Inorganic arsenic (iAs) is a ubiquitous metalloid and drinking water contaminant. Prenatal exposure is associated with birth outcomes across multiple studies. During metabolism, iAs is sequentially methylated to mono- and di-methylated arsenical species (MMAs and DMAs) to facilitate whole body clearance. Inefficient methylation  is associated with increased risk of certain iAs-associated diseases. One-carbon metabolism factors influence iAs methylation, modifying toxicity in adults, and warrant further study during the prenatal period. The objective of this study was to evaluate folate, vitamin B12, and homocysteine as modifiers of the relationship between biomarkers of iAs methylation efficiency and birth outcomes.Data from the Biomarkers of Exposure to ARsenic (BEAR) pregnancy cohort (2011–2012)  with maternal urine and cord serum arsenic biomarkers and maternal serum folate, vitamin B12, and homocysteine concentrations were utilized. One-carbon metabolism factors were dichotomized using clinical cutoffs and median splits. Multivariable linear regression models were fit to evaluate associations between each biomarker and birth outcome overall and within levels of one-carbon metabolism factors. Likelihood ratio tests of full and reduced models were used to test the significance of statistical interactions on the additive scale (α = 0.10).Among urinary biomarkers, % U-MMAs was most strongly associated with birth weight . Larger, more negative mean differences in birth weight were observed among infants born to women who were B12 deficient  or experiencing hyperhomocysteinemia . Generally, mean differences in birth weight were attenuated among infants born to mothers with higher serum concentrations of folate and vitamin B12 (or lower serum concentrations of homocysteine). Effect modification by vitamin B12 and homocysteine was significant on the additive scale for some associations. Results for gestational age were less compelling, with an approximate one-week mean difference associated with C-tAs , but not meaningful otherwise.Tissue distributions of iAs and its metabolites  may vary according to serum concentrations of folate, vitamin B12 and homocysteine during pregnancy. This represents a potential mechanism through which maternal diet may modify the harms of prenatal exposure to iAs.The online version contains supplementary material available at 10.1186/s12940-022-00875-7. As a result of its natural occurrence in soil and rock, globally, at least 200 million individuals are exposed at harmful levels in contaminated groundwater , 2. In ar (VEGF) . Impacts(KCNQ1)) , 11 obseIII, an arsenic species established as the most toxic form in most tissues, including placenta [iAs is metabolized and eliminated from the body through a process that is influenced by micronutrients involved in one-carbon metabolism. This pathway is composed of the folate and methionine cycles and contributes to DNA synthesis, cellular growth, and proliferation . During placenta , 16, 17.Each methylation reaction produces a methylated product and S-adenosylhomocysteine (SAH), which is hydrolyzed to homocysteine , a non-pWe estimate associations between each biomarker and (1) birth weight and (2) gestational age overall and within levels of folate, vitamin B12, and homocysteine using data from the Biomarkers of Exposure to ARsenic (BEAR) pregnancy cohort. Likelihood ratio tests of main effects models against full models incorporating interaction terms were also used to evaluate statistical interactions, or potential effect modification, on the additive scale. We hypothesized that greater bioavailability of folate and vitamin B12 would increase conversion of MMAs to DMAs, thereby attenuating negative associations between biomarkers and birth outcomes.n = 206) provided informed consent for participation. Six of the women providing informed consent were excluded due to a confirmed twin pregnancy (n = 1) or sample collection failure (n = 5), with the final cohort composed of 200 mother-infant pairs.The Biomarkers of Exposure to ARsenic (BEAR) cohort comprises pregnant women who resided in Gómez Palacio, State of Durango, Mexico between August 2011 and March 2012 . ArsenicParticipants completed detailed questionnaires capturing information on demographic characteristics  and sources of iAs exposure . Birth weight was measured at the time of delivery by the physician. Gestational age was estimated using the date of each mother’s last menstrual period. All study procedures were previously approved by the Institutional Review Boards at the University of North Carolina at Chapel Hill and the Universidad Juárez del Estado de Durango, and each participant gave written, informed consent to participate and provide urine samples, drinking water samples, and umbilical cord blood.III) and pentavalent (AsV) species. The limits of detection (LODs) for U-iAs, U-MMAs, and U-DMAs were 0.2, 0.1, and 0.1 μg/L, respectively. These LODs are based on the previously published instrumental LODs [To characterize maternal exposure to iAs, concentrations of iAs and its metabolites were assessed in spot urine samples. These samples were collected at the time of delivery, immediately transferred to cryovials, and stored in liquid nitrogen. Urine samples packed in dry ice were shipped to the University of North Carolina at Chapel Hill for further processing. To account for differences in water intake or differential hydration, the specific gravity (SG) of each urine sample was measured using a handheld refractometer . Additionally, urine iAs (U-iAs) and metabolites (U-MMAs and U-DMAs) were measured using hydride generation atomic absorption spectroscopy (HG-AAS) with cryotrapping . As appltal LODs , 22 and tal LODs . Total uTo characterize fetal exposure to iAs, concentrations of iAs and its metabolites in cord serum  were measured using hydride generation with cryotrapping coupled to inductively-coupled plasma mass spectrometry (HG-CT-ICP-MS), as described previously . This ten = 7) with inefficient amounts of serum (< 200 μL) required for analyses.Folate, vitamin B12, and homocysteine were previously measured in maternal serum samples collected between admission to the hospital and the time of delivery . All samn = 3) or those missing data on one-carbon metabolism factors (n = 7). Unadjusted and adjusted associations between biomarkers of iAs methylation efficiency (%) and first birth weight (grams) and then gestational age (weeks) were estimated overall and within levels of folate, vitamin B12, and homocysteine. Folate, vitamin B12, and homocysteine were dichotomized using clinical cut-offs  and median splits. Using a directed acyclic graph approach, model covariates were selected for if they were: on an open backdoor path and/or (2) based on their a priori status as determinants of low birth weight [birth outcome is either birth weight or gestational age. X1 is the biomarker of iAs methylation efficiency . The β coefficient for Xi is the expected mean difference in birth weight or gestational age corresponding to a 1% increase in the respective biomarker. Zβ is a matrix of potential confounders. Model 1 included the following covariates: maternal age at delivery (years), smoking during pregnancy , and U-tAs (μg/L). Model 2 incorporated the same covariates and folate, vitamin B12, and homocysteine to account for the entire profile of one-carbon metabolism factors. Model 3 incorporated the same covariates as model 1 and excluded women reporting seafood intake within 7 days of providing samples to evaluate potential exposure misclassification resulting from additional sources of organic arsenic species in fish [n = 3) to reduce confounding by gestational age [n = 2), and those missing data on one-carbon metabolism factors (n = 7), resulting in the final inclusion of 188 mother-infant pairs in unadjusted and adjusted models . Models for gestational age excluded mothers missing data on smoking (n = 2) and one-carbon metabolism factors (n = 7), resulting in the final inclusion of 191 mother-infant pairs in unadjusted and adjusted models . The leave-one-out approach was also used in a sensitivity analysis to account for the dynamics of arsenic metabolism, as done in other studies [Biomarker levels in maternal urine or infant cord serum below the LOD were adjusted using the following formula: LOD/ (√2) . Descriph weight   between biomarkers and one-carbon metabolism factors were also assessed on the additive scale using a likelihood ratio test (LRT) to test whether an interaction model fit the data better than a main effects model, with statistical significance defined at α < 0.10. The (1) interaction and (2) main effect model forms were as follows:n = 4), and the average birth weight among male and female newborns was 3453 (range of 2100 to 5120) grams and 3215 (range of 1800 to 4200) grams, respectively. The distributions of iAs, MMAs, DMAs, and total arsenic (tAs) measured in urine (U-) and cord serum (C-) are detailed in Table n = 7), U-MMAs (n = 4), C-iAs (n = 186), C-MMAs (n = 127), and C-DMAs (n = 54). Since fewer than 30% of samples contained measurements above the LOD for C-iAs and C-MMAs, only C-tAs was used to represent fetal exposure. The mean concentrations of vitamin B12, folate, and homocysteine were 130.97 pM, 40.93 nM, and 6.86 μM, respectively. Two women were deficient in folate (< 9 nM), 142 (74%) women were deficient in B12 (< 128 pM), and 14 (7%) women experienced hyperhomocysteinemia (> 10.4 μM).This study includes 200 mother-infant pairs (18–41 years old) participating in the Biomarkers of Exposure to ARsenic (BEAR) pregnancy cohort. Women were recruited in the State of Durango, Mexico, a region characterized by high exposure risk . In brieρ < ±0.40) , while homocysteine was positively correlated (ρ = 0.24). Plasma folate was negatively correlated with homocysteine (ρ = − 0.23) and birth weight (ρ = − 0.15). Birth weight was also negatively correlated with % U-MMAs (ρ = − 0.14). Gestational age was correlated with birth weight (ρ = 0.17) and vitamin B12 (ρ = − 0.13).Generally, biomarkers of iAs methylation efficiency were strongly correlated with each other (Spearman correlation coefficient (ρ) > ±0.60), but the strength of other correlations was weak (40) Fig. . Vitamin, Table Associations ) between biomarkers of methylation efficiency and birth outcomes were first evaluated across all mother-infant pairs. Among urinary biomarkers, % U-MMAs was most strongly associated with birth weight in unadjusted and adjusted models   on the additive scale for homocysteine  and vitamin B12 (in relation to % U-DMAs).These trends were conserved in relation to homocysteine, where the negative association between biomarkers and birth weight was stronger among infants born to mothers with hyperhomocysteinemia or serum concentrations equal to or greater than the median. As an example, shown in Table Models evaluating gestational age were also stratified by levels of one-carbon metabolism factors Table . No meanOne-carbon metabolism factors modify iAs toxicity and the severity of iAs-associated health outcomes among adults , 35, butIII is established as the most toxic arsenic species in most tissues including placenta [Seventy-four percent of mothers in the BEAR cohort were deficient in vitamin B12, a clinical condition independently associated with maternal and neonatal outcomes, including impaired fetal growth . The relplacenta , 16, 17,placenta . Group dplacenta . Given tplacenta  and multplacenta  approachThe present study is among the first to examine homocysteine in relation to iAs-associated lower birth weight. We observed stronger and more negative associations between % U-iAs, % U-MMAs and C-tAs and birth weight in higher maternal homocysteine strata. Homocysteine is a metabolite of one-carbon metabolism that is independently associated with preeclampsia, intrauterine growth retardation, and other adverse pregnancy outcomes , 46. PriAssociations between C-tAs and birth outcomes were larger and more imprecise, as compared to urinary biomarkers. Generally, the association between C-tAs and gestational age was more positive among infants born to mothers with higher vitamin B12 or lower homocysteine levels, but less consistent for associations with birth weight. Larger, more imprecise estimates likely reflect the narrow range of C-tAs (1.15 to 4.0 pg As/mL). Inference is further complicated by the use of left-censored data, given the number of samples below the LOD for cord serum arsenicals.This study is not without limitations. Given its size, the BEAR cohort is underpowered to detect statistical interactions. Moreover, stratified analyses required even smaller sample sizes, decreasing precision. Second, one-carbon metabolism factors were assessed at the end of pregnancy and maternal plasma levels are altered over the course of pregnancy in order to support fetal growth . Early pIn a cross-sectional study, we found that associations between % U-MMAs and birth weight were attenuated by elevated levels of vitamin B12 and folate in maternal serum. These trends were also observed using strata defined by clinical cut-offs. Differences in rates of conversion of MMAs to DMAs may occur according to levels of one-carbon metabolism factors. This study may have public health implications as it positions B vitamins as potential modifiers of the harms of exposure to iAs during pregnancy, but validation is needed given the limitations of our study design. In most arsenic-endemic regions, folic acid fortification is not mandated . VitaminAdditional file 1: Figure S1. Simplified directed acyclic graph (DAG).Additional file 2: Table S1. Results from models fit using the leave-one-out approach.


