In developing countries, maternal and newborn mortality is a major public health issue. Birth preparedness and complication readiness is a method to encourage pregnant women to seek professional birth attendants as soon as possible. The aim of this study was to evaluate practice and factors associated with birth preparedness and complication readiness among women attending antenatal care, southern Ethiopia, in 2019. From September 1st to September 30th, 2019, a facility-based cross-sectional study was conducted. 422 pregnant women were randomly selected and interviewed using a structured questionnaire. Epi-data version 3.1 was used to enter data, while SPSS version 21 was used to analyze it. To find factors associated with birth preparedness and complications readiness, researchers used multivariable logistic regression.From 422 study participants, 205(48.6%)  have birth preparedness and complication readiness practice. Age of respondent ≥ 37 years  and between 25 to 30 ; level of education College and above and secondary school ; previous history of ANC follow up , birth outcome with live birth, and history of birth at health facility  where factors significantly associated with birth preparedness and complication readiness practice. Overall, there was low birth preparedness and complication readiness practices were observed in current study. Age of respondents, level of education, history of ANC follow up, and history of birth at a health facility were factors associated with birth preparedness and complication readiness practice. Governments with other stakeholders should work by focusing on antenatal care and institutional delivery by focusing on older age group mothers with who has no formal education. More than 40% of pregnant women worldwide are at risk of developing serious obstetric problems. According to the World Health Organization (WHO), more than 300 million women in developing countries suffer from short- and long-term problems connected to pregnancy and childbirth . The gloFor the purpose of promoting quick access to skilled maternal health care, the ideas of birth preparation and complication readiness have been integrated. In the event of pregnancy or childbirth complications, it helps expectant mothers and their relatives to get immediate medical care. The processes of preparing for a typical birth while also foreseeing the actions that will be necessary in the event of an emergency are referred to as birth preparedness and complication readiness . BecauseThe essential measures for timely utilization of skilled maternal and neonatal care, especially during pregnancy, labor, and childbirth, include identifying and decreasing delays in seeking, reaching, and acquiring care . One of All through Antenatal care visits in Ethiopian Healthcare Facilities and all levels of hospitals, women were given information about specific pregnancy complications such as a severe headache, abdominal pain, vaginal bleeding, vaginal fluid leakage, and blurred vision, but only half of pregnant women in developing countries receive the suggested minimum of 4 ANC care visits .Birth preparedness and complication readiness are two of the main areas of involvement, with the goal of reducing neonatal and maternal mortality by promoting health-seeking behavior and the use of appropriate skilled people and health-care facilities for delivery . The birAlso, little was known about birth preparedness and complication readiness practice and factors associated with birth preparedness and complication readiness in the study area and the region as well. Therefore, this study aimed to fill the gap by assessing the current status and factors associated with birth preparedness and complication readiness practice among women attending ANC in Yirgalem Hospital, southern Ethiopia.Ethical approval was obtained from Wolaita Sodo University College of health science and medicine Ethical review board. Permission was obtained from Sodo town health office.Oral informed consent was obtained from the parents of the minors and study participants. The purpose and objective of the study was well explained to study participants and they were informed about their full right to withdrawal or discontinue participation at any time they want. In addition privacy and confidentiality of participant was kept.Health facility based cross sectional study was conducted in Yirgalem general Hospital from September 1st to 30th, 2019. Yirgalem general Hospital is located in Sidama zone, Southern Nation Nationality People Republic (SNNPR), Ethiopia. The Hospital has a catchment population of 4.6 million and 260 births per month. Figures from the central statistics agency of Ethiopia published in 2005 indicated that. Yirgalem has an estimated total population of 43,815 of whom 21,975 women and 21,840 are men. Yirgalem town is located at the distance of 260Km from Addis Abeba the capital city of Ethiopia and 40Km from Hawassa the capital city of SNNPR. The study was carried out among pregnant women attending antenatal care.All pregnant womens who came to Yirgalem general hospital for ANC visit were the source population and all pregnant women who fulfill the inclusion criteria and selected pregnant women were a study population.Pregnant women who can hear and communicate and who are volunteering to participate are included and a pregnant women with mental problem or seriously ill/ unable to hear and speak and primigravida women were excluded.The sample size was estimated by using sample size determination for a single population proportion formula. Therefore, the total sample size was calculated with the marginal error of 0.05, with 95% confidence interval and p-value 48.5% from a study conducted in Sodo town, Wolaita Zone, SNNPR, Ethiopia .n = sample sizeZ = standard error with the given level of confidenceD = margin of error 5%Confidence interval 95%P = 48.5%N = Z (a/2)2 p (1-p)                d2        = (1.96)2 0.485(1–0.485)                    (0.05)2                  N = 383A possibility of 10% of the total sample size was considered for non-respondents. And the final sample size was 422.A systematic random sampling technique was used to select 422 pregnant women who attend ANC in Yirgalem Hospital. The first women was selected by simple random sampling; lottery method. Then the next women was selected through systematic sampling technique every 5th interval from the order of mothers who come for ANC; until the desired numbers of the sample were obtained.Face-to-face interviews were used to collect data, and the questionnaire was structured. The questionnaire, which includes socioeconomic and demographic variables, obstetric, health-care-related aspects, information sources, and transportation access, was initially prepared in English and then translated into Amharic. The Johns Hopkins Initiative for International Education in Gynecology and Obstetrics developed the questionnaire for the mother and child health program. The practice of birth preparedness and complication readiness is the result of this research .Birth preparedness and complication readiness: A women was considered as prepared for birth and its complication if she save money for birth-related and emergency expense; identified place of delivery; skill birth attendants; arrange transport to health institution for delivery and obstetric urgent situation, and prearranged blood donor in cases of obstetrics emergency. The 5 variables were transformed on SPSS in to one variable that is “birth preparedness and complication readiness practice” women who found at least 3 of the 5 birth preparedness and complication readiness component were considered as” prepared for birth and its complication” coded as 1. The remain women who found less than three were considered as “not prepared for birth and its complication” and coded as 0. This scoring has been previously used in studies that assessed women’s birth preparedness and complication readiness practice , 13, 14.Data were checked for completeness and entered in to Epi data version 3.1 and SPSS window version 21 was used for analysis. Descriptive analysis was done and the results were presented in the form of narrative and table. bivariable and multivariable logistic regression was done to identify factors associated with birth preparedness and complication readiness. All variables with p-value less than 0.25 in bivariable logistic regression model were entered to a multivariable logistic regression model for controlling possible confounding and odds ratios and their 95% CIs were computed.During the study, data were collected from 422 pregnant women who visited Yirgalem General Hospital for ANC follow-up. The means with standard deviation (SD) of the age of the respondents was (26.3 ± 6.2). The majority of respondents 252 (59.7%) were between the ages of 25 and 30, with 148 (35.1%) having only an elementary education. The majority of the responders 398, (94.3%) were married .Three hundred seventeen (75.1%) of participants had attended ANC during their previous pregnancy, according to obstetrics characteristics. In terms of pregnancy outcomes, 74 (17.5%) of mothers have had an abortion, 256 (60.6%) have had live births, and only six have had still births. 