant setup, where rats were trained to press the lever on one side of the cage until they reached a criterion of a streak of ten correct trials . However, APO-SUS rats made significantly more perseverative errors when the total number of errors during all three reversals was assessed . This suggests that orbitofrontal cortex-dependent reversal learning behavior is impaired in APO-SUS rats, but not as severely as mPFC-dependent working memory and cognitive inflexibility. Taken together, these data indicate that APO-SUS rats show cognitive inflexibility that involves mPFC dysfunction in SZ-relevant tasks.To investigate whether the observed cognitive impairment is also found with behavioral tests dependent on other frontal areas, such as the orbitofrontal cortex, we tested reversal learning in both the T-maze and the operant cagesion Fig. . During als Fig. . A compl36, we next investigated neurobiological features of this brain region. It was recently hypothesized that in SZ PFC interneuron dysfunction is associated with dysmyelination and that this plays a pivotal role in the pathophysiology of cognitive symptoms of the disorder. Myelination of interneurons occurs during mPFC maturation around P21 in rodents. We have recently shown that in the mPFC of P21 APO-SUS rats, there is a reduced inhibitory input onto pyramidal cells, whereas a normal number and morphology of interneurons were observed15. These interneuron abnormalities during adolescence resemble interneuron defects described in SZ patients37. To examine whether the APO-SUS interneuron defect is indeed linked to impaired myelination, we investigated the molecular and cellular correlates of myelin development in the mPFC of APO-SUS and APO-UNSUS rats.Given that the cognitive inflexibility in APO-SUS rats was observed in tasks that are dependent on mPFC functioningPlp), myelin basic protein (Mbp), claudin 11 (Cldn11), myelin-associated OL basic protein (Mobp), myelin-associated glycoprotein (Mag), and myelin OL glycoprotein (Mog) in the mPFC of APO-SUS versus APO-UNSUS rats . PLP immunofluorescence staining also revealed a decrease in myelinated areas in both the infralimbic (IL) and prelimbic (PL) subregions of the mPFC, but not in the BC of adult APO-SUS rats . These results suggest that during the development of the mPFC in APO-SUS rats there is a decrease in myelin-related mRNA expression that persists into adulthood.From P21 onwards, quantitative PCR (qPCR) analysis revealed a decrease in the expression of myelin-related mRNAs encoding proteolipid protein , although axonal density remained unaffected in the APO-SUS versus APO-UNSUS PL . This indicates a reduced number of myelinated axons rather than a reduced total number of axons in APO-SUS mPFC. Furthermore, a significant correlation between the thickness of the myelin sheath (G-ratio) and axon caliber was found in mPFC of both APO-SUS and APO-UNSUS rats . Note that in APO-SUS mPFC myelin thickness, as measured by its G-ratio, as well as myelin structure was normal , suggesting that no demyelination occurred. The lack of demyelination was further confirmed by the absence of myelin debris in macrophages as revealed by Oil Red O staining in the APO-SUS and APO-UNSUS mPFC . Together, these findings suggest that a number of axons do not get myelinated in the APO-SUS mPFC.The observed decreased expression of myelin-related genes in APO-SUS mPFC prompted us to assess the number of myelinated axons and myelin integrity, using transmission electron microscopy at P90. We found a reduced number of myelinated axons in both IL and PL subregions of the mPFC, but not in BC of APO-SUS rats . Therefore, the percentage of non-GABAergic myelinated axons was higher in APO-SUS mPFC , and the reduced number of APO-SUS myelinated axons can be primarily attributed to a reduced number of myelinated GABAergic interneurons in the mPFC of APO-SUS versus APO-UNSUS rats. This notion is strengthened by our finding that the number of myelinated axons of different calibers was reduced in APO-SUS IL; myelinated GABAergic interneurons are known to have a range of different axon calibers11. To confirm that myelin integrity of GABAergic interneurons was not affected, we measured the length of nodes of Ranvier. We found for the myelinated GABAergic axons a similar average node of Ranvier length of 1.18–1.36 μm in APO-SUS and APO-UNSUS IL , in line with previously reported values38. Myelination of GABAergic interneurons in the PFC has been reported to occur only on parvalbumin interneurons39. Immunostaining for parvalbumin and MBP on ultrathin tissue sections indeed revealed a reduced number of myelinated parvalbumin axons in APO-SUS relative to APO-UNSUS IL , confirming parvalbumin interneuron hypomyelination in the APO-SUS mPFC. This was not caused by a decrease in the number of parvalbumin interneurons in the mPFC of APO-SUS rats .We next investigated whether this group of unmyelinated axons in APO-SUS mPFC corresponds to interneurons by performing immunofluorescence co-staining for MBP and GABA on 70 nm ultrathin IL sections. This technique permits visualization of MBP+ myelin sheaths surrounding axons of GABAergic inhibitory neurons, allowing the precise quantification of myelinated inhibitory interneurons at the ultrastructural level Fig. . We fount-test IL: t = 2.501, p = 0.038, df = 7.568 and PL: t = 2.898, p = 0.012, df = 13), suggesting abnormalities in OL lineage progression already when mPFC myelination starts. In adulthood at P90, we found a decrease in the number of OLIG2+ OL lineage cells in the IL and PL of APO-SUS rats . Furthermore, a trend toward a decreased number of OLIG2+NG2+ OPCs in the IL and PL of P90 APO-SUS rats was identified . Additionally, we observed a significant decrease in the number of OLIG2+GPR17+BCAS1+ early-myelinating OLs , indicating that OL maturation is impaired in the adult APO-SUS mPFC. In the APO-SUS BC, we found only a decrease in the number of OLIG2+CC1+ OLs , suggesting that the OL maturation impairment is more severe in the mPFC than the BC of APO-SUS rats.As we identified a lower number of myelinated inhibitory axons, we next examined whether the development of the myelin-producing OLs was impaired in APO-SUS mPFC. Therefore, we assessed the number and maturation state of oligodendroglial cells in APO-SUS and APO-UNSUS mPFC and BC during postnatal development. At P21, when myelination of the mPFC commences, we observed no differences in the number of OLIG2+ OL lineage cells, OLIG2+NG2+ OL precursor cells (OPCs) and OLIG2+CC1+ OLs Fig. , whereast-test t = −0.074, p = 0.944, df = 4) or differentiate . These data indicate that the hypomyelination in APO-SUS mPFC is not due to intrinsic defects of OPC proliferation and differentiation. Rather, the results highlight the critical role of the OL microenvironment in the APO-SUS mPFC that could negatively affect OL maturation and myelination. This notion is strengthened by the fact that during development and in adulthood, we did not observe changes in myelin-related gene expression in brain regions other than the mPFC . Based on these findings, we applied an environmental enrichment paradigm in order to favorably influence the development of OLs, myelination, and cognitive flexibility in the APO-SUS rat model.The development of OLs and the myelination process can be positively influenced by behavioral experiences, such as voluntary exercise, socializing, and environmental enrichmentF = 2.497, p = 0.097, df = 3; APO-SUS standard housing versus environmental enrichment t = −2.960, p = 0.018, df = 8; and APO-SUS standard housing versus APO-UNSUS standard housing t = −2.326, p = 0.045, df = 9); in the environmental enrichment experiment, we have analyzed IL as there were no substantial differences between IL and PL mPFC subregions in our previous analyses . There was a trend toward an increase in the number of OLIG2+GPR17+BCAS1+ early-myelinating OLs in the IL of APO-SUS rats reared in environmental enrichment, as compared to APO-SUS rats housed under standard conditions. The number of early-myelinating OLs in IL of APO-SUS rats housed under environmental enrichment conditions was not different from that of APO-UNSUS rats . The number of OLIG2+CC1+ OLs, however, was not affected by environmental enrichment in the mPFC of both APO-SUS and APO-UNSUS rats . These data indicate that environmental enrichment led to a significantly increased number of OL lineage cells and OPCs, and a trend toward an increased number of early-myelinating OLs, while the number of mature OLs remained unaffected in APO-SUS mPFC.We studied OL lineage progression in APO-SUS and APO-UNSUS rats that were reared under environmental enrichment or standard housing conditions from weaning at P21 onward, and were sacrificed at P90 for histological analysis Fig. . ImmunofF = 4.551, p = 0.029, df = 3; APO-SUS standard housing versus environmental enrichment t = −2.291, p = 0.062, df = 6; and APO-SUS standard housing versus APO-UNSUS standard housing t = −3.302, p = 0.021, df = 5). The G-ratio of APO-SUS and APO-UNSUS myelinated axons was not affected by environmental enrichment , and chi-square test on cumulative axon caliber distribution showed that there were no differences in the distribution of myelinated axon caliber between the groups . However, environmental enrichment significantly increased the number of myelinated axons of 0.2–0.4 μm, 0.8–1.0 μm, and 1.0–1.2 μm axon caliber in APO-SUS mPFC to control levels . We conclude that environmental enrichment during mPFC development restored the number of OL lineage cells, OPCs, and early-myelinating OLs, and improved the myelination of interneurons in the APO-SUS mPFC.We next investigated whether an environmental enrichment-induced rescue of the numbers of OL lineage cells, OPCs and early-myelinating OLs would be sufficient to