Preterm birth is a major healthcare problem and has been rising gradually in the past three decades in China. Yet the ethnic differences in the rates and distributions of preterm birth remain largely unknown in China. This study used data from Yunnan, a multiethnic province, to explore the differences in preterm birth across ethnicities.A population-based observational study was conducted based on data from the National Free Preconception Health Examination Project in rural Yunnan from Jan 1, 2014 to Dec 31, 2018. Pregnancies with at least one livebirth were included in this study. We estimated the rates and 95% confidence intervals (CIs) of overall preterm birth (born < 37 weeks’ gestation), moderate to late preterm birth (born between 32 and <37 weeks’ gestation), very preterm birth (born between 28 and 31 weeks’ gestation), and extremely preterm birth (born < 28 weeks’ gestation) across maternal ethnicity and compared them using log-binomial regressions. Multivariable log-binomial regressions were used to assess the association between maternal ethnicity and preterm birth with adjustment for potential confounders, including year of delivery, maternal age at delivery, education, occupation, pre-pregnancy body mass index, history of chronic disease, history of preterm birth, smoking and drinking alcohol during early pregnancy, and parity and multiple pregnancy of current pregnancy.Among 195,325 women who delivered at least one live baby, 7.90%  were born preterm. The rates of moderate to late preterm birth, very preterm birth, and extremely preterm birth were 6.20% , 1.18% , and 0.52% , respectively. The rates of overall preterm birth, moderate to late preterm birth, very preterm birth, and extremely preterm birth differed across maternal ethnicity. The preterm birth rates in Dai (10.73%), Miao (13.23%), Lisu (12.64%), Zhuang (11.77%), Wa (10.52%), and Lagu (12.34%) women were significantly higher than that in Han women, and the adjusted relative risks were 1.45 , 1.74 , 1.60 , 1.60 , 1.40 , and 1.67  respectively. There was no difference in preterm birth rate between Han women and Hani, Bai, or Hui women.This study found notable differences in the rates of preterm birth and its sub-categories across maternal ethnicities, which were especially higher in ethnic minority women. The findings suggest that greater efforts to reduce ethnic inequalities in preterm birth. Future studies are warranted to understand the drivers of ethnic inequalities in preterm birth in China. Preterm birth is defined as babies born alive before 37 completed weeks of gestation or less than 259 days from the first date of a woman’s last menstrual period by the World Health Organization (WHO) , 2. GlobPreterm birth is considered to be a syndrome initiated by multiple mechanisms, including infection or inflammation, uteroplacental ischemia or hemorrhage, uterine overdistension, stress, and other immunologically mediated processes . The comThe National Free Preconception Health Examination Project (NFPHEP) was launched by the Chinese National Health and Family Planning Commission and Ministry of Finance in 2010 to provide free health examinations and other services before conception for couples who planned to become pregnant in the next 6 months, as well as follow-up in their first trimester of pregnancy and after delivery in rural areas. In the current study, we used data from the NFPHEP in Yunnan, a multiethnic province in Southwest China, to estimate the differences in preterm birth rates in the Han ethnicity and individual ethnic groups.We utilized data from the NFPHEP in rural Yunnan, China. In the NFPHEP, free preconception counseling, medical examinations, and management during pregnancy for all couples who had made their conception plan were provided by well-trained local health workers and obstetricians. Detailed information on the design, organization, and implementation of the NFPHEP has been described elsewhere , 22. BriThe study was terminated when women were enrolled by the NFPHEP in rural Yunnan and had pregnancy outcomes, such as normal birth, preterm birth, spontaneous abortion, induced abortion or stillbirth, between January 1, 2014, and December 31, 2018. This study was approved by the Institutional Review Board of the Chinese Association of Maternal and Child Health Studies and all participants provided written informed consent before enrollment.Preterm birth is the primary outcome of this study. We defined preterm birth as babies born alive before 37 weeks of pregnancy are completed according to the WHO . FurtherAnalyses by ethnic groups were restricted to those with at least 1,000 participants and the ethnic groups with fewer than 1,000 participants were categorized into the “other” group. The ethnicity groups in this study were categorized into Han, Yi, Dai, Hani, Miao, Bai, Lisu, Zhuang, Hui, Wa, Lagu, and other.2 (underweight), 18.5–23.9 kg/m2 , 24.0–27.9 kg/m2 (overweight), and ≥28.0 kg/m2 (obesity). Women were classified as having a history of chronic disease if they had one or more chronic diseases, including anemia, hypertension, heart disease, diabetes, epilepsy, thyroid disease, chronic nephritis, cancer, tuberculosis, hepatitis B, and mental or psychological disorders, whereas women were classified as not having a history of chronic disease if they did not have any of these diseases. Smoking status and drinking status during early pregnancy were both categorized as “yes” or “no” based on whether women smoked or drank alcohol during early pregnancy. Parity was divided into primiparous or multiparous based on current delivery. Type of pregnancy was grouped into singleton or multiple pregnancy based on current delivery.Maternal age at delivery was calculated based on the birth date and delivery date of women and was categorized into six groups: 18–24, 25–29, 30–34, 35–39, 40–44, and 45–49 years. Education level was categorized into primary school or below, junior high school, senior high school, and college and above. Occupation was categorized as farmer or worker. Pre-pregnancy body mass index (BMI) was calculated by dividing weight in kilograms by the square of height in meters and was categorized into four groups: <18.5 kg/mWe summarized the maternal characteristics using the median and interquartile range (IQR) for continuous variables or frequencies (percentages) for categorized variables. The categorized characteristics of women across ethnicities were compared using Chi-square tests. We calculated the numbers and rates of preterm birth and sub-categories of preterm birth overall and separately according to maternal ethnicity. The rates with 95% confidence intervals (CIs) of preterm birth and each sub-category of preterm birth were calculated as the number of liveborn preterm deliveries divided by the total number of deliveries. Log-binomial regression models were used to estimate the crude risk ratios (cRRs) and 95% CIs of preterm births and sub-categories of preterm birth for maternal ethnicity. Multivariable log-binomial regression models were used to estimate the adjusted risk ratios (aRRs) with 95% CIs of preterm birth with maternal ethnicity with adjustment for potential confounders of preterm birth. All descriptive and inferential statistics were estimated using SAS 9.4 . Two-tailed p values less than 0.05 were considered statistically significant.P < 0.05). No difference in history of preterm birth was observed in women across ethnicities. The detailed frequencies and percentages of maternal sociodemographic characteristics, pre-pregnancy BMI, status of smoking and drinking during early pregnancy, history of chronic disease, history of preterm birth, and parity and type of current pregnancy by maternal ethnicity are summarized in There were 197,058 women who had pregnancy outcomes at a reproductive age between 18 and 49 years in rural Yunnan between January 1, 2014, and December 31, 2018. We included 195,325 women aged 18–49 years women in rural Yunnan who delivered livebirths after excluding 1,560 women with abortion or stillbirth and 173 women with missing data with respect to gestational weeks in this period . The medP < 0.001) but higher than that in Yi  (P = 0.001). There was no difference in preterm birth rates between Han women and Hani, Bai, or Hui women. For the rates of sub-categories of preterm birth across ethnicities, Yi women had a lower moderate to late preterm birth rate than Han women (P = 0.013); Bai women had a higher moderate to late preterm birth rate but a lower extremely preterm birth rate than Han women (P < 0.05); Dai, Zhuang, and Lagu women had a higher rate of moderate to late preterm birth and very preterm birth than Han women (P < 0.01); and Miao, Lisu, Wa, and other minority women had a higher rate of moderate to late preterm birth, very preterm birth, and extremely preterm birth than Han women (P < 0.001).The rates of preterm birth and sub-categories of preterm birth in overall and by ethnicity in 2014-2018 are presented in According to the concentration of ethnic minorities in Yunnan, the rates of preterm birth were also marked in maps to clearly show the ethnic and regional disparities in preterm birth . At the In the multivariable regression models adjusted for maternal sociodemographic characteristics, pre-pregnancy BMI, status of smoking and drinking alcohol during early pregnancy, history of chronic disease, history of preterm birth, and parity and type of current pregnancy, preterm birth was significantly associated with ethnicity among women with livebirth in rural Yunnan . ComparePreterm birth is a major healthcare problem affecting 15 million births worldwide every year. We performed a population-based, observational study in rural Yunnan, China, to estimate the rates of preterm birth and sub-categories preterm birth as well as the ethnicity differences in preterm birth. We found that the preterm birth rate was 7.90% in rural Yunnan in 2014–2018, and the rates of sub-categories preterm birth were 6.20% for moderate to late preterm birth, 1.18% for very preterm birth, and 0.52% for extremely preterm birth in this period. The overall