281 (66.6%) of mothers give delivery in a medical facility .The Birth Preparedness and Complication Readiness (BPCR) rate was examining three or more stages from these five components, 205 people (48.6%)  were found to meet the criterion and be ready for birth and its complications .In a multivariable regression analysis, respondents’ age, level of education, pregnancy and childbirth outcomes, attendance at antenatal care during the previous pregnancy, birth outcome with live birth, and birth at a health facility were all significantly related to birth preparedness and complication readiness practice. Birth preparedness and complication readiness practice is higher in older age groups than in younger age groups; age groups 37–44 are four times more likely to practice birth preparedness and complication readiness  than younger age groups, and age groups 25–30 are more than two times more likely to practice birth preparedness and complication readiness  than younger age groups.Another factor that was linked to birth preparations and complication readiness was educational level. When compared to participants with no formal education, those with a college education or more were more than 5 times as likely to practice birth preparedness and complication readiness , Participants with a secondary school education were more than 9 times  as likely as those without a formal education to have birth preparedness practice, and mothers with a primary school education were more than 2 times  as likely as those without a formal education to have birth preparedness and complication readiness practice.History of antenatal care follow up was another significant factor associated with birth preparedness and complication readiness; those who have had previous history of antenatal care follow up are 4.33 times more likely to have birth preparedness and complication readiness practice  than those who have not. One of the birth outcomes that was significantly associated with birth preparedness and complication readiness practice was live birth. When compared to mothers who had no delivery outcome, mothers who had a live delivery were more than three times more likely  to have had birth preparations and complication readiness practice. Another factor that has been connected to birth preparations and complication preparation practice is giving birth in a health facility. Those who have previously given birth in a health institution are more likely to practice birth preparedness and complication readiness  than those who have never given birth in a health facility .Nonetheless, birth readiness and complication readiness are two proven and effective health-care strategies for reducing maternal and child mortality, particularly in countries with a high risk of maternal death and an inadequate health-care system. This study was conducted to assess birth preparedness and complication readiness practice and associated factors among mothers attending ANC in Yirgalem hospital. The overall prevalence of BPCR practice was 48.6%. The findings of this study is consistent with those of a study conducted in Wolaita Sodo, a town in southern Ethiopia, which found 48.5%  and in rIn comparison to a study conducted in Jimma Zone, south west Ethiopia (23.3%) , Dale DiPlan for place of birth 84.4%, identify experienced birth attendants 37.9%, arrange transportation 58.8%, save money 55.2%, and prepared blood donor 24.9% is the most common BPCR practice indications. This finding is nearly similar with a study done in Bench Maji Zone, Southwest Ethiopia 83.7% of mothers identify place of birth, 67.4% identifying skilled birth attendants, 45.2% saving money, 16.8% identification mode of transport and 5.6% arrange blood donor . A study17. The reason for this could be that younger women have never had an obstetrics issue, therefore they do not need to prepare, whereas older women who have had an obstetrics issue in the past do need to prepare for their current pregnancy (not assessed in the study). This The findings contrast with those of a study conducted in Wolaita Zone, Sodo town, which found that younger age groups are more likely to practice birth readiness and complication readiness [Birth preparedness and complication readiness are more common in the older age groups than in the younger age groups; the age group 37 was four times more likely to practice birth preparedness and complication readiness than the age group 18–24. The age group 25 to 30 was more than two times more likely to practice birth preparedness and complication readiness than the age group 18–24. The findings are consistent with research conducted in India among women who were beneficiaries of a rural primary health facility in the Dakshina district of Karnataka eadiness . The obsEducational level was another significantly associated with birth preparedness and complication readiness practice. From the respondents those whose educational level college, University and above are more than 5 time, educational level with secondary school more than 9 time and primary school education more than 2.8 time more likely to have birth preparedness and complication readiness practice than no formal education. The finding of this study was in line with the study done in Wolaita zone, Sodo town, Southern Ethiopia and Robe Woreda, Arisi zone, Central Ethiopia , 16. TheBirth preparedness and complication readiness practice are positively associated with antenatal care follow-up history; those who have had antenatal care follow-up in the past are 4.33 times more likely to have birth preparedness and complication readiness practice than those who have never had antenatal care follow-up. This research is in accord with research conducted in Adigrat, Ethiopia, and Wolaita Zone, Sodo, Ethiopia , 17. TheLive birth was one of the birth outcomes that was found to be substantially linked to birth preparedness and complication readiness practice. Mothers who had a live delivery were more than three times more likely to have undergone birth preparations and complication readiness practice than mothers who did not have a live delivery. When compared to those who have never given birth in a health facility, those who have a history of giving birth in a health facility are 3.09 times more likely to have birth preparedness and complication readiness practice. This study’s findings are comparable to those of a research conducted in the Dale District in Southern Ethiopia . The reaThe current study’s strengths were the high sample size, well-trained research assistants who interviewed the participants, and the BPCR questionnaire, which was adapted from JHPIEGO. However, there are certain limitations to the study. First, it is an institution-based study that only addresses women who visit a health facility; women who do not visit a health facility miss out on assessing their birth preparedness and complication readiness. The study’s cross-sectional design may make it difficult to discern a temporal relationship between the dependent and independent variables.Overall, there was low level of birth preparedness and compilation complication readiness practice. Birth preparedness and complication readiness were found to be strongly correlated with respondents’ age, level of education, age at first pregnancy, outcome face during pregnancy and childbirth, previous antenatal care follow up, and birth at a health institution.The government has to work in collaboration with different stockholder on institutional delivery coverage with health care provider expected to do not miss the opportunity to disseminate all information related BPCR practice.S1 Text(DOCX)Click here for additional data file.