restore myelination of interneurons in APO-SUS mPFC. Ultrastructural analysis revealed that in the APO-SUS mPFC environmental enrichment restored the number of myelinated axons to APO-UNSUS standard housing level, while environmental enrichment had no effect on the number of myelinated axons in APO-UNSUS mPFC . Post hoc testing confirmed a significant improvement in the number of errors until criterion in the third extra-dimensional shift and a reduction of the total number of perseverative errors in APO-SUS rats reared in an environmental enrichment. APO-UNSUS rats reared in an environmental enrichment compared to standard housing-reared APO-UNSUS rats showed a similar number of perseverative errors during extra-dimensional set-shifting, but a decreased error number in the first and an increased error number in the second shift. The number of perseverative errors made during reversal learning was also significantly reduced in APO-SUS rats  reversal learning are found only in a subset of patients and less consistently than abnormalities in extra-dimensional set-shifting58. Thus, APO-SUS rats show cognitive inflexibility with characteristics reminiscent of cognitive impairment in SZ patients, including features suggestive of alterations in mPFC inhibitory circuits.In this study, we have provided direct evidence that in a rat model with SZ-relevant features interneurons are hypomyelinated in the mPFC, and that restoring interneuron myelination through environmental enrichment applied during mPFC development improves cognitive flexibility. We found mPFC-dependent cognitive inflexibility in both spatial working memory and extra-dimensional set-shifting. Spatial working memory is also impaired in SZ patients18. Our data shows that decreased expression of myelin-related mRNAs and proteins starts during adolescence, and persists into adulthood in the APO-SUS mPFC, supporting the hypothesis that in SZ defective myelination occurs during adolescent mPFC development, rather than in adulthood by demyelinating insults. Strengthening this notion is the fact that active demyelination does not occur in the mPFC of APO-SUS rats, and that myelinated fibers in the APO-SUS mPFC have a typical G-ratio and myelin structure, indicating hypomyelination rather than demyelination in the mPFC. Furthermore, ultrastructural analysis revealed a reduced number of myelinated axons in the adult APO-SUS mPFC. Importantly, we found that the population of unmyelinated axons corresponded to parvalbumin interneurons, while the number of parvalbumin interneurons remained the same, demonstrating the occurrence of parvalbumin interneuron hypomyelination in the mPFC of the APO-SUS model with SZ-relevant features. In SZ, parvalbumin interneurons have an intrinsic defect that leads to an impaired inhibitory control and disturbed cognitive functioning37. Interestingly, it has been recently suggested that a myelination deficiency of parvalbumin interneurons may underlie defective inhibitory regulation in the PFC (ref. 14). We here show that in the mPFC of a rat model of SZ parvalbumin interneurons are indeed hypomyelinated.Dysfunctioning of interneurons and defective myelination are both well-replicated neurobiological abnormalities observed in SZ, including decreased myelin-related gene expression in postmortem SZ frontal cortex60. The reduced number of mature OLs in APO-SUS BC was not accompanied by changes in OL differentiation or myelination, and suggests an increased myelin production by mature OLs in this brain region. In contrast, the differences in OL maturation observed in the mPFC do lead to affected myelination. Primary oligodendroglial cell cultures from APO-SUS and APO-UNSUS cortex revealed that the intrinsic capability of APO-SUS OPCs to proliferate and differentiate was still intact. This suggests that APO-SUS oligodendroglia do have the capacity to terminally differentiate and myelinate axons. We therefore conclude that the OL microenvironment within the APO-SUS mPFC plays a pivotal role in inducing the OL lineage progression impairment observed in the APO-SUS rats.A plausible reason for the observed parvalbumin interneuron hypomyelination may be the impaired maturation of OL lineage cells in the mPFC of APO-SUS rats. The increase in the number of early-myelinating OLs at P21 indicates that in APO-SUS mPFC there are abnormalities in OL lineage progression already at the onset of myelination. This was accompanied by decreased myelin-related mRNA expression, suggesting that in the APO-SUS mPFC the OLs produce less myelin-related mRNAs per cell. The fact that at P90 (in adulthood), we found a similar number of OPCs and decreased numbers of early-myelinating, as well as mature OLs points to an impairment of OL maturation, consistent with the significantly reduced expression of all six myelin-related genes examined in the adult APO-SUS mPFC. Likewise, in postmortem PFC of SZ patients, the total number of OL lineage cells is reduced, while no changes in the total number of OPCs have been observed42). For example, in rodents voluntary exercise increases OL proliferation in the PFC61, sociability influences myelination and OL development in the PFC during adolescence43, and environmental enrichment stimulates OLs to produce more myelin45. The effects of behavioral experiences are strongest within the critical period of PFC myelination, i.e., during adolescence43. Importantly, adolescent individuals at high risk for SZ show higher rates of sedentary behavior, less physical activity, and decreased sociability that correlate with cognitive symptoms in later stages of SZ (refs. 62). Furthermore, levels of academic achievement and social interactions prior to disease onset during adolescence are associated with the degree of impairment in working memory and executive functioning during later phases of SZ (refs. 51). This indicates that socializing and cognitive challenges can positively influence cognition during the course of SZ. Indeed, exposing APO-SUS rats to an environmental enrichment paradigm that included voluntary exercise, increased sociability, and opportunity for novelty exploration during adolescent mPFC development restored the number of OL lineage cells. The increased density of OPCs following environmental enrichment suggests that under this condition more OL lineage cells remain in the OPC pool. Other studies that have applied environmental enrichment to treat glial abnormalities in rat brain have reported similar increases in the number of OPCs (refs. 65). Environmental enrichment did not affect in APO-SUS mPFC the total number of mature OLs, but increased the number of early-myelinating OLs as well as the number of myelinated axons to numbers comparable to those in APO-UNSUS mPFC. This is in line with previous studies in rodents, showing that environmental enrichment increases myelin production45. The exact mechanism underlying the myelin increase is at present unclear, but it has been hypothesized that environmental enrichment causes changes in neuronal activity, axon-OPC synapses, growth factor release, neurotransmitter release, excretion of metabolites, and inflammatory factors that can influence oligodendroglial cells and myelination42. Environmental enrichment during mPFC development was also sufficient to rescue cognitive flexibility in the extra-dimensional set-shifting test, clearly indicating that developmental interneuron hypomyelination contributes to the cognitive impairment observed in the APO-SUS rat model of SZ.Behavioral experiences are known to influence the development of oligodendroglia and myelination in the PFC  upon injection of apomorphine were selectively bred as APO-SUS rats. The same selective breeding was performed with the rats that showed a weak apomorphine-induced stereotypy . Apomorphine injection and behavioral selection were only performed with the first 15 generations of APO-SUS and APO-UNSUS rats. In the subsequent breedings, APO-SUS rats displayed SZ-relevant features without this pharmacological treatment. In this study, naive male APO-SUS and APO-UNSUS rats from the 38th–43rd generation were used. Rats were housed in pairs in a temperature- and humidity-controlled room with a 12-h light–dark cycle (lights on at 7.00 a.m.), and ad libitum access to water and standard laboratory chow , unless otherwise indicated. Animal experiments were approved by the Animal Ethics Committee of Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands, and were conducted in accordance with Dutch legislation .During the continuous delayed alternation test, APO-SUS and APO-UNSUS rats were kept on a food restriction schedule. A limited amount of standard laboratory chow was given to maintain the rats at 85–90% of their free-feeding weight. Rats were habituated for 20 min to a T-maze with food pellets scattered around the maze . Food pots (4 × 4 ×4 cm) were present 4 cm from the end of each arm. The animals were trained to perform a minimum of seven trials within 20 min , after which the continuous delayed alternation paradigm started. Rats were tested during the light phase and performed nine trials per day  for 10 days. During the start trial of each session, a food pellet was present both in the left and the right arm of the T-maze. The rat was placed in the start box and upon lifting of the guillotine door the rat was allowed to move freely around the maze until one of the food pellets was retrieved. After that the rat was placed back in the start box for the delay period of either 10 or 60 s. In the first test trial only the arm that the animal did not visit during the start trial was baited. During following trials, a food pellet was present only in the arm that was not baited during the previous trial. Hence, rats had to alternate between the left and right arms of the T-maze Fig. . Trials 33 and described