preterm birth rate decreased from 8.93% in 2014 to 6.63% in 2018; however, it increased in Lagu (from 9.02 to 9.29%), Wa (from 8.54 to 8.69%), Miao (from 11.36 to 14.83%), and Zhuang (from 11.40 to 12.77%) women from 2014 to 2018. This study found significant differences in rates of preterm birth and sub-categories preterm birth across maternal ethnicity. After adjusting for potential confounders, Yi women had a decreased risk of preterm birth compared with Han women, and Dai, Miao, Lisu, Zhuang, and Wa women had an increased risk of preterm birth compared with Han women.The preterm birth rate in rural Yunnan reported in our study 7.90%) was slightly higher than the pooled rate (7.04%) estimated for China in 2015–2016 reported by one previous meta-analysis 0% was sl, and anoThere are few studies exploring the distribution of preterm birth and sub-categories of preterm birth in ethnic minorities and Han women in China. This study using data from NFPHEP in Yunnan, a multiethnic province in Western China, found significant differences in rates of preterm birth and sub-categories preterm birth across maternal ethnicity. We found that the preterm birth rates ranged from 6.15 to 13.23% across maternal ethnicities, with the highest rate in Miao women (13.23%) followed by Lisu 12.64%) and Lagu (12.34%). We also found that the trends in preterm birth rates from 2014 to 2018 differed across maternal ethnicity and an increased rate was observed in Lagu, Wa, Miao, and Zhuang women. In addition, there were also differences in the rates of sub-categories of preterm birth across maternal ethnicities, with the highest rates of both moderate to late preterm birth (9.62%) and very preterm birth (2.51%) observed in Miao women and the highest rate of extremely preterm birth observed in Lagu women (1.21%). The preterm birth rates of several ethnic minorities in rural Yunnan, such as Miao (13.23%), Lisu (12.64%), and Lagu (12.34%), were much higher than the global rate (10.6%) in 2014 % and Lag. In ChinThe large sample size of this study provided, to our knowledge, the first direct comparison of preterm birth rates among major minority groups in Western China within one survey, using the definition of preterm birth and its sub-categories according to the WHO. However, some limitations in this study should also be acknowledged. First, this study was conducted in Yunnan, a multiethnic province in Southwest China, which differs from other provinces in population structure and ethnic composition, perhaps leading to limited national representativeness. Thus, future studies are warranted to further explore the association between ethnicity and preterm birth in China to achieve the Millennium Development Goal for maternal and reproductive health. Second, this study was unable to separate spontaneous preterm births from iatrogenic preterm births because the mode of delivery was simply grouped into vaginal or cesarean delivery in the NFPHEP. Third, several factors possibly associated with preterm birth were not included in this study, such as environmental and nutritional factors, socioeconomic status, and lifestyle.In summary, we found that the preterm birth rates ranged from 6.15 to 13.23% across maternal ethnicities in rural Yunnan. Notable differences in rates of preterm birth and its sub-categories were observed across maternal ethnicity, especially higher in ethnic minority women. We recommend further research to understand the drivers of ethnic inequalities in preterm birth, together with policy changes to improve the equity of access to maternal and child health services for women from minority ethnic groups in China.The original contributions presented in this study are included in the article/This study was approved by the Institutional Review Board of the Chinese Association of Maternal and Child Health Studies and all participants provided written informed consent before enrollment.GC and ML searched the literature, designed the study, analyzed the data, interpreted the results, and drafted the manuscript. YY, CK, and HY collected the data, interpreted the results, and revised the manuscript. GC, JL, and ML conceived the study, designed the study, supervised the study, interpreted the results, and revised the manuscript. All authors approved the final version of the manuscript.


Using BCl3 only Lewis adduct formation with Lewis basic sites in the template is observed. While combining BBr3 and the template containing an amide linker only led to amide directed ortho C−H borylation, with no pyridyl directed meta borylation. The amide directed borylation is selective for the ortho borylation of the aniline derived unit in the template, with no ortho borylation of the phenylacetyl ring – which would also form a six membered boracycle – observed. In the absence of other aromatics amide directed ortho borylation on to phenylacetyl rings can be achieved. The absence of meta‐borylation using two templates indicates a higher barrier to pyridyl directed meta borylation relative to amide directed ortho borylation and suggests that bespoke templates for enabling meta‐directed electrophilic borylation may be required.Two templates used in  Two templates used in metal catalysed directed meta functionalisation led to either no borylation, or only ortho borylation under electrophilic borylation conditions. In the absence of the complex template amide directed ortho borylation onto phenylacetyl groups is shown to be possible. In this area C−H borylation is one of the most developed transformations due to the synthetic versatility of C−B containing units.ortho‐C−H borylation methodologies have been reported.[BX4] that effects C−H borylation.ortho‐to NH alkyl group also was omitted as based on previous studies borylation conditions were envisaged that would only proceed via the borenium cation formed from activation of the pyridyl→BX3 moiety. Initially BCl3 was utilised as it is reported that BCl3 does not affect amide directed electrophilic borylation,[BCl4]) is endergonic,meta C−H borylation using 1/BCl3. Therefore, the addition of AlCl3 to 2 was explored to make borenium cation formation exergonic and thus lower the overall barrier to meta borylation,3 appears to displace BCl3 from the amide, as only the pyridyl→BCl3 resonance is observed post AlCl3 addition (at δ11B=8.4 ppm). More insight into the relative stability of the pyridyl‐BCl3 and amide(O)→BCl3 adducts was forthcoming from the exposure of 2 to “wet” (non‐purified) solvent/chromatographic work up which produced compound 3  now observed attributable to the pyridyl→BCl3, Figure S1). This confirms the expected stronger binding of BCl3 to pyridyl relative to amide.The formation of borenium cations from pyridyl→BCl2][BBr4] boreniums could be accessed selectively (over [amide‐BBr2][BBr4]) we explored the controlled addition of BBr3. One equivalent of BBr3 was added to a DCM solution of 1. Analysis of the 11B NMR spectrum showed four resonances at δ11B=−1.3, −7.4, −11.5 and −24.3. These resonances can be assigned as follows: the δ11B=−7.4 is in the region expected for pyridyl→BBr3 adducts, the δ11B=−11.5 is closely comparable to benzoyl→BBr3 adducts3 moiety, the δ11B=−24.3 is consistent with [BBr4]−, while the broad resonance at δ11B=−1.3 is assigned as the product from amide directed C−H borylation. This indicates that BBr3 reacts unselectively with the Lewis basic sites in 1 thus can affect amide directed ortho borylation even with only 1 equiv. of BBr3. Indeed, heating the reaction mixture to 60 °C in a sealed tube resulted in disappearance of three of the resonances in the 11B NMR spectrum with only δ11B=−7.4 ppm (pyridyl→BBr3) persisting and a new broad resonance appearing at δ11B=0.9 ppm, the latter is in the region for acyl‐coordinated aryl−BBr2 species in 6‐membered boracycles.To determine if [pyridyl‐BBr11B NMR spectra), >2 equivalents of BBr3 are required to achieve complete conversion of 1. Therefore, ca. 3 equivalents of BBr3 were added to 1, which led to a complex mixture of species in the 1H NMR spectrum at room temperature. The 11B NMR spectrum exhibited mostly the same resonances as observed when one equivalent of BBr3 was used, however the resonance at δ11B=−24.3 (due to BBr4−) was no longer present and instead a broad resonance at δ11B=+22.0 was observed attributed to a halide transfer equilibrium between BBr3 and BBr4− (vide infra). Heating this reaction mixture to 60 °C resulted in complete conversion to a single species in the 1H NMR spectrum that was consistent with compound 4 , 2.9 and −7.8; the δ11B=2.9 resonance is assigned as the C−H borylated unit in species, 4. Note this species changes chemical shift in the presence of excess BBr3 due to reversible bromide abstraction (vide infra). The species at δ11B=−7.8 is as expected for a pyridyl→BBr3 moiety. To further confirm this assignment and enable full characterisation crystals suitable for X‐ray diffraction analysis were grown by layering a DCM solution of 4 with pentane. The resultant solid‐state structure confirmed that ortho borylation had occurred via amide direction producing a 6‐membered boracycle, while the pyridyl group forms a Lewis adduct with BBr3. Ortho borylation occurred exclusively on the aniline derived phenyl and results in planarization of part of the directing template (displacement maximum of 0.023 Å from the plane of O1−B1−C12−C11−N1−C10). Locking the template in the conformation required for the 6‐membered boracycle reduces the flexibility of the template and may help prevent meta borylation from occurring as post ortho borylation, (and using excess BBr3) prolonged heating of 4 does not lead to any further C−H borylation. This indicates that preventing amide directed ortho borylation is essential when using amide containing templates and BBr3, this is closely related to the findings of Yu with Pd catalysed meta‐functionalisation.As two boron atoms are incorporated into the product  under a range of conditions, with complex mixtures formed from which the only boron containing species that can be assigned with confidence being due to pyridyl→BX3 adducts . The absence of C−H