However, the proteolytic cleavage of HER2, or HER2 shedding, induces the release of the target epitope at the ectodomain (ECD) and the generation of a constitutively active intracellular fragment (p95HER2), impeding the effectiveness of anti-HER2 therapy. Therefore, identifying key regulators in HER2 shedding might provide promising targetable vulnerabilities against resistance. In the current study, we found that upregulation of dolichyl-phosphate N-acetylglucosaminyltransferase (DPAGT1) sustained high-level HER2 shedding to confer trastuzumab resistance, which was associated with poor clinical outcomes. Upon trastuzumab treatment, the membrane-bound DPAGT1 protein was endocytosed via the caveolae pathway and retrogradely transported to the ER, where DPAGT1 induced N-glycosylation of the sheddase — ADAM metallopeptidase domain 10 (ADAM10) — to ensure its expression, maturation, and activation. N-glycosylation of ADAM10 at N267 protected itself from ER-associated protein degradation and was essential for DPAGT1-mediated HER2 shedding and trastuzumab resistance. Importantly, inhibition of DPAGT1 with tunicamycin acted synergistically with trastuzumab treatment to block HER2 signaling and reverse resistance. These findings reveal a prominent mechanism for HER2 shedding and suggest that targeting DPAGT1 might be a promising strategy against trastuzumab-resistant breast cancer.Human epidermal growth factor receptor 2–targeted (HER2-targeted) therapy is the mainstay of treatment for HER2  DPAGT1), encodes an endoplasmic reticulum (ER) integral membrane enzyme, belonging to the polyprenyl-phosphate N-acetyl hexosamine 1-phosphate transferase (PNPT) family (Dolichyl-phosphate N-acetylglucosaminephosphotransferase 1  family . DPAGT1 ) family . Althoug) family –25, it r. Importantly, inhibition of DPAGT1 resensitized trastuzumab-resistant HER2+ breast tumors. Therefore, these findings identified DPAGT1 as a key regulator of HER2 shedding and suggested that DPAGT1 inhibition might be a promising strategy against trastuzumab-resistant HER2+ breast cancer.In the present study, we found that DPAGT1-mediated N-glycosylation of ADAM10 sheddase at N267 was required for the stability, maturation, and activation of ADAM10. This occurred through the protection of ADAM10 from ER-associated degradation (ERAD), which led to robust HER2 shedding, trastuzumab resistance, and poor clinical outcomes+ breast cancer (with “early stage” defined as lymph node–positive or tumor size over 5 cm in diameter) before they received neoadjuvant trastuzumab therapy. The serum HER2-ECD level was determined to stratify the subgroups from patients with sensitive or poor responses using the median as the cutoff value for high or low serum HER2-ECD expression (P < 0.001), and that, among the patients with complete response (CR) or partial response (PR) after trastuzumab therapy, 71% (22 of 31) had low serum HER2-ECD, while 73% (22 of 30) of the patients with progressive disease (PD) or stable disease (SD) expressed high serum HER2-ECD levels (https://doi.org/10.1172/JCI164428DS1). These results indicated that high serum HER2-ECD level was associated with poor trastuzumab response and worse progression in HER2+ breast cancer.To identify potential regulators of HER2 shedding in trastuzumab resistance, peripheral blood samples and breast cancer biopsies were collected from 61 patients with locally advanced or early stage HER2pression . StatistDPAGT1 resulted in the most significant reduction of HER2-ECD level in the culture medium and inhibition of trastuzumab-induced cell death  in these patients (DPAGT1 mRNA expression correlated positively with the serum HER2-ECD in the resistant group (P = 0.012) but not in the sensitive group (P = 0.073)  group with the highest levels of HER2-ECD levels and the half of the sensitive (CR/PR) group with the lowest HER2-ECD levels were selected for RNA-Seq. Among these biopsies, RNA samples of 27 biopsies that met the quality and dose demand for RNA sequencing were finally used. The RNA-Seq analysis indicated that 378 genes were upregulated while 359 genes were downregulated in the resistant group compared with that in the sensitive group . Furtherll death , which spatients . Interes= 0.073) . These r+ breast cancer specimens  HER2pecimens . IHC stacurrence . Significurrence . Importareatment . It was t cancer . In addi cancers  . These results suggested that the N-glycosylation activity of DPAGT1 was indispensable for HER2 shedding. Consistently, the trastuzumab-resistant SK-BR-3 and BT-474 cell lines exhibited elevated expression levels of DPAGT1, p95HER2, and HER2-ECD compared with those in the parental cells  and extracellular regulated kinase (ERK1/2)  , A and B(ERK1/2) , A and B of HER2 . Therefon = 8/subgroup). The mice in each subgroup were given an i.p. injection of either IgG or trastuzumab once a week. Notably, the SK-BR-3/DPAGT1 tumors grew much faster than those of the other 2 groups, regardless of trastuzumab treatment, indicating that DPAGT1 promoted tumor growth and induced trastuzumab resistance in vivo , but not CLTA (encoding clathrin), substantially impaired trastuzumab-induced retrograde transport of DPAGT1  its trafficking from the ER to the Golgi apparatus, (b) protein processing in Golgi, and (c) its sheddase activity at the plasma membrane , 47. We DPAGT1 increased, while silencing DPAGT1 reduced, the endogenous preADAM10 and matADAM10 levels in SK-BR-3 cells  . Indeed,n the ER . Althougreatment . These rreatment , suggestNext, we explored the mechanism underlying ERAD-mediated ADAM10 degradation. Multiple steps are involved in the process of ERAD, including that the defective protein being recognized by ERAD substrate recognition factors and specifically ubiquitinated by the ER-associated E3 ligase, and then the adaptor protein adaptor subunit of ERAD E3 ubiquitin ligase (SEL1L) links the ubiquitinated substrates and recruits the valosin-containing protein  complex to promote retrotranslocation by which substrate proteins are extracted from the ER and degraded by the cytosolic 26S proteasome , 50. ConHRD1 substantially reduced the polyubiquitination level of ADAM10/N278Q-267Q and ADAM10/4NQ, but had little effect on the ADAM10-N278Q polyubiquitination , autocrine motility factor receptor , and membrane associated ring-CH-type finger 6 (MARCHF6), have been identified to be involved in ERAD-mediated degradation –53. Our tination . Moreovetination . Taken tHRD1, SEL1L, or VCP potently increased the ADAM10-4NQ and ADAM10-N278/267Q expression  using the clinical trastuzumab-resistant HER2mination . Similarmination . Althougmination . Patholoreatment . Coadminreatment . Therefooutcomes . Howeveroutcomes .