below.Upon the start of behavioral training, rats were food restricted and received 5–8 g of food per 100 g of rat daily. Rats were pre-exposed once to grain reward pellets  in the home cage. Operant conditioning chambers  were situated in light and sound-attenuating cubicles equipped with a ventilation fan. Each chamber was equipped with two 4.8-cm-wide retractable levers, placed 11.7 cm apart and 6 cm from the grid floor. A cue light  was present above each lever. At the same wall, a reward pellet could be delivered in a magazine between the levers and a house light  was located on the same wall. The lever presentation and cue light illumination sides were counterbalanced between rats. Operant extra-dimensional set-shifting and reversal learning procedures were programmed, and analyzed in MED-PC V. Procedures are based on previous reportsDuring pretraining, the rats were first habituated to the operant cage and received 50 food pellets with an inter-trial interval varying between 10 and 50 s. During the second training phase, one of the levers was extended during the entire session and each lever-press resulted in the delivery of a reward pellet. After the criterion of 50 lever presses was reached, this was repeated with the other lever. During the third training phase, rats were familiarized with the house light and the insertion of the levers into the chamber, and were required to press within 10 s to receive a food pellet. Upon pressing, the lever retracted, a reward pellet was delivered, and the house light remained illuminated for another 4 s. If the rat did not press the lever within 10 s, the lever retracted and the house light went off. In each pair of trials, the left or right lever was presented once, and the order within the pair of trials was random. Five of these retractable lever-press training sessions were performed, each consisting of 90 trials with 20 s inter-trial intervals. Immediately after the last session of retractable lever-press training, without being removed from the operant chamber, the rats performed a side bias test. During the side bias test both levers were presented and rats received a food pellet upon pressing either the left or the right lever. Upon pressing, the levers retracted, a reward pellet was delivered, and the house light remained illuminated for another 4 s. In case the rat did not press a lever within 10 s, the levers retracted and the house light went off. Subsequently, a food pellet was delivered only upon pressing the lever that was not chosen during the previous trial. In case the rat pressed the same lever as during the previous trials no food pellet was delivered, the levers retracted and the house light turned off. Trials continued until the rat pressed the lever that was not chosen during the initial trial. After pressing both levers, a new set of trials commenced and seven sets of trials were applied. The number of responses on each lever was used to determine side preference.Upon completion of pretraining, the rats were trained to perform visual cue discrimination. The cue light was randomly presented above the left or right lever for 3 s, then both levers extended and the house light turned on. The rat was required to press the lever above which the cue light was illuminated within 10 s in order to receive a reward pellet. When rats reached the criterion of ten subsequent correct trials, the session finished. During consecutive phases of the experiment, rats were either required to perform an extra-dimensional set-shift or a reversal learning procedure. During extra-dimensional set-shifting, the first 20 trials of the session consisted of the ‘old rule’, in which rats had to press the lever above which a cue light was illuminated, and from trial 21 onward, rats were required to press their non-preferred lever (as indicated by the side bias test) irrespective of the cue light illumination Fig. . During Rats were weaned at P21 and placed in environmental enrichment or standard housing conditions. For environmental enrichment, rats were housed in groups of ten animals in 100 × 54.5 × 48 cm cages with cage enrichment in the form of toys, running wheels, tunnels, and nesting places. Enrichment was of different colors and textures, and was changed three times a week to promote exploration behavior and rats were handled once a week. Rats in standard housing were housed in pairs in standard cages with standard enrichment in the form of a rat retreat. Rats in standard housing and environmental enrichment conditions were housed in the same room. Rats were either sacrificed at P90 for immunohistochemistry or transmission electron microscopy experiments, or did the operant attentional set-shifting task starting at P60.Naive P0, P7, P14, P21 (±1 day), P60 (±1 day), P90 (±1 day), P120 (±1 day), and P365 (±14 days) APO-SUS and APO-UNSUS rats were sacrificed by direct decapitation, and brains were isolated, frozen on dry ice, and stored at −80 °C. Brains of P0, P7, and P14 were freshly dissected, while brains of P21, P60, P90, P120, and P365 were micropunched. Micropunching was performed in a cryostat (Leica) at −15 °C and the Paxinos and Watson rat brain atlas was used to aid dissection. mPFC was collected with a 1.20 or 2.00-mm punch needle (Harris) from 300 µm coronal sections at Bregma 4.00–2.20. CC was microdissected at room temperature (RT) and striatum (STR) punched with a 2.00-mm punch needle from Bregma 1.60 to −0.20. BC was punched with a 2.00-mm needle at Bregma 8.08–6.10. Dissected tissues were stored at −80 °C until further analysis. Tissue samples were homogenized using Trizol (Sigma) and RNA was extracted with chloroform, precipitated with isopropanol and glycogen (Fermentas), washed with 75% ethanol, dissolved in MilliQ H2O, and stored at −80 °C until further analysis.69) after which normalized mRNA expression levels were calculated.For qPCR analysis, RNA samples were treated with DNase I (Fermentas) and cDNA was synthesized using the Revert Aid H-minus first strand cDNA synthesis kit (Thermo Scientific). cDNA was diluted in MilliQ H2O and stored at −20 °C. qPCR samples were pipetted using a robot (Corbett Robotics) and contained 2.0 μL diluted cDNA, 0.8 μL 5 μM forward primer, 0.8 μL 5 μM reverse primer, 5 μL SybrGreen mix (Roche), and 1.8 μL MilliQ H2O. qPCR was performed with a Rotor Gene 6000 Series (Corbett Life Sciences) using a three-step paradigm with a fixed gain of eight. Forty five to 50 cycling steps of 95, 60, and 72 °C were applied and fluorescence was acquired after each cycling step. Primers were designed with NCBI Primer-Blast or Primer Express 2.0 and synthesized by Sigma  using 20 (mPFC and BC) or 25 (CC) strokes in a glass potter, and protein levels were determined with a BCA assay (Thermo Scientific). A total of 80 µg of mPFC, 5 µg of CC, and 30 µg of BC protein were used for SDS–PAGE and western blotting onto PVDF membranes. Blots were blocked in 1× PBS, 0.1% Tween, and 10% non-fat milk  and incubated with MBP antibody  at 1:4000 or 1:1000, and GAPDH antibody  at 1:5000 in 1× PBS, 0.1% Tween, and 5% milk at 4 °C overnight. Secondary antibodies /PO and GAR/IgG(H + L)/PO, Nordic Immunology) were incubated for 2 h at RT at 1:5000, and images were acquired using Lumi-Light Western blotting substrate (Sigma) in an ImageQuant LAS-4000 digital imaging system  and analyzed using FIJI.For immunohistochemistry, naive APO-SUS and APO-UNSUS rats of P21 (±1 day) and P90 (±1 day) were perfused with 2% paraformaldehyde (PFA). Perfused brains were removed, postfixed overnight, and placed in 30% sucrose in PBS for 3–5 days, frozen on dry ice and stored at −80 °C until further processing. Coronal cryosections of 10–14 μm were collected in a cryostat (Leica) and rehydrated in 1× PBS, 0.1% Triton X-100, or for OLIG2 NG2 immunohistochemistry in 1× PBS, 0.05% Tween-20. For OLIG2-CC1 staining antigen retrieval was performed in a microwave using citric acid-based antigen unmasking solution (Vector). Tissue was blocked in 4% BSA, 0.1% Triton X-100 or 5% NGS/NDS/NHS, 1% BSA, 1% glycine, 0.1% lysine, and 0.4% triton X-100 for 1 h at RT. Primary antibodies were anti-OLIG2 , anti-CC1 , anti-NG2 , anti-GPR17 , anti-BCAS1 , or anti-PLP  and incubated overnight at 4 °C. Secondary antibodies were 488, 555, 568, and 647-conjugated anti-rabbit or anti-mouse  or TRITC-conjugated anti-mouse , and incubated for 1–2 h at RT. Hoechst  was added as a nuclear counterstain in MilliQ H2O, rinsed with 60% isopropanol, and incubated in 60% isopropanol with 0.3% Oil red O  for 15 min at RT, and rinsed with 60% isopropanol and MilliQ H2O. As positive control for Oil red O staining, a mouse spinal cord lesion with active demyelination induced by experimental autoimmune encephalomyelitis was used. Sections were mounted in Fluoromount  and 20× images were obtained using an Axioscan (Zeiss) and analyzed with FIJI. Regions of interest were drawn and all cells in the region of interest were counted manually in one to three brain sections for OLIG2+NG2+, OLIG2+GPR17+BCAS1+, and OLIG2+CC1+ cells. OLIG2+ cell counts were obtained in the OLIG2−NG2 or OLIG2−CC1 images. Positive areas for PLP and Oil red O were quantified using a threshold of 25/255 and 0/133, respectively. For PLP images, background was subtracted with a rolling ball radius of 50 pixels.For electron microscopy, naive animals of P90 (±1 day) were perfused with 2% PFA/2% glutaraldehyde and perfused brains were removed, postfixed overnight in 2% PFA/2% glutaraldehyde, and stored at 4 °C in PBS/0.01% azide until further processing. Sagittal sections of 100 μm were collected using a vibratome (Leica), fixed with 2% osmium tetroxide, and contrast was obtained with 5% uranyl acetate. Following ethanol dehydration, sections were embedded in epon resin and ultrathin (70–100 nm) sections were obtained with an ultramicrotome (Leica). Sections were contrasted using lead citrate and 40 non-overlapping 26,000× images were obtained in each region of interest. Myelinated axons were counted in all 40 images per brain region, and the G-ratio and axon caliber of all myelinated axons perpendicular to the field of view were measured in FIJI. The percentage of axon surface was calculated using 49 equally (200 µm) spaced crosses superimposed over ten randomly picked images. The percentage of crosses that touched an axon was calculated.38 with adjustments. Sections were immersed in 100 mM glycine in milliQ water, washed with 1× PBS, and primary antibodies diluted in 2% BSA in 1× PBS were applied at 4 °C overnight. Sections were then washed with 1× PBS, secondary antibodies  were added for 30 min at RT, and sections were subsequently washed with 1× PBS and coverslipped using Fluoromount (Thermo Fisher). Images were obtained from IL layer V, 300 µm from the CC, using a wide-field fluorescent microscope with a 63× oil lens (Leica) and Zen 2  software. The number of MBP+GABA+ or MBP+parvalbumin+ axons, as well as the total number of MBP+ axons was quantified. Because of the reduction in the number myelinated axons in APO-SUS mPFC, we calculated the percentage of MBP+ axons that was also GABA+ or PV+. Nodes of Ranvier were measured in Zen software in MBP+GABA+ axons that were horizontal to the field of view.Naïve APO-SUS and APO-UNSUS rats of P90 (±1 day) were perfused with 2% PFA/2% glutaraldehyde and perfused brains were removed, postfixed overnight in 2% PFA/2% glutaraldehyde, and stored at 4 °C in PBS/0.01% azide until further processing. Sagittal sections of 100 μm were collected using a vibratome (Leica), and IL was dissected according to Figure 81 of the Paxinos and Watson Rat Brain Atlas. IL samples were dehydrated in ethanol , embedded in LRwhite resin (Electron Microscopy Sciences) at RT for 14 h, and polymerized in LRwhite resin in airtight capsules at 60 °C for 2–3 days. Ultrathin (70–100 nm) sections were obtained with an ultramicrotome (Leica), collected on gelatin-coated glass slides, and stained with anti-MBP  and anti-GABA , or anti-parvalbumin (PV27 Swant 1:300) antibodies using a protocol based on refs. n = 3 replicates per condition. The tissue was homogenized in mixed glia culture medium (GlutaMAX (Invitrogen), 10% fetal bovine serum (Thermo Fisher), 1% Pen-Strep (Thermo Fisher), and 1% non-essential amino acids (Thermo Fisher)) and cells were kept in this medium on 1:10 poly-L ornithine-coated T75 flasks at 37 °C 5% CO2. Medium was refreshed after 7 and 13 days in culture, and at day 14 OPCs were purified using a shaking protocol. Mixed glia cultures were shaken at 7 × g for 1 h to remove microglia cells, then cultures were shaken again at 7 × g for 18 h to purify OPCs. Supernatant containing OPCs was placed on petri dishes  for 3 × 5 min to eliminate astrocyte contamination. OPCs were then plated on PLO-coated coverslips in 24 well plates. After 4 h, medium was changed to either proliferation medium (DMEM-F12 (Invitrogen), 0.5% B27 (Sigma) 1% Pen-Strep (Thermo Fisher), 0.05% fibroblast growth factor , and 0.05% platelet derived growth factor BB ), or differentiation medium (DMEM-F12 (Invitrogen), 0.5% B27 (Sigma), 1% penicillin–streptomycin (Thermo Fisher), and 0.05% of T3 ) for 4 days. After 4 days, cultures were stained with O4 antibody  1:100) for 1 h and fixed in 2% PFA for 7 min. Subsequently, OPCs were incubated with anti-SOX10 (R&D Systems AF2864 1:100) and anti-MBP (Abcam ab7349 1:200) primary antibodies for 1 h at RT, washed with 1× PBS, and incubated with secondary antibodies  for 1 h at RT, washed, and mounted with fluoromount. Images were obtained in an Axioscan (Zeiss).Mixed glia cultures were obtained from cortices of P1 APO-SUS and APO-UNSUS rats, t-test and, when appropriate, with Benjamini–Hochberg correction for multiple comparisons in IBM SPSS Statistics 24. Chi-square test was performed on axon caliber frequency data and a linear regression with two-way ANOVA was used to test statistical significance of the regression between G-ratio and axon caliber. For the environmental enrichment experiment, immunohistochemistry and electron microscopy data analysis were done using one-way ANOVAs, and effects were assessed with independent samples t-tests, and analysis of the set-shifting task was done using multivariate ANOVAs. For all analyses, outliers were discarded beforehand as indicated by the Grubbs outlier test using Graphpad quickcalcs 2018, and the level of significance was set at p = 0.05. All data were visualized using GraphPad Prism 6.01.For qPCR, western blot, immunohistochemistry, electron microscopy, delayed alternation, set-shifting, and reversal learning data analysis, statistical significance was calculated using the independent samples Further information on research design is available in the Supplementary InformationReporting Summary


Identification of causal factors that influence fetal growth and anthropometry at birth is of great importance as they provide information about increased risk of disease throughout life. The association between maternal genetic polymorphism MTHFR(677)C>T and anthropometry at birth has been widely studied because of its key role in the one-carbon cycle. MTHFR(677) CT and TT genotypes have been associated with a greater risk of low birth weight, especially in case of deficient intake of folic acid during pregnancy. This study aimed to analyze the association between the maternal MTHFR(677)C>T genetic polymorphism and anthropometry at birth in a population with adequate folate consumption. We included 694 mother–newborn pairs from a prospective population-based birth cohort in Spain, in the Genetics, Early life enviroNmental Exposures and Infant Development in Andalusia (GENEIDA) project. Women were genotyped for MTHFR(677)C>T SNP by Q-PCR using TaqMan© probes. Relevant maternal and newborn information was obtained from structured questionnaires and medical records. Results showed that maternal MTHFR(677)C>T genotype was associated with newborn anthropometry. Genotypes CT or CT/TT showed statistically significant associations with increased or decreased risk of large-for-gestational-age (LGA) or small-for-gestational-age (SGA) based on weight and height, depending on the newborn’s sex, as well as with SGA in premature neonates. The relationships between this maternal genotype and anthropometry at birth remained despite an adequate maternal folate intake. Anthropometric measurements of infants not only allow for assessing intrauterine growth, but also help to identify infants with a greater risk of suffering developmental disorders or diseases in adult life. Fetal ultrasound biometry measurement as well as birth anthropometry parameters  are used as indicators to evaluate intrauterine fetal growth . The terFetal growth and SGA or LGA results are influenced by different factors including maternal socio-demographic characteristics, health status, nutritional intake, environmental exposures, genetic background and gene-environment interactions ,7,8.Among nutrients, folate stands out for its role in fetal growth and placental development. Folate is the natural occurring state of folic acid (synthetic form) or vitamin B9, an essential water-soluble vitamin that must be obtained from diet. The main sources of folate are citrus fruits, legumes, green vegetables and liver. A daily dose of 400 micrograms of folic acid supplement is recommended to future mothers from the periconceptional period to prevent neural tube defects as well as low birth weight and preterm birth that are known to be associated to insufficient folate consumption during early pregnancy , as it pOn the other hand, alteration in folate levels can occur as a result of genetic poylmorphisms in gene coding for enzymes involved in its metabolism, which may lead to changes in their catalytic activity. One of these enzymes is methylenetetrahydrofolate reductase (MTHFR), which is often assessed in epidemiological studies due to the direct relationship between its activity and the levels of folate and also because of its implication in hyperhomocysteinemia ,16. FolaWe hypothesized that key genetic polymorphisms responsible for folate availability influence fetal growth in populations with adequate folate intake. Therefore, the aim of the present study was to assess the association between the maternal MTHFR(677)C>T genetic polymorphism and anthropometry at birth in the Genetics, Early life enviroNmental Exposures and Infant Development in Andalusia (GENEIDA) birth cohort. This consists of a Mediterranean population of pregnant women supplemented with folic acid that reached the recommended folate dose during pregnancy.This study was conducted on 694 mother/child pairs from the GENEIDA birth cohort. Pregnant women were recruited in the “Hospital El Poniente” in El Ejido, Almería (Southeast Spain) during the period from April 2014 to November 2017. Women were recruited in the first trimester of pregnancy (12–13 weeks of gestation) and then followed-up in the third trimester of pregnancy, at delivery. The inclusion criteria were: 16 years old or older, intention of giving birth at the reference hospital mentioned above, singleton pregnancy, pregnancy without assisted reproductive technology, lack of any chronic disease or being under medical treatment and absence of any language barrier.