borylation was supported by work up with pinacol/NEt3, which revealed no species containing C−Bpin moieties were formed (by 11B NMR spectroscopy). Therefore, the failure of template 5 in meta borylation under standard electrophilic borylation conditions is not due to preferential ortho borylation but is presumably due to a high energy barrier to electrophilic borylation using BX3via a 12 membered transition state.With no 1 only one ortho borylation product is formed, compound 4, with no alternative ortho borylation product, compound B, observed . To the best to our knowledge amide directed electrophilic borylation has not been reported to date for these types of substrates.It is notable that during the amide directed borylation of 6 with 2.5 equivalents of BBr3 resulted in a species containing a resonance in the 11B NMR spectrum at δ11B=−11.0, in the region for an amide(O)→BBr3 adduct, thus it is tentatively assigned as 36‐BBr  – the by‐product of C−H borylation . However, two new resonances were observed in the in‐situ11B NMR spectrum suggesting two boron centers are incorporated into the product. Under these conditions the two new resonances in the in‐situ11B NMR spectrum are at δ11B=+10.2 and +42.8, which we assign as an equilibrium between 8 A and 8 B, with the BBr3 associated with either O or N in 8 B as it is not removed in‐vacuo. Note the 11B chemical shifts were dramatically affected by the equivalents of BBr3 used in this reaction (vide infra).The reaction of 8 A/8 B. The solid‐state structure  Å, within the combined van der Waals radii for B and Br (Σ=3.75 Å)vide infra for more detailed structural discussion). To further examine the proposed equilibrium between 8 A and 8 B, variable temperature NMR spectroscopy studies were conducted. Incremental cooling of a DCM solution of 8 A/8 B  showed a gradual change in the 11B NMR spectra whereby the boron centre in the cationic component was shifted gradually downfield from δ11B= 37.5 to δ11B ca. 42 ppm (in the range expected for an ArylB(OR)Br species),11B=−16.6 to δ11B=−25 ppm  to probe the effect on both 11B resonances ; while the cationic component moves to a limiting δ11B=+43.2 (consistent with the three coordinate boron centre in 8 A), the second resonance shifts closer and closer to that for free BBr3 , as expected for a fast exchange of bromide between BBr3/BBr4− and an increasing quantity of BBr3.Crystals of the borylated product suitable for X‐ray diffraction studies were grown by slow diffusion of pentane into a DCM solution of e Figure , bottom  at 3.474 Å, withi at 3.474 Å, withi6/BBr3 compound 7 was reacted with BBr3. While the Lewis adduct 37‐BBr forms rapidly, using 2.5 equivalents of BBr3 and heating at 60 °C  resulted in incomplete conversion of 37‐BBr to the borylated species 9 A/9 B, after 16 hours. High levels of conversion (by NMR spectroscopy) to 9 A/9 B required 3 days of heating. It should be noted only 9 A is shown in Figure 9 A (to form 9 B) as observed for 8 A/8 B. It is noteworthy that the borylation of 7 is slower than the borylation of 6, this is attributed to steric clash between the −NMe2 moiety and the −CMe2 in 37‐BBr (and the borenium derived from 37‐BBr). Such interactions will presumably lead to rotation around the Me2C−C(O)NMe2 bond to orientate the NMe2 unit to reduce clash with the CMe2 unit and thus position the boron centre unfavourably for C−H borylation (disfavouring formation of the key transition state – which itself maybe higher in energy due to the unfavourable interactions between NMe2/CMe2 when the carbonyl is positioned appropriately). Regardless, the formation of 8 A/8 B and 9 A/9 B confirms that carbonyl directed electrophilic borylation tolerates CH2/CMe2 groups in the linker. Therefore the preference to form compound 4 over B is attributed to a lower kinetic barrier to borylate the aniline derived unit in carbonyl directed borylation (which has been previously observed to undergo borylation at room temperature).[2. Despite this borylation still proceeds on the aniline derived unit to form 4.With an understanding of the products formed from 9 A was confirmed by X‐ray diffraction studies  and 3.487(5) Å for 8 A and 9 A, respectively. Notably, due to the planar N1−C8−O1−B1 unit 9 A has one of the methyls of the −NMe2 orientated between the two CMe2. Thus, the structure of 9 A shows minimal deviation of planarity between the plane of the cyclic boronate ring and the −NMe2 (max. 0.026 Å), whereas the −NMe2 moiety in 8 A is deviated by upto 0.423 Å from the plane of the boracycle. Here, the entire −NMe2 unit in 8 A is bent out of the plane possibly due to packing effects (as short contacts of 2.92–2.98 Å are observed between the −N(CH3)2 and BBr4− anion) of the cyclic boronate framework with the C−NMe2 moiety remaining planar (angles around N Σ=359.9° for both 8 A and 9 A). Additionally, the angles around C8 in both 8 A and 9 A sum to to 359.9 and 360.0°, respectively. Other notable features include the C−O bond in the cations which at 1.336(6) Å is lengthened relative to an uncoordinated amide C=O bond .8 A and 9 A, respectively, slightly shortened relative to that in an uncoordinated amide .8 A and 9 A can be considered to have iminium character as well borocation character .8 A/9 A into bench stable products familiar to synthetic chemists was targeted. Attempts to form the pinacol‐protected product derived from 8 A was successful using pinacol and NEt3, with the product having a δ11B=30.7 ppm. However, in our hands we could not isolate this product sufficiently pure due to its instability on silica. Isolation of the pure ortho borylated products as bench stable compounds was achieved by protecting at boron with 1,8‐diaminonaphthalene  to form −BDan protected 10 and 11 in 65 % and 34 % isolated yields, respectively, via a one‐pot procedure  to produce reasonable yields of 13 post protection. The meta (to the acetylamide unit) bromo derivative leads to two inequivalent ortho C−H positions, but borylation is only observed at the less hindered ortho site. Again, the electrophilic borylation step is slower relative to that of 6, this time due to the electron withdrawing bromo group para to the C−H borylation position. While requiring longer reaction times/higher temperatures (than 6), the successful formation of 13 and 14 nevertheless demonstrates that challenging substrates (in terms of substrates that are deactivated towards SEAr) can be borylated with high selectivity and reasonable yield using this methodology. Finally, variation in the nitrogen substituents was investigated, with bulkier iPr substituents used in place of methyl. This led to formation of 15 in reasonable yield, with the C−H borylation step proceeding to high conversion within 24 h at 60 °C .Finally, several additional substrates related to meta‐C−H functionalisation were not able to effect meta directed C−H borylation via electrophilic borylation under a range of conditions. Thus bespoke  covalently attached directed groups may be required to enable meta‐selective electrophilic borylation. Removing any Lewis basic groups in the template that could affect ortho C−H borylation (via 5 or 6 membered boracycles) is one obvious next step emerging from this work. Indeed, the template with an amide unit can be used to effect amide directed ortho borylation which proceeds selectively on the aniline derived aryl unit in preference to the phenylacetyl unit. Phenylacetyl units were shown to be amenable to carbonyl directed ortho borylation with BBr3 on heating. This process tolerated electron withdrawing substituents and groups at all three positions on the aryl unit.In summary, two close analogues of directing templates effective in transition metal catalysed The authors declare no conflict of interest.1As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


The objective of this study was to investigate the application of these two strategies in women of different ages. (2) Methods: Analysis was carried out on 5477 frozen embryo transfer cycles of women in different ages. All the cycles were divided into three groups according to the age of the included women: <35, 35–39, and >39. The live birth rate (LBR) and multiple birth rate (MBR) between the SBT and DBT among these groups, respectively, were compared. (3) Results: In the women < 35 group, the LBR was similar in SBT and DBT, but the MBR was higher in DBT than SBT. In women 35–39, with >10 oocytes retrieved, the MBR in DBT was significantly higher than SBT, but there was no significant difference in LBR between the two groups; with ≤10 oocytes retrieved, the LBR in DBT were significantly higher than SBT, but the MBT was not significantly different between the two groups. In women > 39, the LBR and MBR were lower in the SBT than DBT, but the differences were not statistically significant. (4) Conclusions: Selective SET is appropriate for most young women, whereas older women are advised to make individualized choices based on the number of oocytes retrieved and blastocyst quality. The wide application of assisted reproductive technology (ART) has made great progress in the treatment of infertile couples. A single delivery of a healthy child is the ideal outcome of ART. However, multiple embryos were transferred to increase the clinical pregnancy rate and live birth rate per embryo transfer cycle over the past several decades, which has led to higher rates of multiple pregnancy and a higher incidence of pregnancy complications. In the past 30 years, the overall rate of twin pregnancy increased by 50~70%, and the rate of triplet pregnancy increased by 400% . MultiplAt present, single blastocyst transfer (SBT) and double blastocyst transfer (DBT) are widely used in clinical practice. The objective of this study was to investigate the application of these two strategies in women of different age groups who first underwent a frozen blastocyst transplantation cycle in the Reproductive Center of the First Affiliated Hospital of Anhui Medical University from October 2018 to October 2021.This study was initiated and conducted after receiving approval from the Committee of Medical Ethics