+ breast cancer  and anti-ADAM10  antibodies. The IHC scoring was performed in the 170 HER2+ breast cancer slides using 5 random 200 × tumor fields per slide by 2 independent pathologists who were blinded to the clinical outcomes. The staining of DPAGT1 and ADAM10 were graded with 4 scores, strong 3+, moderate 2+, weak 1+, and negative 0. Specimens with scores of 3+ or 2+ were defined as having high expression, while those with scores of 1+ or 0 were defined as having low expression. Slides considered controversial in DPAGT1 or ADAM10 expression were assessed and determined by a third pathologist. Details of the IHC method are provided in the Supplemental information.IHC staining was performed on the 170 HER26) expressing vector, DPAGT1, or DPAGT1-N185A were orthotopically injected into the mammary fat pads of BALB/c nude mice (n = 8/group). Drugs were administered 2 weeks after tumor inoculation. Trastuzumab (20 mg/kg) was injected i.p., while TM (6 μg) was administered i.t. weekly for 5 weeks. Tumor volumes were measured weekly. For the relapse model (n = 15/group), all SK-BR-3 cells were stably transduced with the luciferase gene. Tumorectomy was performed 2 weeks after cell injection, and tumor resection was confirmed a week later by failed detection of luciferase signal using an In vivo Imagining System . Trastuzumab treatment was subsequently administered for 8 weeks , and tumor relapse was traced by the luciferase signal.Immune-deficient BALB/c-nude mice  and NOD-SCID  obtained from Gempharmatech Co., Ltd. were used for examination of the in situ growth of SK-BR-3 tumors and the s.c. growth of patient-derived xenograft (PDXs), respectively. Mice were housed in barrier facilities on a 12 hour light/dark cycle. In the in situ models, SK-BR-3 cells  and transplanted s.c. into the left armpit of anesthetized NOD-SCID mice. When the tumors reached 1.0–1.5 cm in diameter, they were implanted into new mice and passaged a minimum of 3 times to establish model stability. Treatment was started at a tumor size of approximately 200 mm3. Groups of 8 mice were randomized to receive either IgG  as control, trastuzumab , TM , or trastuzumab  combined with TM , once a week for 8 weeks. Serum was gathered from 3 random mice in each group before euthanization and subjected to HER2-ECD detection. The formed tumors were then used for validation of HER2 shedding, HER2 signaling, cleaved caspase-3, proliferation index, or apoptotic index.In the PDX model, fresh HER2Additional methodological information is provided in Supplemental Materials and Methods.2 test, 2-tailed Student’s t test, and 2-way ANOVA. Multivariate statistical analysis was performed using a Cox regression model. P < 0.05 was considered statistically significant.Statistical analyses were performed using the SPSS version 19.0 statistical software package (IBM Corp.). Statistical tests for data analysis included the log-rank test, χEthics approval (#B2022-459-01) and prior patient consent were obtained from the Institutional Research Ethics Committee of Sun Yat-sen University Cancer Center to use the clinical specimens for research purposes. Animal experimental procedures were approved by the IACUC of Sun Yat-sen University (No. L102042020000C). The study conformed to the principles set out in the Declaration of Helsinki.+ breast cancer biopsies have been deposited in the National Center for Biotechnology Information Sequence Read Archive (SRA) database (PRJNA860770). The gene expression profile of each sample was analyzed quantitatively using the Cuffdiff suite from Cufflinks (v2.2.1)  via the iProX partner repository with the data set identifier PXD035378.The RNA-Seq data of the 27 HER2(v2.2.1) . The masMY, YL, LK, SH, and LH carried out most of the experimental work; they collected and analyzed the data. MY, YL, SM, and XH conducted the RNA-Seq, qRT-PCR, ELISA, IF staining, and PLA assays. SH, LK, PL, YX, DS, and X Lin collected tissues and patient information and conducted IHC and survival analysis. MY, LH, SM, XC, and X Lu conducted the immunoblotting analysis, plasmid constructions, and IP assays. MY, SH, YL, and BC conducted animal studies. YO, LH, and BC conducted cell culture. MY, SM, YL, LH, and X Lu performed the in vitro studies. JL, CL, and LS raised the concept, design the experiments, wrote the manuscript, and supervised the project. The order of the co–first authors was assigned based on their efforts and contributions to the study. All author read and approved the final manuscript.


Objective In around 85% of vaginal births, the parturients undergo perineal lacerations and/or episiotomy. The present study aimed to determine the incidence of lacerations and episiotomies among parturients in 2018 in a habitual-risk public maternity hospital in southern Brazil, and to determine the risk and protective factors for such events.Methodology A retrospective cross-sectional study. Data were obtained from medical records and analyzed using the Stata software. Univariate and multivariate logistic regressions were performed. Values ofp < 0.05 were considered significant.Results In 2018, there were 525 vaginal births, 27.8% of which were attended by obstetricians, 70.7% by obstetric nurses, and 1.5% evolved without assistance. Overall, 55.2% of the parturients had some degree of laceration. The professional who attended the birth was a significant variable: a greater number of first- and second-degree lacerations, as well as more severe cases, occurred in births attended by nurses . Positions at birth that did not enable perineal protection techniques (expulsive period with the “hands-off” method), when analyzed in isolation, determined the risk; however, in the final regression model, this relationship was not confirmed. Although reported in the literature, there were no associations between the occurrence of laceration and age, skin color, or birth weight. In 24% of the births, episiotomy was performed, and doctors performed 63.5% of them.Conclusion Births attended by nurses resulted in an increased risk of perineal lacerations, of varying degrees. In turn, those assisted by physicians had a higher occurrence of episiotomy. During vaginal birth, some degree of perineal trauma can occur in ∼ 85% of parturients,Perineal lacerations are classified in degrees, according to the injured anatomical structures. First-degree lesions are restricted to the skin and mucosa; in second-degree lesions, the perineal muscles are affected. In third-degree lesions, the anal sphincter is compromised; these lesions are subdivided into: 3A, if less than 50% of the thickness of the external anal sphincter has been compromised; 3B, if more than 50% of the thickness of the external anal sphincter is injured; and 3C, if the internal and external sphincters are affected. In fourth-degree lesions, the rectal epithelium is also injured.Lacerations have short- and long-term negative impacts on women's lives.The risk factors include: parity;2);The protective factors mentioned in the literature include: perineal massage with saline-heated compresses in the perineum during the second stage of labor;Seeking to prevent the occurrence of perineal trauma, since 2018, the World Health OrganizationEpisiotomy, which represents the second most frequent type of perineal trauma, is defined by an incision made in the perineum during the expulsive period, aiming to increase vaginal diameter, facilitating birth.Since the beginning of the last century, episiotomy vecame part of the birth care routine, being performed on most parturients, despite the lack of studies.In recent years, with the “naturalization” of vaginal birth, the use of episiotomy has come to be considered “obstetric violence.” However, like any other medical surgical procedure, episiotomy has precise indications and a recommended surgical technique, which, if correctly executed, effectively protects the parturient from this important outcome of vaginal birth.The present study aimed to demonstrate the incidence of spontaneous lacerations and episiotomy in a southern