® Predesigned SNP Genotyping Assays  was used for the detection of the specific MTHFR(677)C>T (rs1801133) SNP in a QuantStudio 6 Flex Real-Time PCR System. As quality control, each series of amplifications included three negative controls without the DNA template to test for DNA contamination and three previously genotyped samples to test genotyping consistency.Blood samples were collected in the second trimester of pregnancy. Each sample (4 mL) was centrifuged at 2500 rpm for 15 min and the buffy coat (containing white blood cells) was carefully removed and stored at −20 °C until analysis. DNA was extracted using the “X-DNA Extraction Kit  following the manufacturer’s instructions. DNA integrity was checked by gel agarose gel electrophoresis. Quantification of DNA was carried out by spectrophotometry using the NanoPhotometerTM P-Class . A total of 20 ng of dried-down genomic DNA was used for genotyping. Specific TaqMan3) [Gestational age and anthropometric measurements  at birth were obtained from medical hospital records. Weight index was calculated as the ratio of birth weight (grams) to height (cm3) .Size for gestational age was estimated based on Carrascosa et al. 2004  using neA suitable alternative to identify SGA was using a chest circumference ≤ 30 cm as cut-off point.Information on covariates was obtained from questionnaires administered by personal interviews in the first and third trimester of pregnancy, and medical records. Covariates were selected according to clinical relevance and literature review of previous birth cohort studies in which association between genetic polymorphisms and birth outcomes was analyzed.Information on diet was obtained using an adapted version of the food consumption of the Andalucian population based on a previously validated food frequency questionnaire (FFQ) ,23. In tp < 0.001, respectively. Validation analysis of the questionnaires was also carried out in a selected subsample of women (n = 43) who filled-in three 24-h dietary recalls in addition to the FFQs. The limits of agreement (LoA) for folate and vitamin B12 were 90.70% and 93.02%, respectively.A reproducibility and validation study of the FFQ was developed within the same population of pregnant women. The reproducibility analysis was conducted in a sample of 690 pregnant women who provided food consumption data for the first and third trimester of pregnancy. The Spearman correlation coefficients for folate and vitamin B12 intake estimated by the two FFQs were 0.486 and 0.385 Maternal covariates were obtained from hospital medical records  and questionnaires administered by trained interviewers in first and third trimester of pregnancy. These covariates were maternal height, self-reported pre-pregnancy weight and weight at week 36 of pregnancy, which allowed for calculation of weight gain during pregnancy and body mass index (BMI). Data collected on sociodemographic characteristics included maternal age, maternal education level, employment data, family income, marital status, parity, planned pregnancy, and lifestyles  during the pregnancy.The R Project for Statistical Computing  was used for imputation of missing values of covariates. Multivariate imputations by chained equation in R (MICE package) were used to replace missing information . Ten impStatistical analyses were performed using the IBM SPSS Statistics v.21 software. General characteristics of the study population were described using geometric mean, arithmetic mean, standard deviation and 95% confidence interval for continuous variables and absolute  and relative frequencies (percentage) for categorical variables. Analyses were stratified by newborn sex.The Student’s t test was used to compare means of continuous variables with a normal distribution. The non-parametric Mann–Whitney test was used to compare quantitative variables that failed to fit a normal distribution. The Chi-squared test was used for qualitative variables comparison  and also to examine whether genotype distribution was in Hardy–Weinberg equilibrium.Bivariate and multinominal logistic regression analyses were used to evaluate association  between MTHFR(677)C>T maternal genotypes and anthropometric variables for gestational age (SGA versus AGA and LGA versus AGA). Homozygous CC was the reference genotype used. OR greater than 1 was considered a positive association (risk effect) whereas OR values less than 1 indicated negative association (protective effect). All models were stratified by newborn sex.p-values < 0.1.Statistical significance was assumed for p > 0.1).The distribution of maternal genotype and allele frequencies is shown in p < 0.01). The remaining newborn anthropometric measures failed to show significant differences. Mean total folate and vitamin B12 intakes of pregnant women were within the recommended range during pregnancy. When compared by sex, mean maternal vitamin B12 intake during the first trimester of gestation was significantly lower for female newborn infants (p = 0.055).The main clinical and sociodemographic characteristics of the study population are shown in p = 0.006).p < 0.05) male infants born to mothers carrying the T allele had a protective effect against LGA condition  p < 0.1). The same was observed when maternal genotype was CT or TT for female  p < 0.1) and male infants  p < 0.05).The association between anthropometric measures of newborns  stratified by sex and maternal MTHFR(677)C>T genotype is shown in p < 0.05).Maternal genotype TT was also a protective factor for being born LGA according to height, but only in female infants  p < 0.1; OR = 1.60 CI 95% ), although statistical significance did not remain in the adjusted model. In the case of female premature infants, an increased risk of being born SGA was found for the maternal CT genotype  p < 0.1) or CT/TT  p < 0.1).Although male infants had an increased risk of being born SGA according to weight index when their mothers had the heterozygous CT genotype (p > 0.1)Head and chest circumferences did not show significant associations with the maternal genotype C>T genetic polymorphism was associated with infant anthropometry at birth and with a higher risk of SGA in female premature infants.The frequency of maternal MTHFR 677T allele of our population (38%) was in agreement with those reported for other populations, which also found no differences in the distribution of genotypes and allele frequencies by sex . NewbornThe common MTHFR genetic polymorphism (677)C>T rs1801133) leads to the synthesis of a thermolabile enzyme, which may lead to high levels of homocysteine in the blood (hyperhomocysteinemia) that have been considered a risk factor for several adverse outcomes at birth. Previous studies have shown that the cytosine (C) to thymine (T) transition at position 677 within exon 4 of the MTHFR gene, which results in an alanine to valine amino acid substitution in the protein, is responsible for reducing enzyme activity in the homozygous TT and heterozygouw CT allelic variants by 60% and 30% of the CC genotype, respectively ,29. This leads toIn the absence of adequate folate availability, methionine production decreased, homocysteine accumulates and methylation reactions are compromised. Thus, folate, as a donor of carbon units in the one-carbon cycle, participates in the synthesis of nucleic acids as well as in the methylation of DNA sequences, including key placental genes. Folic acid supplementation during early pregnancy is intended to prevent the possible pathological conditions that a folate deficiency could cause ,36,37. TThe association between the maternal MTHFR(677)C>T genetic polymorphism and adverse birth outcomes has been previously reported in different populations, especially in pregnant women with inadequate folate intake. A recent meta-analysis showed that the maternal MTHFR(677)-T allele, and specifically carriers of the TT genotype, were associated with an increased risk of preterm birth and with low birth weight under most of the genetic models . That stIn our population, only 1% of pregnant women did not intake a folic acid supplements before or during the first trimester of pregnancy. The vast majority (99%) of pregnant women received a supplement of 400 or 500 micrograms of folic acid per day, which in almost all cases also included 2 to 2.6 micrograms of vitamin B12. The same folate intake was maintained during the third trimester of pregnancy. Thereby, the mean total folate intake was considered adequate, as it falls within the recommended dose of 600–1000 micrograms per day (679.79 µg/day (95% CI 669.78–689.80) during first trimester and 655.30 µg/day 95% CI 643.27–667.33 during the third trimester). This means that 77 and 65% of women had total folate intakes over the minimum recommended dose during the first and third trimester, respectively. Vitamin B12 total intake was always above the recommended dose of 2.6 µg/day during pregnancy. A daily folic acid intake of 400 µg per day has been reported to be sufficient to override the effects of unfavourable MTHFR genotypes on homocysteine and plasma folate levels . This maAltered methylation pattern in placental genes involved in fetal growth could be one of the biological mechanisms that would explain the association between the MTHFR(677)C>T genetic polymorphism and anthropometric measures at birth. Hypomethylation of DNA sequences of that gene, which usually are imprinted, could lead to overgrowth. That is, placental hypomethylation may indiscriminately activate various imprinted and not imprinted genes, including those involved in fetal growth. Likewise, in utero exposure to adverse environmental conditions might impair DNA methylation through altered availability of methyl donors provided by the one-carbon pathway and this effect could be sex-specific. Drake et al., observed that maternal smoking was associated with sex-specific alterations in fetal liver vitamin B12, plasma homocysteine and expression of enzymes in the one-carbon cycle in fetal liver . These mAlterations in DNA methylation have been proposed as the possible link between MTHFR(677) unfavorable variant and increased risk of pregnancy complications Del Gobblo et al. . That stp < 0.01 and r = 0.12, p < 0.05, respectively), and a percentage of agreement of 72% and 55%, respectively [The current study has a number of strengths and limitations. The major limitation was the semi-quantitative assessment of the maternal dietary folate intake obtained by questionnaires. We do not have data on biomarkers in biological samples, which otherwise is seldom used in cohort studies in which information of food and vitamins consumption is commonly obtained through food frequency questionnaires. The questionnaire we used was adapted from one previously validated in a Mediterranean population of pregnant women. In this validation study significant correlations were observed between total folate and vitamin B12 intake estimated by FFQ and plasma folate and vitamin B12 concentrations C>T genetic polymorphisms is associated with infant’s anthropometry at birth. Considering the potential effects of low folate availability due to diet and/or genetic background, it is necessary to continue emphasizing the importance of following the dietary recommendations during pregnancy, or at least to inform pregnant women on the risk of not following such recommendations. It is crucial to raise awareness on this issue, even in populations where folic acid supplementation has been implemented for years.