from Anhui Medical University (No. 2017002).This was a retrospective, single-center cohort study that included all women without a fresh embryo transplant in our center who underwent their first resuscitation cycle blastocyst transfer from October 2018 to October 2021. A total of 5477 cycles were enrolled in the study after excluding patients with various uterine malformations, adenomyosis, severe endometriosis, recurrent miscarriages, and preimplantation genetic diagnoses of the blastocyst.In this study, the patients received either a long or short regimen used routinely in our center for ovarian induction, and the detailed process is described in our previous article . When 1–Under a microscope, the oocytes in follicular fluid were picked up and cultured for a short time in vitro in fertilization medium , and they then underwent ICSI or IVF insemination based on sperm quality. At 14–18 h after IVF/ICSI insemination, the fertilized oocytes were assessed by the presence of pronucleus, and then cleavage embryo culturing was continued for two days in cleavage medium  with a subsequent two or three days of blastocyst culturing in blastocyst medium . Finally, the obtained blastocysts were selected and cryopreserved in −196 °C liquid nitrogen using the vitrification method  to be followed by thawed embryo transfer.Three months later, one or two warmed blastocysts were transferred into the uterus under ultrasound guidance. More operation procedures are described in the literature .According to Chinese experts’ consensus, elective SBT is recommended for all couples in the first transplantation cycle, whereas SBT is strongly recommended for patients with a history of cesarean section. In addition, personal wishes and demands should be taken into account in the determination of the number of blastocysts transplanted. After full communication with patients and careful assessment of patients’ basic conditions, DBT was performed in patients who strongly requested and were willing to accept all risks caused by multiple pregnancy.In this study, high-quality embryos at the cleavage stage were defined as embryos with 7–9 blastomeres at equal size on day 3 with no or less than 15% Fragmentation. “High-quality blastocysts” refers to embryos whose grades were above 3BB on day 5 or above 4BB on day 6 as determined using the blastocyst grading system according to Gardner’s criteria .Two weeks after embryo transfer, a positive serum β-hCG level (≥10 IU/L) was defined as a biochemical pregnancy. Thirty days after embryo transfer, the presence of a gestational sac on an ultrasound scan was defined as clinical pregnancy. In addition, preterm birth was defined as delivery between 28 weeks and less than 37 weeks of gestation. Preterm birth was further subdivided into very early preterm (<28 weeks), early preterm (28–33 weeks), and late preterm (34–36 weeks) .Biochemical pregnancy rate = Number of biochemical pregnancies cycles/Number of transplant cycles × 100%; Clinical pregnancy rate = Number of clinical pregnancy cycles/Number of transfer cycles × 100%; Multiple gestational sacs rate = Number of multiple gestational sacs cycles/Number of intrauterine pregnancy cycles × 100%; Live birth rate = Number of live birth cycles/Number of transplant cycles × 100%; Multiple birth rate = Number of multiple birth cycles/Number of live birth cycles × 100%; Preterm birth rate = Number of preterm birth cycles/Number of live birth cycles × 100%.t-tests, and the χ2 test was used to enumerate data. p < 0.05 was considered to be statistically significant. Binary logistic regression analysis was used to verify the independent influencing factors of the live birth rate and preterm birth rate.Statistical analysis was carried out by using GraphPad Prism 8.0 and SPSS 23.0 software. The measurement data were expressed in terms of mean ± standard deviation, and the enumeration data were expressed as a percentage. The measurement data were tested by using two independent sample p < 0.05).Among the 5477 cycles included in this analysis, there were 3323 cycles of SBT and 2154 cycles of DBT. The comparison between the two groups showed that the rates of biochemical pregnancy, clinical pregnancy, and live birth in the SBT and DBT groups were similar. However, the rate of multiple gestational sacs, multiple birth, and preterm birth in the DBT group were significantly higher than those of the SBT group (p > 0.05), with the majority of preterm births being late preterm and only a few cases of very early preterm births (p < 0.05).Furthermore, a breakdown of the preterm birth data revealed that the mean gestational age and the rates of late, early, and very early preterm births were similar in both groups , Medium (35 to 39 years old), and Aged (over 39 years old). p < 0.05).In women aged < 35 years, the rates of biochemical pregnancy, clinical pregnancy, and live birth were similar in the SBT and DBT groups, but the rates of multiple gestational sacs, multiple births, and preterm births were significantly higher in the DBT group than in the SBT group (p < 0.05).In women aged 35–39 years, the rates of biochemical pregnancy, clinical pregnancy, and live birth in the DBT group were significantly higher than those of the SBT group, and the rates of multiple gestational sacs, multiple birth, and premature birth were also significantly higher than those of SBT group (p > 0.05). The rate of multiple births was lower in the SBT group (Group E) than in the DBT group (Group F), but the differences were not statistically significant (p > 0.05).In women aged over 39 years, there were no significant differences in the rates of biochemical pregnancy, clinical pregnancy, live birth, and preterm birth between SBT and DBT in transplant cycles  and an unsuccessful birth group (MU), and the basic characteristics of the two groups are reported in p < 0.05). There was a 1-fold increase in the live birth rate for each additional oocyte retrieved, a 5-fold increase in live birth rate with high-quality blastocysts compared to no high-quality blastocysts, and a 1.5-fold increase in live birth rate with DBT compared to SBT. In addition, the number of blastocysts transferred was significantly associated with preterm birth (p < 0.05), and DBT showed a 2.7-fold increase in the preterm birth rate compared to SBT.Subsequently, a logistic regression analysis was carried out with live birth and preterm birth as dependent variables and indicators with statistical differences as independent variables. The results are shown in p < 0.05), but the rates of multiple births and preterm birth were not significantly different between the two groups (p > 0.05). Among the women with more than 10 oocytes, the rates of biochemical pregnancy, clinical pregnancy, multiple gestational sacs, multiple birth, and preterm birth in the DBT group were significantly higher than those in the SBT group (p < 0.05), but there was no significant difference in the live birth rate between the two groups (p > 0.05).To further clarify the strategy of blastocyst transfer in this age group, women aged 35–39 years with at least one high-quality blastocyst were grouped according to the number of oocytes retrieved  and the number of blastocysts transferred. The clinical outcomes of the four treatment cycles in the LSBT (Group G), LDBT (Group H), HSBT (Group I), and HDBT (Group J) groups are shown in This study had a large sample size of 5477 patients undergoing blastocyst transfer cycles to assess whether clinical outcomes differed between SBT and DBT at different age segments. Due to the selection criteria for the number of blastocysts transplanted in our center, the proportion of patients with a history of cesarean section in DBT group was much lower than that of the SBT group (2.92% vs 15.53%). However, the rate of preterm birth in DBT group was significantly higher than that of SBT group (34.77% vs 13.15%). It is believed that having a history of cesarean section had little effect on the occurrence of preterm birth and no selective bias on the subsequent analysis.In our current analysis, we indicated that although the clinical outcomes of single or double blastocysts were similar in young women receiving embryo transfer for the first time (age < 35), the DBT group had significantly higher rates of multiple pregnancy and preterm birth. Therefore, for young women undergoing the blastocyst transplant for the first time, selective SBT was undoubtedly the most appropriate choice. This is consistent with the 2017 guidelines of the American Society for Reproductive Medicine (ASRM) that recommend selective SBT for all embryos, whether fresh or thawed, in the first transplant cycle of a patient .For older women, the situation is more complicated. The age-related decline in the ovarian reserve not only reduces the number of oocytes retrieved but also reduces embryo implantation and pregnancy rates , and theIn women aged over 39 years, DBT did not increase the rate of multiple pregnancy or preterm birth while increasing the rate of live birth, which is consistent with previous results in the literature . TherefoIn conclusion, this study suggests that selective SET is appropriate for young women (under 35 years), which increases the safety of ART treatment without decreasing the live birth rate and minimizes the health risks faced by these women. However, due to the age-related decline in embryonic development potential, the treatment effect of ART will also decline in women with advanced age. Therefore, we do not recommend selective SBT for every woman over 35 years of age; rather, we recommend that these women make an individual choice based on the number of oocytes retrieved and blastocyst quality. In addition, this study is a retrospective study, which means it inevitably has a certain selection bias. Although the inclusion and exclusion criteria were strictly stipulated, there are still certain limitations in the screening of enrolled patients. Future multi-center and multi-book prospective studies are needed to further investigate the effect of the number of blastocyst transplants on clinical pregnancy outcomes.