Brazilian habitual-risk public maternity hospital, and to analyze the risk and protective factors associated with the occurrence of perineal lacerations.The present is a cross-sectional, retrospective study that analyzed births occurred in 2018 at Maternidade Santa Isabel, Hospital Casa de Saúde (HCS), in the municipality of Santa Maria, state of Rio Grande do Sul, Brazil. The study was approved by the Ethics in Research Committees of HCS and Universidade Franciscana under number 3.041.714.We included for analysis all vaginal births, including those that occurred upon arrival at the maternity, such as at its gateway, for example. The only exclusion criteria was the need for a cesarean section. Data were obtained from the electronic medical records. The following variables were included for analysis: age and skin color of the mother, gestational age, parity, birth position, professional responsible for the birth, the need for instrumental delivery, newborn birth weight, and the need for episiotomy. The birth positions included: lithotomy, squatting, semi-Fowler, four supports, vertical, birth seat, as well as other positions. The present study considered the “hands-off” positions: squatting, four supports, vertical, semi-Fowler and birth seat, and, as a position that enables the performance of the “hands-on” method: the classic lithotomy. The main outcome analyzed was the occurrence of perineal laceration, classified into: grade 1: lesion to the skin and mucosa; grade 2: injury reaching the perineal muscles; grade 3: lesion affecting even the anal sphincter complex; and grade 4: involvement of the rectal epithelium.p < 0.05 was accepted.The data were analyzed using the Stata  software, version 14. Initially, the normality of the variables was verified by the Shapiro-Wilk test. The continuous variables were expressed as median, minimum and maximum values, and the categorical variables were expressed as percentages. Univariate and multivariate logistic regression analysis were performed to identify the possible risk and protective factors (independent variables) associated with perineal laceration (dependent variable). A significance level ofDuring the study period, there were 741 births at Maternidade Santa Isabel. Of these, 525 (70.8%) were vaginal births, which were included for analysis in the present study. The main maternal data, birth position, and newborn birth weight are shown inThe median age of the mothers was of 23 years; the youngest was 14 years old, and the oldest, 43 years old, and the median gestational age was of 39.5 weeks (range: 27.6 to 41.6 weeks). Regarding skin color, there was no record of it for 29% (151) of the sample. Among the remaining sample, 64.2% were white and yellow, and 6.8% were black and brown. Most mothers (57.1%) were multiparous. The positions most adopted at birth were lithotomy (58.1%) and semi-Fowler (28.8%). The majority of births were attended by nurses 371 (70.7%), and 146 (27.8%) were performed by medical doctors. In total, 8 (1.5%) parturients gave birth at the hospital entrance, without professional assistance, and 6 (1.1%) births were instrumented by forceps. For 24% (126) of parturients, a mid-lateral episiotomy ewas necessary: 63.6% were performed by a medical doctor, and 36.5%, by a nurse. Most newborns had an adequate birth weight, and only 4.5% weighed more than 4,000 g.The perineum was considered intact, that is, not submitted to episiotomy and with no lacerations in 114 (21.7%) women, 8.9% of them primiparous, and 31.3% of them multiparous. Most parturients were multiparous (82.4%).The maternal and obstetric characteristics of the 290 parturient women who presented perineal lacerations are described inp > 0.25) . The other variables analyzed did not show an association with the occurrence of lacerations (> 0.25) .p < 0.25). The professional who performed the birth, in this case, a nurse , and the lack of performance of an episiotomy  were the variables that remained associated, increasing the chance of perineal lacerations during vaginal birth.Aiming to analyze the occurrence of perineal trauma and the associated risk and protective factors in a habitual-risk public maternity hospital, the present study found, in a sample of 525 parturients, an incidence of 55.2% of perineal lacerations. Of these, 1.4% were classified as severe, grades 3 and 4. In the analysis of the risk and protective factors, we observed that delivery performed by nurses and the lack of performance of an episiotomy were the factors that remained significantly associated with the occurrence of perineal lacerations in the final model.The frequency of perineal lacerations found in the present study (55.2%) is in line with hat is expected according to a report by the American College of Obstetricians and Gynecologists (ACOG),n = 290), 87.5% had their birth attended by nurses. It was in this group of professionals that the lacerations classified as severe occurred. In the present study, some degree of laceration was 2.89 times more likely to occur in births attended by nurses than in those attended by medical doctors. These data are in line with the work performed by Ott et al. (2015).Of the total number of parturients who presented some degree of laceration . However, most were grade-1 lacerations, which less traumatic than episiotomy.Regarding episiotomy, the rate found, of 24%, was still far from that recommended by the WHO,In the univariate analysis, there was also an association between perineal laceration and the positions adopted at the end of the expulsive period. Gåreberg et al. (1994)In the present study, there was no association of the parturient's skin color and perineal lacerations. Perhaps this data was influenced by a high rate of women (28.9%) whose skin color was not included in the medical records. However, when we analyzed the parturients without any degree of laceration, we found that 36% of black women had a whole perineum compared with 22% of white women. These findings were very similar to those of the study by Howard et al. (2000).We were not able to demonstrate an association between perineal laceration and an instrumentalized delivery. This may be due to the low number of parturients that needed such an intervention in the present study, since several studies have reported a strong association.No association was observed between the patients' age or parity and birth weight. However, the relationship between birth weight and the occurrence of lacerations is consolidated in the literature.Spontaneous lacerations occurred in half of the analyzed vaginal births, especially in those in which an episiotomy was not performed, and in those attended by a non-medical professional. Although high, this rate is acceptable according to the ACOG. It should be noted that most were grade-1 lacerations, which are less traumatic than an episiotomy. The episiotomy rate was above that recommended by the WHO, and the procedure was performed more frequently when birth assistance was provided by an obstetrician. It was not possible to demonstrate the association between perineal laceration and the parturient's age, skin color, as well as the birth weight of the newborn. Therefore, regarding all the questions that have been made for decades on the practices adopted at birth, the present study provides a panorama of the situation in some Brazilian hospitals.