Frequent coaching was associated with increased adherence to evidence-based essential birth practices among birth attendants but not with improved maternal and perinatal health outcomes in the BetterBirth Trial, which assessed the impact of a complex intervention to implement the World Health Organization's Safe Childbirth Checklist. To promote sustainable behavior change, future coaching-based interventions may need to explore cost-effective, feasible mechanisms for providing more frequent coaching delivered with high coverage among health care workers for longer durations. Frequent coaching was associated with increased adherence to evidence-based essential birth practices among birth attendants but not with improved maternal and perinatal health outcomes in the BetterBirth Trial, which assessed the impact of a complex intervention to implement the World Health Organization's Safe Childbirth Checklist. To promote sustainable behavior change, future coaching-based interventions may need to explore cost-effective, feasible mechanisms for providing more frequent coaching delivered with high coverage among health care workers for longer durations. Coaching can improve the quality of care in primary-level birth facilities and promote birth attendant adherence to essential birth practices (EBPs) that reduce maternal and perinatal mortality. The intensity of coaching needed to promote and sustain behavior change is unknown. We investigated the relationship between coaching intensity, EBP adherence, and maternal and perinatal health outcomes using data from the BetterBirth Trial, which assessed the impact of a complex, coaching-based implementation of the World Health Organization's Safe Childbirth Checklist in Uttar Pradesh, India.For each birth, we defined multiple coaching intensity metrics, including coaching frequency (coaching visits per month), cumulative coaching , and scheduling adherence (coaching delivered as scheduled). We considered coaching delivered at both facility and birth attendant levels. We assessed the association between coaching intensity and birth attendant adherence to 18 EBPs and with maternal and perinatal health outcomes using regression models.Coaching frequency was associated with modestly increased EBP adherence. Delivering 6 coaching visits per month to facilities was associated with adherence to 1.3 additional EBPs . High-frequency coaching delivered with high coverage among birth attendants was associated with greater improvements: providing 70% of birth attendants at a facility with at least 1 visit per month was associated with adherence to 2.0 additional EBPs . Neither cumulative coaching nor scheduling adherence was associated with EBP adherence. Coaching was generally not associated with health outcomes, possibly due to the small magnitude of association between coaching and EBP adherence.Frequent coaching may promote behavior change, especially if delivered with high coverage among birth attendants. However, the effects of coaching were modest and did not persist over time, suggesting that future coaching-based interventions should explore providing frequent coaching for longer periods. The coaching process helps individuals use their existing skills, resources, and training to improve their performance and achieve personalized goals.–Although some studies have reported associations between increased intensity of coaching-related activities and improved quality of care,BOXCoaching Forma: coaching delivery method, including the coach's identity and experience level  and the strategies the coach used to generate behavior change Coaching Qualitya: coach's ability to correctly and consistently use coaching strategies to generate behavior changeCoaching Frequency: number of coaching sessions delivered over a specific duration of time Coaching Durationa: time period during which coaching is delivered Cumulative Coaching: accrual of exposure to coaching over time that is determined by both coaching frequency and coaching duration aCoaching intensity domain not covered in this analysis.One coaching-based intervention designed to improve the quality of care provided to mothers and newborns during facility-based childbirth is the BetterBirth Program. Although this intervention did not reduce maternal morbidity or maternal and perinatal mortality in a recent matched-pair, cluster-randomized trial conducted in Uttar Pradesh, India, it increased birth attendant adherence to 18 essential birth practices (EBPs) believed by experts to prevent or successfully manage complications during facility-based deliveries from an average of 7.9 in the control arm to an average of 11.1 in the intervention arm.In this article, we used data from the intervention arm of the BetterBirth Trial to assess the relationship between coaching frequency, cumulative coaching, and scheduling adherence with birth attendant adherence to EBPs and maternal and perinatal health outcomes. By investigating multiple dimensions of coaching intensity, we aimed to provide insights into the optimal coaching regimen for future coaching-based interventions.By investigating dimensions of coaching intensity, we aimed to provide insights into the optimal coaching regimen for future interventions.The BetterBirth Program was designed to promote the use of the World Health Organization's Safe Childbirth Checklist (SCC), a 28-item tool intended to assist birth attendants in performing EBPs. To ensure EBPs are completed in time to avoid potential complications, these items are organized into 4 “pause points”: (1) on admission, (2) just before pushing, (3) within 1 hour after birth, and (4) before discharge. When using the checklist, birth attendants can either first read the item and then complete the task or first provide care and then review the checklist to confirm that all tasks for that pause point have been completed.–The BetterBirth Program used an engage-launch-support model, which has been previously described in detail.Birth attendants, who were primarily auxiliary nurse midwives or general nurses in the labor and delivery wards, received peer-to-peer coaching from study staff who were nurses with training in childbirth and at least 2 years of experience in delivery. These coaching relationships were designed to be collaborative and were not designed to replace the existing traditional supervision structure. In practice, coaches' training, age, and years of experience were similar to that of the nurse birth attendants, who comprised approximately 78% of the birth attendant population. However, about 16% of the birth attendant population were auxiliary nurse midwives, who were older and had fewer years of formal training but more years of experience, and approximately 7% were lady medical officers, who were trained as physicians.Coaches' clinical skills were assessed via interviews during the recruitment process. Coaches received 5–7 days of skills-based training, which emphasized coaching skills, including relationship building; verbal and nonverbal interpersonal communication; handling difficult persons or situations; observation, listening, and speaking skills; and prioritizing and setting goals with frontline health workers. The training program for coaches also included a physician-led review of government guidelines around skilled birth attendants.,,,Coaching followed an “opportunity-ability-motivations-supplies” framework adapted from previous behavior change models.Birth attendant coaching was scheduled to occur twice per week during the first through fourth months of the intervention, once per week during the fifth and sixth months of the intervention, once every 2 weeks during the seventh month, and once per month in the eighth month for a total of 43 visits per health facility. Coaching visits were intended to last for the entire duration of a daytime shift (8 am to 5 pm). Additional details on the content of coaching sessions, perceptions around the credibility of the peer-coaching program, and the quality of the relationships between coaches and birth attendants have been published elsewhere.A parallel peer-to-peer coaching also occurred at the facility leadership level with facility leadership being coached by other physicians or public health professionals to help improve their abilities to provide leadership and supervision. In this manner, coaching was intended to support rather than substitute for existing supervision structures. Facility leadership coaching followed a similar, but less intensive, schedule for a total of 23 visits. To promote long-term sustainability, each site also designated