Seasonality of births is a worldwide phenomenon, but the mechanisms behind it remain insufficiently explored. Birth seasonality is likely to be driven by seasonal changes in women’s fecundity (i.e. ability to conceive), which is strongly influenced by their energetic status. We tested whether birth seasonality is driven by high workload and/or low access to food using 200 years of birth data, from 1782 until 2004, in an agricultural rural Polish community. First, we analysed the time series of births and within-annual variance in births, a proxy for the extent of seasonality. Secondly, we tested the hypothesis that a high agricultural workload and/or low access to food decreases number of births. We found seasonality of births throughout more than 200 years of observation in an agricultural Polish population, with a dominant birth seasonality in January and February which gradually shifted towards June in the late twentieth century. The observed pattern does not support the hypothesis that birth seasonality resulted from women’s energetic status. We discuss the possible reasons why our results do not support the tested hypothesis and some implications for our understanding of the birth seasonality. Many factors have been suggested to be related to birth seasonality, either directly or indirectly2, including (i) social factors such as seasonal patterns in marriages, holidays, temporal migration3 and the mother’s socio-demographic characteristics6, (ii) environmental factors including, for example, temperature and photoperiod7, and (iii) energetic factors that principally affect female fecundity8, including workload and food availability. Observed birth seasonality most likely results from interactions among environment, biological factors, and human volitional control4, all of which may vary among populations.Human births show seasonal variation and this is a worldwide phenomenon. Two general patterns were identified: (i) a European pattern with an excess in births during spring and summer, and a secondary peak in September and (ii) an American pattern with a trough in April–May, and a peak in September2, since ovarian function is sensitive to female energetic conditions. In the past, farm work was highly seasonal, labor-intense, and mainly done by hand, thus requiring the participation of most family members, including women. Periods of restricted energy availability, either because of low energy intake or high energy expenditure, are associated with declines in estrogen and progesterone levels, less frequent ovulations, longer cycles2 and consequently a lower probability of conception9. For example, Lese, a horticulturalist population living in Ituri Forest of the Democratic Republic of the Congo, showed a clear pattern of birth seasonality and backdated conceptions, with fewer conceptions occurring during months when women had a negative energy balance (May–July) and a higher number during the season of positive energy balance (September–November)10.Identifying the mechanisms behind birth seasonality may be easier when analysing a population with a homogenous way of living relative to nationwide analyses that combine different socio-economic groups and lifestyles. In agricultural populations, seasonal workload and nutrition seem to be the most important factors affecting birth seasonality through influencing a probability of conception11. High energy expenditure during harvest and haying in summer months remained in a contrast to late fall when work was less intense, and winter when women were not involved in agricultural work. Such variation has the potential to affect the seasonality of births through the suppression of ovarian hormone levels2. However, despite the evidence that workload suppresses ovarian function, few studies have shown that seasonal workload may also influence birth rates.It has been shown previously for a Polish rural women that seasonal increases in intensity of physical activity corresponded to a reduction in ovarian progesterone levels by almost 25%11, we expected fewer births in April and May followed by an increase in June and July.We analysed over 200 years of birth data, from 1782 until 2004, for an agricultural rural Polish community to investigate the seasonality of births and conceptions. Seasonality of birth was never before analysed in a dataset covering such a long period of time and for a population originrating from a Central European region. We analysed the data in two steps. First, we analysed the time series of births and within-annual variance in births, a proxy for the extent of seasonality. Secondly, we tested the hypothesis that high agricultural workload correlates with a decreased ability to conceive. Because the study population is agricultural with a high workload in July and August, followed by lower workload and high food abundance in September and OctoberThe data contained 26,874 birth records between January 1st 1782 and December 31st 2004, with an average of 121 per year or 10 per month , live births peaked early in the year, but between 1948 and 1992 this gradually moved towards April and June Fig. B. A shif14. Our study population might behave differently on that matter because it remained agricultural during the twentieth century with a minimal access to a modern contraception and a delayed experience of the demographic transition relative to other European countries. It is noteworthy, however, that some studies reported a lack of seasonality in the past, e.g. a lack of birth seasonality between 1851 and 1901 was documented for Escazú, Costa Rica16. Hence, while some studies reported loss of birth seasonality in the twentieth century, it is not a consistent pattern across populations and there are very few studies in which long-term changes in seasonality were analysed.We found no changes in the extent of birth seasonality with time Table A,B, whic8, but, as outlined by Ellison et al.8 three particular situations should be distinguished: (i) energy status, i.e. energy stored as fat or glycogen which could be mobilized when needed, (ii) energy balance, i.e. the difference between energy input and expenditure, and (iii) energy flux, i.e. the total rate of energy turnover. Conception is more likely to occur when women are in positive energy balance and not constrained by high energy expenditure17. Previous research from our study population11 showed that energy flux of women was important for ovarian function. During high workload months—July and August—average progesterone levels were low and rose in September and October when work was less intense, which suggests that energy flux can impact ovarian function independently from energy status and energy balance. However, low sex steroid levels do not prevent conceptions, but only reduce their probability which means that women may take longer to conceive.By backdating, we showed a maximum in the seasonality of successful conceptions in April–May and a minimum from October until February. It is known that human ovarian function is sensitive to energetic condition7. In fact, in our study population, we observed relatively low levels of progesterone during January and February, despite the fact that women during these months had low levels of energy expenditure33. These winter months are characterised by short daylight and, in the past, very limited exposure to artificial light.Another factor which may influence conceptions is photoperiod. Longer days and higher light intensity are related to a lower melatonin production which could lead to higher sex hormone levels19. Vitamin D deficiency in pregnancy is associated with adverse maternal–fetal outcomes, e.g.: preeclampsia, gestational diabetes mellitus, preterm delivery, recurrent pregnancy loss, low birth weight, being small for gestational age, and low immunity20. With very limited access to (or even lack of) food such as oily fish, which contains vitamin D and lack of supplements of vitamin D in the past, humans had to rely on the vitamin D produced in the skin through exposure to sunlight.Our study, together with the previous ones, raises the question if there is an optimal time for conceptions and the ensuing births in a Central European population? One option is that a conception during spring with a birth occurring in January and February, i.e. similarly to what we found in our study, could be optimal, because it may be beneficial for both the mother and her offspring. In spring people have higher exposure to sunlight and that is necessary for the vitamin D production, which is crucial for successful reproduction, but its level is highly dependent on seasonal variation of ultraviolet B radiation (UVB)21. Between 1769 and 1850 in Finland, the survival probability to one year of life was highest for children born between August–October comparing to other months of the year22. However, the maximum in births occurred between March and April, dropped in June–August, followed by another peak in September and hence in Finland, births did not concentrate around months with the highest infant survival. To the best of our knowledge, the reasons for the discrepancies in birth seasonality between studies remain unidentified.Furthermore, with conceptions in the spring, the third trimester of pregnancy occurring during late fall/winter would provide good developmental conditions for a fetus, because this period is characterised by a relatively high food availability and low energy expenditure due to a lack of heavy agricultural work. Pregnant women with a positive energy balance could more successfully support a developing fetus during the third trimester when energy demands are high. Hence, the seasonality of conceptions and births could be driven by third trimester energy balance rather than the energy balance influencing ovarian function. Indeed, a seasonality of births that optimised infant survival probability was described in Matlab, Bangladesh where the maximum of conceptions was observed in March–April and the maximum of live births was in November, and the proportion of stillbirths was lowest in November23. Intensive lactation in women with negative energy balance results in a longer period of ovarian suppression24. In our study population, traditionally women breastfed for about 12 months25 and this could well contribute to the seasonality of births. Resumption of menstrual cycles during winter when women had high availability of food and low energy expenditure was more probable than during other times of a year.Another factor that can affect birth seasonality is lactation. In the past, lactation was the best way of keeping a child alive after birth. A chance of survival of children born in winter could also increase because their mothers had more time to look after them . The resumption of ovarian function after lactational amenorrhea is also energy sensitive26. However, while this could affect the seasonality of firstborns and in populations with low number of children per family, it is unlikely that a seasonality of first-order births drives the seasonality of all births in a population with a continuously high fertility. Data used for this study come from a population with one of the highest birth rates in Poland (18.3/1000 people vs 9.3/1000 in Poland in 2020)27 and hence there were few first-borns relative to their later-born siblings. A second cultural factor that can affect birth seasonality is associated with religion: the liturgical calendar prohibited sexual intercourse during Lent28, which can explain some of the observed changes in birth seasonality in the latter period of the time series. Even though in the early twenty-first century our study population remains very Catholic, it is well possible that the gradual secularization of society and the loosening of traditions affected or changed the birth seasonality in our population.The observed birth seasonality in our study population could also be due to the influence of cultural factors, such as a seasonality of marriages. Marriages, especially in the past, were clustered with two peaks during the year in February and November12. Hence, the birth peak in January could to some extent reflect a reporting bias. However, this bias cannot fully explain the long-lasting birth peak until February and the gradual decline in birth rate towards autumn  and the highest risk for spontaneous abortion was observed in late August32, which would cause a birth minimum in December, a pattern which is also not consistent with our results , Polish farmers had to rely mostly on products which they were able to produce themselves39, because access to food products in shops in villages was limited. Significant changes in the way of life were observed during last 25 years of XX century37. All methods were carried out in accordance with relevant guidelines and regulations. The study was approved by the Jagiellonian University Bioethical Committee.Agricultural life has been characterized by seasonality, including a seasonal access to food. The diet of rural people was very simple, based on potatoes, cabbage, beans, milk , rye, barley, buckwheat groats and beetrootsWe performed the analyses with the number of live births and successful conceptions using complete years between January 1st 1782 and December 31st 2004  the temporal trends in the time series of the number of births, (ii) their within-annual standard deviation (sd) and (iii) their within-annual coefficient of variation . Both, the sd and cv reflect within-annual change, and hence are a proxy for seasonality, thereby allowing to test for changes in the extent of seasonality with time, but the cv has the advantage that it corrects for temporal trends in the number of births, which can be important because the sd often increases with the mean.40 using the function ‘gls’ of the package ‘nlme’41 in R42. In these models, to determine the statistical ‘significance’ of predictor variables, we used model selection based on the second order Akaike Information Criterion (AICc): better fitting models are indicated by their lower AICc values, with models within 4 ΔAICc being plausible and becoming increasingly equivocal up to 14 ΔAICc, after which they become implausible44.For these analyses, we followed the general linear modelling approach, as explained in2, standard normalized to a mean of 0 and a sd of 1. We checked robustness of the findings by repeating all analyses fitting the more flexible general additive models (gamms), allowing to explore non-linear shapes of the associations. To this end, we used the function ‘gamm’ of the package ‘mgcv’ Woods, S. 2017. Generalized Additive Models. An Introduction with R (Second Ed.). Chapman & Hall, Boca Raton, Fl. . In all models, we accounted for possible heteroscedasticity with the ‘weights’ function, which improved the model fits for births . We visually inspected model residuals for other assumptions, e.g. Q-Q plots45, and checked for influential datapoints with the function ‘CookD’ from the package ‘predictimeans’46.All data followed a normal distribution Fig. A–C and w48. In brief, wavelet analysis decomposes time series data using functions (wavelets) simultaneously as a function of both period and time48. Because the time component is continuous, the approach requires no assumptions regarding when changes occur or arbitrary compartmentalizing of the time series. Hence, this approach is useful, for example, to detect whether the seasonality changes over time48.In the second analyses, we identify whether births showed recurring seasonality using wavelet analyses. Wavelet analyses are an approach used in many fields, including ecology, demography, epidemiology, economics and engineering, to detect trends in non-stationary time series, i.e., systems which properties change through time42 using the function ‘analyse.wavelet’ from the package ‘WaveletComp’49 and quantified a periodicity between 6 months and 2 years, with 1 year indicating an annually recurring seasonal signal. Performing the analyses using weekly or monthly intervals gave consistent conclusions (results not shown) and for the ease of interpretation we present the monthly analyses. To avoid the results being driven by local peaks, we smoothened and detrended time series following standard procedures49. To determine statistical significance, we then compared the data’s periodicity with that of 1000 ‘white noise’ datasets with significance at the 5% level. Once we detected a statistically significant seasonal signal, we identified the month(s) of peak births based on the monthly counts.For the wavelet analyses, we fitted a Morlet wavelet in RSupplementary Information.