Marginalized populations experience disproportionate rates of preterm birth and early term birth. Exposure to per- and polyfluoroalkyl substances (PFAS) has been reported to reduce length of gestation, but the underlying mechanisms are unknown. In the present study, we characterized the molecular signatures of prenatal PFAS exposure and gestational age at birth outcomes in the newborn dried blood spot metabolome among 267 African American dyads in Atlanta, Georgia between 2016 and 2020. Pregnant people with higher serum perfluorooctanoic acid and perfluorohexane sulfonic acid concentrations had increased odds of an early birth. After false discovery rate correction, the effect of prenatal PFAS exposure on reduced length of gestation was associated with 8 metabolomic pathways and 52 metabolites in newborn dried blood spots, which suggested perturbed tissue neogenesis, neuroendocrine function, and redox homeostasis. These mechanisms explain how prenatal PFAS exposure gives rise to the leading cause of infant death in the United States. Mechanisms of the impact of PFAS  on adverse birth outcomes remain largely unknown. Here, authors identified tissue neogenesis, neuroendocrine function, and redox homeostasis as imprints of prenatal PFAS exposures and reduced gestational age in the newborn metabolome. The annual rates of these adverse birth outcomes are consistently highest among Black Americans1. Preterm birth (PTB) and early term birth (ETB) are leading risk factors for morbidity and mortality during infancy, childhood, and early adulthood5. A reduced length of gestation is also linked to cardiovascular disease, diabetes, neurodevelopmental disorders, cancer, and other prevalent chronic health conditions across the life course11. The gestational age at birth is influenced by a complex interplay of psychosocial, behavioral, nutritional, and biological determinants, plus mounting evidence suggests environmental exposures potentiate the risk of PTB and ETB16. Recent work has demonstrated per- and polyfluoroalkyl substances (PFAS) are commonly present in utero, which may explain poor fetal growth and development20. However, most environmental epidemiologic studies have focused on white, highly educated populations and little is known about marginalized populations. Further, the underlying molecular mechanisms elicited by PFAS within the fetus’ metabolic, endocrine, and immune systems remain poorly understood.In 2020, there were an estimated 364,487 infants born preterm  and 1,003,260 infants born early term  in the United States (US)21. Legacy PFAS, including perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorooctane sulfonic acid (PFOS), and perfluorohexane sulfonic acid (PFHxS), share in common a lipophobic carbon-fluorine chain, hydrophilic functional group, and proteinophilic attraction towards albumin and various fatty acid binding proteins24. These chemical properties result in long half-lives and foster persistence in the environment, which through a variety of exposure pathways lead to bioaccumulation in humans25.PFAS are anthropogenic surfactants used by industries throughout the world and have a long history of use by the US Department of Defense26. Similarly, PFOA, PFOS, PFNA, and PFHxS are detected in nearly all maternal serum samples collected in the late first trimester or early second trimester in the Atlanta African American Maternal-Child Cohort27. During pregnancy, a proportion of the maternal PFAS body burden is able to cross the placental barrier into the uterus where the fetus is exposed 31. The direct effects of prenatal PFAS exposure on reduced length of gestation have been examined in several human populations34. However, the inconsistent and limited data among marginalized groups warrant more research, plus an unfulfilled public health priority is to hone the causal pathways for these exposure-outcome relationships. Our group and others have proposed interference with homeostatic processes from such environmental exposures in utero leads to a cascade of bioenergetic perturbation, endocrine disruption, and oxidative stress production, which may synergistically promote adverse birth outcomes38.In the US population, 99% of pregnant people have detectable levels of PFOA and PFOS in their bloodmeet-in-the-middle (MITM) analysis may help to characterize the molecular signatures of PFAS in utero, biological processes integral to fetal programming, and adverse phenotypes in early life37. Specifically, MITM allows for environmental exposures and health outcomes to be linked to metabolomic profiles, the global set of metabolites and systemic responses to internal doses of exogenous and endogenous substances40. Intermediate biomarkers and biological pathways for exposure-outcome relationships have been identified in several environmental epidemiologic studies using the MITM framework, including exposure to air pollution, tobacco smoke, and PFAS and fertility, PTB, and small-for-gestational age (SGA), respectively42. To our knowledge, no investigations have taken this approach to understand mechanistically how prenatal PFAS exposure influences the newborn metabolome and, in turn, how these responses are associated with gestational age at birth outcomes.Untargeted metabolomics by high-resolution mass spectrometry combined with a 43. Additionally, we analyzed newborn dried blood spots (DBS), a minimally invasive biospecimen used for screening within 48 h of birth, with high-resolution metabolomics and the MITM framework to identify and measure the underlying metabolites and pathways.In a prospective birth cohort, we sought to profile the neonatal metabolome for molecular signatures of maternal PFAS concentrations during early to middle pregnancy and gestational age at birth outcomes among African American mother-newborn dyads in Atlanta, Georgia. Based on prior work, we hypothesized that prenatal PFAS exposure interferes with gestational length and fetal growthHere, we show that an increase in maternal serum PFAS concentrations was prospectively associated with ETB and medically indicated early birth prior to full-term. The newborn DBS metabolome revealed perturbations in biological pathways involving amino acids, bioactive lipids, and enzymes, coenzymes, and cofactors underly the PFAS and gestational age at birth outcome relationships. We further characterized the molecular network by identifying salient metabolites in the newborn circulatory system, including L-DOPA, linoleic acid, and β-NAD.n = 58; 22%) or with obesity , were parous , and did not use tobacco  or marijuana . At enrollment, the average participant age was 25.6 years (SD = 5.2) and 163 (61%) of the mothers were in the first trimester. Participants predominantly had a high school education or less , public health insurance with Medicaid , and an income level 132% or lower times that of the Federal Poverty Level .The characteristics of 267 African American pregnant people and newborns included in our study are summarized in Table There were 139 (52%) newborns assigned female sex at birth. The average gestational age at delivery was 38.7 weeks (SD = 2.0), with a total of 118 (51%) healthy and full-term, 31 (12%) preterm, and 82 (31%) early term. Among the early births (PTB or ETB) prior to full-term, 82 (69%) followed spontaneous labor and 31 (26%) followed medically indicated induction or C-section Fig. .2-transformed PFAS concentrations (ng/mL) were weakly to moderately correlated with each other  Table .2-unit increase in PFOA concentrations, the odds of ETB were 1.59  compared to healthy full-term birth. The odds ratio (OR) of ETB was also significantly increased among those in the 2nd quartile  and 4th quartile  of PFOA concentration versus the referent. Concentrations of PFOA categorized as the 3rd quartile increased the odds of ETB, but did not reach statistical significance (p > 0.05). Quartiles of PFHxS concentrations demonstrated a similar dose-response relationship with PTB and medically indicated early birth. Log2-transformed and quartile PFOA concentrations were associated with moderate increases in the odds of spontaneous labor. Finally, gestational age at delivery was inconsistently associated with PFAS concentrations.The effects of maternal PFAS concentration on gestational age at birth  and labor and delivery  outcomes relative to healthy, full-term birth are presented in