a childbirth quality coordinator who was a well-respected facility-based staff member but not necessarily a supervisor or manager and was intended to serve as a long-term, facility-based coach and project champion. In practice, this role was often filled by a head nurse, medical officer in-charge, or pharmacist. This champion was intended to continue the practice of peer-coaching and not replace existing supervision practices.This implementation package was evaluated in a matched-pair, cluster-randomized trial that enrolled 120 primary-level health facilities in Uttar Pradesh, India, a region with high maternal  and neonatal  mortality.All facilities enrolled in the BetterBirth Trial were required to conduct at least 1,000 deliveries per year, have at least 3 birth attendants trained at the level of auxiliary nurse midwife or higher, have no concurrent quality improvement or research programs, and have district and facility leadership who were willing to participate. Forty-six facilities were primary health centers, 56 were community health centers, and 18 were first referral units.At each facility, registers were used to document the admission date for each woman in labor and any instances of facility-based mortality and morbidity. Data on 7-day health outcomes were obtained using a call center, which contacted mothers and their families between 8 and 42 days postpartum, followed by home visits if neither the woman nor a family member was reached by phone after 22 days postpartum.In a convenience sample of births occurring in 30 facilities  located in the Lucknow hub, which is in central Uttar Pradesh, additional direct observations of deliveries were conducted to collect data on birth attendant EBP adherence. Trained independent nurses observed and recorded EBP adherence using standardized data collection tools. Visits from independent data collectors occurred in addition to the coaching visits, which occurred in all intervention facilities. Unlike coaches, who used the opportunity-ability-motivation-supplies framework to improve birth attendant performance, study nurses who served as independent observers did not serve as coaches and did not intervene in clinical care. Data collection on EBP adherence occurred during 3 of the 4 pause points: on admission to facility, just before pushing, and within 1 hour after birth. However, practical considerations related to the timing and duration of labor prevented all births from being continuously observed from admission through discharge. Consequently, not all EBPs were observed for each birth. For intervention facilities, nurse coaches recorded the date of each coaching visit as well as the unique ID code for each birth attendant who was coached during that visit.,We considered 2 types of outcomes: birth attendant EBP adherence and maternal and perinatal health outcomes. EBP adherence was measured as the number of EBPs that a birth attendant successfully completed of the 18 practices that the World Health Organization recommends as essential for all mothers and newborns .29,30 PrMother-baby dyads were included as a birth in our EBP analysis if they occurred at 1 of the 15 intervention facilities where EBP adherence data were collected, occurred after the start of coaching at that facility, and were directly observed during admission to facility, just before pushing, and within 1 hour after birth such that adherence to all 18 practices was recorded. Our analysis was conducted exclusively among intervention sites to focus on likely effects of birth attendant coaching without potential confounding from other components of the complex intervention.As in the main trial, our primary health outcome was a composite outcome of events occurring within 7 days after delivery that included severe maternal morbidity, defined as self-reported complications including seizures, loss of consciousness for more than 1 hour, fever with foul-smelling vaginal discharge, hemorrhage, or stroke; maternal mortality; or perinatal mortality, defined as stillbirth or death within the first 7 days of life. A secondary composite health outcome consisting of only 7-day maternal or perinatal mortality was also considered.Mother-baby dyads were included as a birth in the health outcomes analysis if they occurred in an intervention facility after the start of coaching, if mothers consented to follow-up, and if data on 7-day outcomes were obtained. As in the main trial, dyads were included in the 7-day outcome analysis even if they were transferred to a higher-level facility before delivery. Because the timing of direct observations of birth attendant adherence to EBPs (that occurred 0–8 and 13–17 months after the start of coaching) differed somewhat from the timing of call center activities (that continued from 0–13 months after the start of coaching), the EBP adherence sample is not a subset of the health outcomes sample. However, some births appear in both samples.For each birth, we calculated metrics that reflected multiple domains of coaching intensity, including coaching frequency, cumulative coaching, and scheduling adherence. These metrics were based on the dates of the peer-to-peer birth attendant coaching visits that had occurred at a given facility before each birth. For coaching frequency, we assigned each birth a coaching intensity equal to the number of coaching visits occurring at that facility in the 30 days before the admission date (visits in the past month).Because we hypothesized that the impact of coaching on birth-related outcomes would be stronger when we considered the intensity of coaching provided to the birth attendants who conducted the deliveries rather than to the facility as a whole, we also created coaching frequency metrics that reflected coaching delivered at the birth attendant level. In this study, it was not possible to identify which birth attendant conducted a specific delivery, so we created coaching metrics that reflected the delivery of coaching among all birth attendants working at a single facility. These metrics included the average number of visits in the past month among birth attendants, the percentage of birth attendants receiving at least 1 visit in the past month, and the standard deviation of coaching visits in the past month among birth attendants. We hypothesized that facilities would experience more benefits from coaching if birth attendants had, on average, a greater number of visits in the past month, higher coaching coverage (percentage of birth attendants receiving at least 1 visit in past month), and a more equal distribution of coaching visits among birth attendants (lower standard deviation in visits among birth attendants in the past month).Supplemental Tables.All metrics reflecting coaching delivered at the birth attendant level were calculated under the assumption that the birth attendants listed in the coaching database reflected a complete list of birth attendants employed by the facility over the course of the intervention. These metrics did not consider staff turnover, which was assumed to be minimal over the intervention period. We also explored coaching frequency metrics calculated over a 1-week, rather than a 1-month, time horizon. However, since these 2 time windows produced similar results, we have presented only the results for the 1-month time horizon. Results for the 1-week time horizon can be found in the For cumulative coaching, we assigned each birth a coaching intensity equal to the total number of coaching visits accrued at the facility between the start of program and the admission date . As with coaching frequency, we believed that (a) coaching delivered at the birth attendant level would have a greater impact on birth attendant behavior change and health outcomes than coaching delivered at the facility level and (b) facilities with higher coverage of coaching at the birth attendant level would experience greater benefits from coaching. Therefore, for each birth we also calculated the mean number of visits accrued among birth attendants between the start of the program and the admission date and the standard deviation of coaching visits among birth attendants.Scheduling adherence was defined according to the prescribed frontline coaching schedule of attaining at least 2 visits per week during the first 4 months of the intervention, at least 1 visit per week during the fifth and sixth months, at least 1 visit every 2 weeks during the seventh month, and at least 1 visit per month during the eighth month. Current scheduling nonadherence was a binary variable reflecting whether the date of admission occurred on a day when the facility had deviated from this schedule. Cumulative scheduling nonadherence reflected the total number of nonadherent days accrued between the start of program and the date of admission. For example, if a facility had been 3 days late for its first coaching visit and 4 days late for its second coaching visit, then subsequent births would receive a cumulative scheduling nonadherence value of 7.For each birth, we calculated the mean number of visits accrued among birth attendants between the start of the program and the admission date and the standard deviation of coaching visits among birth attendants.Because the BetterBirth Program prescribed high-frequency coaching early in the intervention and gradually reduced the frequency of coaching over time, there were strong correlations among coaching frequency metrics, cumulative coaching metrics, and time since the start of the intervention. We reported the mean and standard deviation for each coaching metric and explored correlations between coaching metrics graphically and using Spearman correlation coefficients. To assess associations between each metric of coaching intensity and the outcomes of interest, we used generalized linear models