The survival of HIV-infected infants depends on early identification and initiation on effective treatment. HIV-exposed infants are tested at 6 weeks of age; however, testing for HIV sooner  can identify in utero infection, which is associated with rapid progression. Infant early diagnostic virologic tests often have long turnaround times, reducing the utility of early testing. Point-of-care (POC) testing allows neonates born in health facilities to get results prior to discharge. This study aimed to understand the views of mothers and health workers regarding the use and acceptability of POC birth testing.Beginning in 2018, Zimbabwe offered standard HIV testing at birth to high-risk HIV-exposed infants; as part of a pilot program, at 10 selected hospitals, POC birth testing (BT) was offered to every HIV-exposed infant. In order to understand experiences at the selected sites, 48 interviews were held: 23 with mothers and 25 with health workers, including 6 nurses-in-charge. Participants were purposively sampled across the participating sites. Interviews were held in English, Shona, or Ndebele, and transcribed in English. Line-by-line coding was carried out, and the constant comparison method of analysis was used to identify key themes for each respondent type.Findings were organized under four themes: challenges with BT, acceptability of BT, benefits of BT, and recommendations for BT programs. Overall, BT was well accepted by mothers and health workers because it encouraged mothers to better care for their uninfected newborns or initiate treatment more rapidly for infected infants. While the benefits were well understood, mothers felt there were some challenges, namely that they should be informed in advance about testing procedures and tested in a more private setting. Mothers and HCWs also recommended improving awareness of BT, both among health care workers and in the community in general, as well as ensuring that facilities are well-stocked with supplies and can deliver results in a timely way before scaling up programs.Mothers and health workers strongly support implementation and expansion of birth testing programs due to the benefits for newborns. The challenges noted should be taken as planning guidance, rather than reasons to delay or discontinue birth testing programs.The online version contains supplementary material available at 10.1186/s12887-022-03601-x. The development of the research question and outcome measures were informed by previous research on patient priorities, experiences, and preferences. The direct outcome of the research is to assess patient preferences and acceptability of a new technology by asking patients (in the case of children: their families) and health care workers directly. However, patients were not involved in the design, recruitment, or conduct of the study. Findings will be shared with the Ministry of Health, who can share results with health facilities and patient populations. Participants are included in the acknowledgments.Many HIV-exposed newborns are not tested for HIV or are tested too late to receive timely clinical intervention; long turnaround times for virologic tests compound the issue.Point-of-care HIV testing has been shown to greatly reduce turnaround time for infant HIV diagnostic tests at 6-8 weeks of age; point-of-care HIV testing for newborns offers the possibility of rapid testing and result return, often prior to hospital discharge after birth.There have been very few studies of birth testing, even fewer on point-of-care birth testing, and almost none on the use of point-of-care birth tests in routine settings across districts. Quantitative studies have suggested high uptake and effectiveness.Qualitative studies of early infant diagnosis have shown high acceptability among mothers and health care workers; however, some concerns have been raised about acceptability of birth testing and implementation of point-of-care testing in routine settings.Point-of-care birth testing was well accepted by mothers and health workers in Zimbabwe because it encouraged mothers to better care for their uninfected newborns or initiate treatment more rapidly for infected infants.This study provides additional findings related to a maternal desire to be informed in advance about testing procedures and tested in a more private setting, as well as the need to ensure that facilities are well-stocked with supplies and community awareness of diagnostic options is high.Mothers and health workers strongly support implementation and expansion of birth testing programs due to the benefits for newborns. The study provides evidence to support the use of point-of-care birth testing in routine health care settings. The challenges noted should be taken as planning guidance, rather than reasons to delay or discontinue birth testing programs.Prevention of mother-to-child HIV transmission has greatly reduced the number of children born with HIV, but in 2020, there were still 150,000 cases of perinatal transmission globally; despite greater availability of pediatric treatments, 37% of HIV-exposed infants are not tested in the first 2 months of life  program was readily accepted because when it was introduced… it came with less burden than DBS because DBS is strenuous plus it has long turn around…” (HCW)Many HCWs felt that BT POC testing was more convenient than standard dried blood spot (DBS) testing, with quicker results, but several highlighted that the convenience was only possible with enabling factors such as sufficient staff, training, and the availability of birth testing resources.“I’m going to wait for my six weeks visit [to test the baby again], for now I will just give my baby the [prophylactic] medication then they will repeat the test at six weeks as told.” (Mother)“They (mothers) do accept [the test results],… but then that’s when questions are like: ‘my child has become positive; isn’t the baby having my antibodies?’ otherwise when the baby is negative there are no questions, but if positive they will ask…” (HCW)Mothers generally trusted the infant test result, and most did not plan to get a confirmatory test elsewhere, but stated they would wait to have the child tested again at 6 weeks as instructed. This was especially true for mothers of infants who tested HIV-negative. As one HCW highlighted, if the infant tests positive, mothers may raise questions about the test’s accuracy more so than if the baby tests negative.“I was happy with that (birth testing) because they will have intervened early by protecting it so that it does not show that the baby has been diagnosed with HIV or not, because they had explained that if the baby is HIV positive and give it the required medication then the baby will be as healthy as other babies you will not notice any difference. As a parent I will be knowing how to protect the baby, what type of food I should give the baby and giving medication on time.” (Mother)Mothers explained that during birth testing they were provided information on how to care for the infant according to the received result, and with this knowledge, it encouraged them to properly care for their child.“We had a challenge: we said exclusive breastfeeding so the mother was not sure whether the baby will test negative or positive so some were just doing mixed feeding but ever since we started POC birth testing the mothers will be eager to know what to do which will make their babies negative and they will be having that zeal so that their babies remain negative...” (HCW)Both mothers and HCWs highlighted that when mothers knew their infant was HIV-negative at birth, it can motivate the mother to try to keep the infant HIV-negative by closely following care instructions including medication administration, returning for appointments, and proper feeding.“If the baby is supposed to be on medication, we may do so early instead of waiting for 6 weeks. It may be too late to wait for 6 weeks, and the child would have been affected so much.” (Mother)Most mothers and HCWs said one of the main benefits of birth testing is the ability to initiate an HIV-positive infant on treatment early, rather than waiting until the usual early infant diagnostic test was performed at age 4-6 weeks.“… what we knew way back was that a child gets tested at 6 months so when you are told a new thing now at first you are a bit puzzled because you don’t know the effects because it’s like an immediate thing which has just come so your mind spins and you don’t know how to accept it and you don’t know what to do…” (Mother)Many mothers and HCWs discussed challenges with information about birth testing being provided to mothers too late. Several mothers noted that they would have preferred to learn about birth testing in advance, such as during ANC visits, rather than at delivery.“There are some people who still have to get consent from the partner… it becomes a challenge now because the husband might not be available at the time you want to do the test and the mother would tell you I cannot do this without the father’s consent.” (HCW)“… if you are married and if you are in a relationship whereby you communicate before you do anything, you inform each other like are you ok with it… it [birth testing] is one of those things.” (Mother)Both mothers and HCWs stated that some mothers may need and/or want to first discuss testing the child with their husband or partner. Some respondents described situations where the partner was not available at the time of delivery as a reason that the newborn was not tested at birth.When she [a mother] gives birth to a baby who test positive, … she has to go home with drugs for the baby. It becomes difficult for her to adjust when the baby has tested positive, instead of us giving her time to think over it and come back. … People are not getting time to decide when the baby is positive, they are given medication for the baby, no time to think about it, to decide whether to give the baby [the medications] or not. I think that’s one of the challenges the mother won’t be prepared psychologically.” (HCW)“A few respondents commented that mothers may not be in a mental state to care for an HIV-positive newborn right after delivery, including needing to be ready to take home antiretroviral treatment (ART) medication at discharge. Both mothers and HCWs noted this as a possible disincentive to accept birth testing, but mothers generally thought it was not enough of a reason to not test.“… I was happy that it was done privately right. Where we were, no one knew where I was going. …” (Mother)“I couldn’t ask [all my questions] because there was no time and I wanted to maintain privacy since we were in the ward.” (Mother)Several mothers were appreciative that the testing was done in a private location away from other patients, and many respondents commented on the importance of privacy. For one mother the fact that the testing was not conducted in a private location, prevented her from asking all her questions as she wanted to maintain privacy.“They [mothers] are willing, but to wait for 1 h waiting for results they won’t be comfortable… they will wait because they don’t have a choice, but you will see that they are not happy waiting.” (HCW)While most mothers said testing the newborn and receiving the results took less than 2 h, HCWs raised concerns that some mothers may not be willing to wait the additional time it takes for testing for a variety of reasons, such as transportation schedules or impatient family members waiting for the mother.