Fig. p-value < 0.05), PFOS , PFNA , and PFHxS , respectively , ETB , spontaneous early birth , PTB , and gestational weeks , respectively  prior to full-term among African American pregnant people and their newborns. Furthermore, the neonatal metabolome had an intermediate role for this relationship and offered insights into the underlying molecular network, as presented in Fig. 27. Relative to other prospective cohorts in the US, maternal serum PFAS concentrations collected during early pregnancy are comparable to those observed in Chemicals In Our Bodies and Illinois Kids Infant Development Study, and lower than those observed in Project Viva, LIFECODES, and Healthy Start Study, which have a majority white and higher SES study population47.In comparing pregnant people in the Atlanta African American Maternal-Child Cohort versus participants matched on sex, age, and race in NHANES, the detection rate and geometric average of PFOA, PFOS, and PFNA are similar while PFHxS is significantly higher in our study population52. Similarly, the relationship between various PFAS measured in pregnant people and gestational age of newborns has been difficult to replicate across environmental epidemiologic studies54. A potential explanation for these observed differences is the proportion of PTB and ETB were higher in the present study relative to prior work, which mirrors the current trends of maternal and child health outcomes among African American families. Our findings provide credence to a relationship between prenatal PFAS exposure and adverse birth outcomes in populations routinely excluded from environmental epidemiologic studies.While a growing body of evidence suggests exposure to PFAS increase the risk of PTB, as supported by our findings, other studies linking spontaneous or medically indicated early birth in relation to serum PFAS levels have proven inconclusive55. Our analyses suggest maternal PFAS levels may affect fetal P450 and other drug-metabolizing enzymes, as made evident in the newborn DBS metabolome, and are associated with PTB and ETB, respectively. Such exposures may interfere with developmental transitions in CYP expression, which occur throughout gestation and postnatally, thereby influencing the disposition of PFAS56. Since mitochondrial P450 catabolize cholesterol, these enzymatic mechanisms may also point to perturbed steroidogenesis, bile acid synthesis, and vitamin D3 activation, which our group and others have shown to be affected by PFAS67.When compared to adults, the developing fetus is exposed to a higher dose of environmental chemicals due to immature size and has insufficient enzymatic capacity to protect against xenobiotics69. We found maternal serum PFOA and PFHxS levels affected neonatal vitamin B3 metabolism, which was associated with medically indicated early birth prior to full-term. Additionally, the vitamin B3 metabolite β-NAD was attenuated by PFOS and PFHxS. Taken altogether, a reduced length of gestation may arise in part from prenatal PFAS exposure targeting vital pathways for energy metabolism.In early life and beyond, bioenergetic processes rely on vitamin B3 to sustain the reduced nicotinamide adenine dinucleotide phosphate (NADPH) pool, which is involved in mitochondrial oxidative phosphorylation, redox homeostasis, and protein synthesis and function74. PFAS generate an excessive amount of ROS in vitro and deplete antioxidant reserves in vitro and in vivo78. We found several antioxidative metabolites, including benzoic acid and S-lactyolglutathione, were associated with maternal serum PFHxS concentrations and gestational age, which may be in response to exposure. In addition, oxidative stress biomarkers have been linked to an increased risk of preterm birth79. Our metabolomic findings need to be validated in other fetal and neonatal populations, but illustrate the potential for oxidative stress to serve as a shared link between prenatal PFAS exposure and reduced length of gestation.Biopterins are cofactors for the biosynthesis of neurotransmitters, hormone precursors, and vasodilators plus indicate oxidative stress since reactive oxygen species (ROS) limit their bioavailability37. Complex systems of amino acid transporters in the placenta tightly regulate the flux of amino acids between mother and fetus to ensure the demand for tissue neogenesis is met and so byproducts of protein metabolism are removed81. It has been shown that PFAS exposure during gestation reduces amino acid transporter expression and system activity, which subsequently limits fetal growth and development. For example, there is reduced expression of SNAT4, an isoform of the system A amino acid transporter, in the placentas of pregnant mice exposed to PFOS82. In the future, these early life mechanisms of amino acid metabolism may serve as therapeutic targets for reduced length of gestation caused by PFAS in utero.We observed most of the intermediate metabolomic signatures between PFAS in pregnant people and gestational age at birth outcomes were related to amino acids, consistent with our prior work using maternal serum metabolomics85. Perturbations to this amino acid during pregnancy have been implicated in chronic inflammation disease states among mothers and adverse neurodevelopmental outcomes among offspring86. Similarly, tyrosine has a prominent role in the dopaminergic system of the placenta-brain-axis. Tyrosine hydroxylase synthesizes the dopamine precursor, levodopa (L-DOPA), from a combination of tyrosine, oxygen, and biopterin to support fetal neurodevelopment and survival87. Our results suggest the indirect effects of PFAS on reduced length of gestation may occur through neuroendocrine pathways, and follow-up research should prioritize the investigation of related chemical messengers among premature and early newborns.Prenatal exposure to PFAS may potentiate risk of PTB and ETB by disrupting the metabolism of tryptophan and tyrosine. Tryptophan results in serotonin, kynurenine, tryptamine, and indole production, which take part in neurological, hormonal, and immunological processes during pregnancy and afterwards92. Specific to fetal growth and development, perturbed bioactive lipids may contribute to the mechanisms underlying the relationship between PFAS toxicity in utero and reduced length of gestation.The newborn DBS metabolome was enriched with metabolic pathways for glycerophospholipids and leukotrienes along with arachidonic acid, linoleic acid, and 15-cyclohexyl pentanor prostaglandin F2α metabolites. In other populations with occupational, community, and background exposure, dyslipidemia is commonly present regardless of age, possibly suggesting PFAS affect a lipid pathway conserved across the life course94. These three processes have the potential to drive bioenergetic perturbation and oxidative stress production during gestation, which may be antecedent molecular events to adverse birth outcomes. One proposed mechanism is upregulated PPAR signaling from PFAS exposure alters arachidonic acid metabolism and its downstream metabolites, which leads to oxidative stress and inflammation, based on experimental work in rodents using metabolomics, proteomics, and transcriptomics98. Pregnancy may represent a sensitive window for PFAS to interact with eicosanoids and their precursors because of their role in the onset of labor, which has long been recognized by clinicians99. The application of targeted methods is needed to validate and expand upon the results presented here, particularly the roles of bioactive lipids and inflammatory processes that precede or concomitantly occur with a reduced length of gestation.The relationship between serum PFAS levels and lipids has been primarily attributed to suppressed mitochondrial fatty acid β-oxidation, attenuated farsenoid x receptor (FXR) via bile acids, and activated peroxisome proliferator-activated receptors (PPAR)The exact role of prenatal PFAS exposure, as well as their overall significance, in driving maternal and child health disparities among African Americans remains unclear. Perhaps, exposure to PFAS compounds the physiological effects of gendered racial stress and discrimination during pregnancy, ultimately leading to an early birth. In consideration of the mechanisms identified here, more research is needed to understand the potential joint effects of environmental chemicals and non-chemical