and accounted for clustering at the facility level by estimating standard errors using the empirical variance with an exchangeable working covariance structure.For EBP adherence, we estimated the change in the number of EBPs that birth attendants adhered to associated with each coaching intensity metric using an identity link and a normal distribution. For binary health outcomes, we estimated the risk ratios associated with each coaching intensity metric using a log link and a binomial distribution.For all models, we used robust score tests to assess the statistical significance of model parameters.Because the effects of behavior change interventions often fade over time,Finally, we assessed whether the association between coaching intensity metrics and EBP adherence or health outcomes changed over the course of the intervention by adding an interaction between each coaching metric and months since the start of the intervention to our models. Because of the strong collinearities between coaching metrics and months since the start of the intervention, several models produced statistically significant interaction terms that were not interpretable. To ensure interpretability, we reported results for these interaction models only if both the time-by-coaching interaction term and the overall effect of coaching based on the joint null hypothesis that both the main effect of coaching and its interaction term were zero, were statistically significant at the α=.05 level.Data on EBP adherence at intervention facilities were collected for 3,283 births. We excluded 262 births that occurred before the start of the coaching intervention and 938 that were not observed for all 3 pause points for a final sample of 2,083 births . Health Supplemental Table).Fidelity to the coaching schedule was very high. By the end of the intervention, of the 60 facilities, 53 (88%) of facilities reached the target of 43 total coaching visits, 6 (10%) reached 42 visits, and 1 (2%) facility reached 37 visits. However, fidelity at the facility level did not necessarily translate into delivery of high-coverage coaching among birth attendants. Although birth attendants received, on average, 10 coaching visits by the end of the intervention, 34% received fewer than 5 visits. Fidelity at the facility level to the coaching schedule did not always translate into delivery of high-coverage coaching among birth attendants.In our primary model, all 4 coaching frequency metrics were significantly associated with increased EBP adherence . On averP<.01; test for overall significance of coaching: P=.04; Supplemental Tables show results from additional models). This cumulative coaching measure was associated with increased EBP adherence during the early months of the intervention when coaching occurred very frequently, but the positive association did not persist after coaching visits ceased  compared to the control arm  12 months into the intervention but no significant changes in health outcomes.Health outcomes were generally not associated with coaching.–Previous articles have reported dose-response relationships between coaching intensity and health worker behavior change,This is the first article that simultaneously investigated multiple domains of coaching intensity on care worker behavior change.,Our finding that frequent coaching was associated with modest improvements in EBP adherence is similar to previous reports. A recent meta-analysis found that strategies commonly used to improve health care worker performance including supervision, training, and group problem solving, are associated with improving health care worker performance by 1.0, 6.4, and 13.6 percentage points, respectively.In this study, all facilities were primary-level health facilities located in Uttar Pradesh, and all coaching was provided by external peer coaches according to the opportunity-ability-motivations-supplies framework.,–,Other programs that have used coaching as a Safe Childbirth Checklist implementation strategy have reported extremely variable EBP adherence at end line (range=32%–93%).Our data provide several practical insights for those seeking to implement or study future coaching-based interventions. First, even high fidelity to a facility-centric coaching schedule will not necessarily ensure that individual health care workers receive adequate coaching coverage. In our study, the unequal distribution of coaching among birth attendants likely reflect preferential behavior on the part of the coaches, dynamics related to the timing of shifts in birth facilities or are the result of staff turnover. Interventionists should specify and monitor coaching delivered at both the facility and the health care worker levels to identify whether these processes are taking place.Second, our study suggests that high-frequency coaching can improve health worker adherence to EBPs, but maintaining high-frequency coaching is a resource-intensive intervention. Future interventionists may wish to explore cost-effective methods for maintaining high-frequency coaching over longer periods of time, such as recruiting internal coaches, coaching more consistently on health systems at a facility-level, or combining in-person coaching visits with remote coaching methods.Although high-frequency coaching can improve health worker adherence to EBPs, it is a resource-intensive intervention.Third, identifying optimal coaching regimens requires designing interventions that have uncorrelated variation in coaching frequency and cumulative coaching. This could be achieved, for example, by conducting a 3-armed trial with 1 control arm, 1 arm receiving evenly spaced coaching visits over a set duration of time, and a third arm receiving the same total number of coaching visits delivered over the same duration of time but following a tapered schedule similar to that of the BetterBirth Program's.Finally, statistically significant improvements in quality of care indicators such as EBP adherence do not necessarily translate into meaningful improvements in health outcomes. In general, changes in quality of care indicators will be more likely to predict improvements in health outcomes if researchers choose quality of care indicators that are a valid surrogate for the primary health outcomes of interest, have been empirically demonstrated to have a causal relationship with the health outcome, if the magnitude of the change is clinically meaningful on the absolute scale  rather than on the relative scale , and if the quality of care indicators reflect most or all major determinants of the primary health outcome.Our analysis has several limitations. As discussed above, the BetterBirth Program's coaching schedule created strong correlations among coaching intensity metrics, which complicated the interpretation of some findings. Second, this analysis focused on 2 domains of coaching intensity, coaching frequency and cumulative coaching, but was unable to assess other domains, including coaching quality, which was unmeasured, or coaching form, which did not vary across sites. Therefore, we cannot comment on which aspects of coaching are most effective, and it is possible that alternative coaching models, such as those that coach on clinical quality rather than on adherence to certain tasks, may be more effective than the model used here. We also did not capture information on the duration or timing of individual coaching visits.Third, although we sought to minimize bias by adjusting for facility-level characteristics, residual confounding is possible if unmeasured facility or birth attendant characteristics that were associated with the outcomes also impacted coaches' behavior. For example, reports suggest that, to minimize travel time, coaches would provide difficult-to-reach facilities with visits on back-to-back days. If less accessible facilities experienced worse outcomes, we would expect this practice to bias our results against coaching frequency metrics. Coaches may have also been more likely to provide coaching to the facilities or birth attendants who were most motivated and receptive to their help and less likely to provide coaching to relatively junior nurses, who often staff evening, night, and weekend shifts. We would expect both of these processes to bias our results in favor of coaching.Fourth, assessment of EBP adherence outcomes was based on directly-observed sessions that took place during daylight hours and may not reflect adherence at night or when birth attendants were not observed. Finally, because we were unable to link individual birth attendants to individual births in our dataset, we assessed the effectiveness of coaching delivered at the birth attendant level using facility-level aggregated metrics that did not consider staff turnover within the health facility. We would expect this measurement error to result in an underestimation of the direct benefits of providing coaching to individual birth attendants.Frequent coaching was associated with increased adherence to essential birth practices among birth attendants in the BetterBirth Trial. The effect size was greater for coaching delivered at the birth attendant level compared to coaching delivered at the facility level. Cumulative coaching metrics were not associated with essential birth practice adherence, suggesting that the short-term effects of high-frequency coaching may not translate into sustained effects of cumulative coaching over time. Future coaching-based interventions seeking to promote sustainable change may need to consider identifying sustainable, cost-effective models for providing more frequent, high-coverage coaching for longer periods. Coaching was generally not associated with health outcomes, suggesting that additional coaching and other implementation strategies may be needed to achieve the desired health impact.