“There is a challenge at times there will [be] 3 or 4 kids who will need [birth testing] and older kids 6 weeks who will need testing so the machine will be one. People will take time waiting to be tested so some end up going away.” (HCW)HCWs also explained that the wait time for results could be over an hour if there were stock outs of necessary supplies, a backlog of other samples, or other challenges with the POC machine. HCWs felt these delays might hinder mothers’ confidence in the program or affect receipt of the child’s result if the mother or another caregiver could not wait and was unable to return to the facility.“There are none [risks] except I was hurt a bit when they pricked the baby and it cried when they were taking blood - but there was nothing bad about it though.” (Mother)While most HCWs were not concerned about testing a newborn, a few were worried that a newborn was too young to be tested. Among the HCWs who expressed concerns, most felt that knowing the newborn’s status was more advantageous and outweighed any concerns about the infant’s age and fragility. A couple of mothers noted that it was difficult to see their child pricked and hear them cry, but for these mothers knowing the newborn status right away was more important than the momentary discomfort.I think [information should be given] through health education of the mothers during the ANC because they really need to know what is going to happen.” (HCW)““The partner can be involved when they come with their partners for the routine ANC visits and we talk to them we allow them to ask questions and by the time they deliver maybe the partner would actually agree because they have information.” (HCW)Participants gave recommendations for health system, facility, and community levels. Several HCWs suggested including birth testing information as part of health education during ANC visits to help mothers plan for any additional time to wait for test results, process information, and ask questions, as well as allow mothers to discuss testing the child with their partners prior to delivery. One HCW suggested providing mothers with written information materials they could take home.“Our infrastructure remains a challenge… because when you want to do birth testing soon after delivery you cannot do it because our set up is very poor, there will be other patients in the same room…and we cannot divulge anything. …” (nurse-in-charge)At the individual facility level, both mothers and HCWs stressed that birth testing needs to be discussed in a private location, not in open wards; however, HCWs stated that their facilities may need additional private spaces for counseling, testing, and providing results.“… we also need to conduct teachings, workshops, tutorials so that every health worker knows that there is birth testing so that even if a client comes and asks I have heard about this, any health workers in that facility must be able to explain and give information to that client.” (HCW)In addition, HCWs recommended that all facility staff, in particular nurse midwives and other maternal/postnatal department staff, should be educated about birth testing so that any staff member can identify and inform mothers about birth testing.“The people who are providing the cartridges should be aware of the number of babies that are born every month, maybe an average at this hospital, so that maybe they give enough resources to cater for all of them.” (HCW)HCWs were unified in their recommendations to ensure that BT programs maintain a consistent and adequate supply of birth testing materials, and most notably cartridges, to ensure that all newborns can be tested and tested in a timely manner.“… we sometimes have too many specimens to do at one [time], those that need testing at birth and the other ones being tested at 6 weeks all to be done with one machine, … it becomes a very big burden such that you don’t finish running the specimens in single a day and some people will be waiting and they will be complaining you are taking too long to produce the results. ….” (nurse-in-charge)To avoid backlogs and reduce wait times, HCWs suggested providing facilities with more than one POC machine and/or providing POC machines to smaller facilities.“I think there should be community talks, like health education, health workers going to community impacting knowledge about the benefits of birth testing …” (HCW)Several HCWs also suggested educating the general community about birth testing to increase mothers’ awareness and acceptance of birth testing.Overall, mothers and HCWs strongly recommended continuing and expanding birth testing programs due to the related benefits, such as early initiation. They noted the challenges as program aspects to be considered, not a reason to delay or discontinue testing programs.Mothers and health care workers were overwhelmingly accepting of POC BT, even as concerns about the program were noted, especially around the need for improved information to patients. Mothers noted ongoing structural challenges with health systems, such as the lack of private space in hospitals in which to discuss tests and results, and noted the need for introducing the topic of birth testing and point-of-care both to a wider audience in the community, and at appointments prior to delivery. Mothers also worried about the age and fragility of newborns to have a blood draw; however, no respondents believed this outweighed the need and benefit of having the test. Further, most mothers were grateful to receive test results sooner in order to act sooner to prevent transmission in the postpartum period or initiate treatment as soon as possible.Health care workers equally saw a benefit of being able to provide test results quickly, especially as they previously had challenges contacting patients after discharge. Health care workers agreed with the need to raise correct information and awareness about BT generally in the community, but also noted the need to ensure consistent supplies of equipment  before scaling up programs, in order to ensure that every patient offered a test was actually able to receive one and get results in the promised timely way. Considering the benefits, mothers and health care workers, including in-charge nurses, were in favor of continuing, expanding, and promoting POC programs, including offering BT.Findings from this study suggest that, while scaling up POC BT programs, attention should be paid to the health system aspects that are necessary for its success: specifically, ensuring private areas for patient testing and discussion, ensuring sufficient numbers of trained staff to run tests, strengthening forecasting and supply chain systems for testing equipment, and improving the provision of information to patients. As much as possible, health care workers should include discussions about birth testing and point-of-care options in antenatal care visits and allowing mothers time  to ask questions about the test and be reassured about its safety and minimal pain and invasiveness, especially for newborns.In other studies, both with POC and not, BT has been found to be generally acceptable among mothers and HCWs , 24. SimCompared to conventional birth testing, POC birth testing offers a unique opportunity to ensure that infants are tested and lab results returned more quickly. A recent study from Zambia which evaluated a conventional BT pilot, reported a median turnaround time of 53 days ; a pilotOne of the main challenges, regardless of testing method, is ensuring that infants who test HIV-positive are linked to care in order to begin treatment. The conventional testing study in Zambia found that only a third of infants diagnosed with HIV were linked to care , but theOne of the initial concerns raised with BT was whether parents would return in the 6-8 week window period for the follow-up test as recommended once they had already received a birth test. Quantitative data from the same study as presented here found a 46% return for subsequent testing within 8 weeks after an initial POC negative birth test . The conOn the health system side, this study identified the challenge of adequate staff and supplies, which has been reported elsewhere in the literature . ChallenThis study provides important insight into the experiences with POC BT but has some limitations. As this was not a longitudinal or comparative study, participants were not asked to directly compare laboratory-based to POC testing, nor birth testing directly to early infant (6-week) HIV testing. Health care workers spoke frequently about the advantages of POC over laboratory-based testing, as many had experience in both. Most mothers in the study only had the experience of POC BT, unless they had other children who were tested, most likely receiving conventional testing at 6 weeks, and possibly at different clinics under different circumstances. As it was possible to have a conventional BT or a 6-week POC test, the experiences of POC and BT together may be conflated. Although efforts were made to include a selection of participants from across multiple regions in the country, the selected facilities were only hospital level, and patients at other types of medical facilities (where the program might expand to) may be different. Further, future research would benefit from interviews with ministerial-level policymakers and administrators about potential challenges at the national or regional level. Lastly, because acceptance of BT was so high, we were unable to interview mothers who refused testing, and this minority of mothers may have differing views than the mothers who accepted testing. This is an important group, as they are the most vulnerable to not only poor clinical outcomes, but also social isolation; future studies may need different, targeted methods to gain perspectives from these families.Because BT is so new, and POC BT even newer, this study provides important information about its acceptability among various stakeholders. Unlike previous pilot studies, this study’s strengths included collecting data at 10 hospitals in a wide range of regions, providing further confidence in the near-universality of acceptability, which echoes findings about POC in general. The study also provides one of the first analyses of acceptability of birth testing in a routine (non-study) setting, and as offered to every exposed dyad, rather than only those considered at highest risk of mother-to-child transmission. While there is consensus around the need to offer HIV testing as early as possible to those exposed, the long-term clinical advantage of early testing and treatment will need to be examined separately.As POC BT is introduced in a country or region, the specific needs of the mothers and health care workers should be taken into consideration, as there may be differences in the experiences and concerns across countries. Future research should also seek to understand the views of the general community on HIV and birth testing and to identify the best strategies for raising community awareness and providing accurate information about new diagnostic options. Programs will need to implement strategies to improve linkage to care for infected infants as well as follow-up for subsequent testing for infants initially testing negative , as wellPOC BT not only improves timeliness and clinical outcomes, but it is overwhelmingly acceptable to mothers and health care workers. Mothers’ concerns did not deter from testing, and more rapid results allowed for more immediate health-promoting actions. Most had positive experiences and wanted to share knowledge about the ability of the test with their communities. HCWs noted the need to strengthen supply chains in order that those seeking care receive it, as well as improve information flow about available diagnostic options to patients. The use of POC BT should be scaled up wherever possible, to reach the most at-risk infants, and to improve their health and survival.Additional file 1.