stressors on perinatal health outcomes.37. As a step to validate and expand upon such findings, we used newborn DBS samples to make inferences about how the fetal biological system is affected by PFAS exposure in utero, a major advancement over studies that have relied on cord blood. Our study also serves as a proof-of-concept for integrating high-throughput -omics, newborn screening protocols, and environmental molecular epidemiology, which will be increasingly important as public health and translational science advance toward a precision medicine model. Third, our phenotypic classification of gestational age at birth outcomes was based on medical record abstraction by medical personnel, minimizing the chance of misclassification that might affect studies based on self-report. Our research protocol also considered the phenotypic classification of births as early (PTB or ETB) or full-term based on early pregnancy dating with further phenotypic classification of early births following spontaneous labor or medically indicated labor, allowing us to distinguish these different pathways to early birth in our analyses. There is a recognized need for further research into factors increasing the risk of ETB as this is an understudied birth outcome and infants born in this category disproportionately experience excess morbidity relative to full-term infants100. Lastly, our study population included exclusively African Americans, a population that is sorely underrepresented in the extant literature.A combination of systems biology methods and tools, sampling techniques, and biostatistical approaches strengthened our study. First, we leveraged an untargeted high-resolution metabolomics platform with the MITM framework, which together positioned us to identify and measure the links among exposures, molecular signatures, and outcomes. Our group initially examined maternal metabolomic perturbations associated with prenatal PFAS exposure and fetal growth outcomes in the Atlanta African American Maternal-Child Cohort102. The newborn DBS samples and panel of gestational age at birth outcomes were measured during the same time period, which hinders causal inference for the metabolomic signal-outcome associations. Specific to the newborn DBS samples collected via heel-stick, critics have raised concerns over potential uneven cell distribution, cell lysis, hematocrit variation, and pre-analytical clotting when analyzing this biomatrix using untargeted metabolomics105. Although the untargeted DBS metabolomics analysis was normalized by the size and weight of the DBS, we were unable to conduct additional, potentially more optimal normalization methods, including adjustment for hemoglobin, specific gravity, protein, or potassium due to limited volume of available sample106. Finally, there are fewer assumptions required for using the MITM framework compared to formal mediation analysis and the results should be interpreted with caution.Our study also had several limitations. Only the individual effects of PFAS were included in this study, but mixture effects are plausible and alternative PFAS with lower detection rates and/or shorter half-lives may contribute to the observed associations. There is a potential for interaction of the studied PFAS with other environmental chemicals from co-exposure, which may be antagonistic, synergistic, or additive, and mask the true health effects of PFAS. Future population-based studies are needed to investigate environmental chemical mixtures and emerging PFAS, for which there are thousands, in both the intrauterine and extrauterine environments. Our limited sample size also necessitates a larger-scale study, which newborn DBS sampling could facilitate. There was a relatively small number of pregnant people who experienced PTB, which may explain why we did not observe a significant dose-response relationship when participants were categorized by exposure quartiles. Furthermore, the pathogenesis of adverse birth outcomes is likely multifactorial and influenced by genetics, socioeconomic status and behaviors, diet, environment, and notably for minority racial and ethnic groups, structural factors such as social inequality and systemic racism. As a result, we cannot rule out the possibility of residual confounding. By the same token, there is an ongoing discussion in environmental epidemiology about how maternal BMI is associated with serum PFAS concentrations during pregnancy. Based on the body of literature available today, we treated maternal BMI as a confounder, although more research is needed to clarify the direction of effect between adiposity and PFAS in pregnant populationsin utero influence PTB and ETB risk. Additional research is needed to validate our findings in other populations and to investigate the potential of these metabolic mechanisms and biomarkers for detection, treatment, and prevention strategies in public health and clinical settings.Taken altogether, our study provides novel evidence and supports the current mechanistic understanding of how PFAS 108. Participants were eligible for inclusion if they self-identified as African American or Black, and were born in the US, between 18 and 40 years old, pregnant with a singleton, fluent in English, and had no chronic medical conditions. All participants provided written, informed consent to participate in the study, which was approved by the Institutional Review Board at Emory University . Participant data are confidential and proprietary information to the ATL AA cohort.Participants from the Atlanta African American Maternal-Child Cohort (ATL AA hereafter) were included in the present analysis. This ongoing, prospective birth cohort enrolls pregnant African Americans between 6 and 17 weeks gestation at Emory Midtown Hospital and Grady Hospital, which serve socioeconomically diverse populations in Atlanta, Georgia, and extends dyad follow-up through age six. Additional information regarding the cohort profile and data collection is described in detail elsewheren = 6) from our analysis. The remaining 267 dyads were included in the PFAS metabolome-wide association studies (MWAS), gestational age MWAS, and prenatal PFAS-gestational age analysis.A total of 279 mothers and newborns enrolled in the ATL AA cohort had serum and DBS samples, respectively. At the time of our analysis, there were 273 participants with complete data available for PFAS measurements and DBS metabolomics Fig. . The liv27. Tobacco smoke is also associated with perturbations in the maternal serum metabolome and adverse birth outcomes42. Altogether, this information guided our construction of a directed acyclic graph  was calculated from the first clinical visit’s weight and height measurements during early pregnancy and categorized as follows: underweight <18.5 kg/m2, normal weight 18.5– < 25 kg/m2, overweight 25– < 30 kg/m2, and with obesity ≥30 kg/m2. Since PFAS undergo trans-placental transfer throughout the course of pregnancy, the gestational week when the serum samples were collected from the mothers was included as a continuous covariate in the models which included PFAS as the exposures. Finally, neonatal sex was believed to affect the exposures and outcomes, and thus adjusted in the models.A thorough review of the literature was completed a priori to identify additional covariates that potentially confound the relationships between our exposures and outcomes. In previous analyses conducted in the ATL AA Cohort, we found maternal body mass index (BMI), use of tobacco and marijuana, education, parity, and age are significant predictors of prenatal PFAS exposureg) to obtain the serum fraction, which was used to quantify PFOA, PFNA, PFOS, and PFHxS, and subsequently stored at −80 °C. The analytical procedures and methods to quantify PFAS have been described in detail elsewhere109. Briefly, after randomization, the sera were spiked with isotopic internal standards followed by solid-phase extraction and analysis by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Individual PFAS species were quantified by isotope dilution calibration.Whole blood samples were collected from pregnant participants between 6 and 17 gestational weeks via venipuncture in an antecubital vein. These samples were transported to the National Institute of Environmental Health Sciences (NIEHS) Children’s Health Exposure Analysis Resource (CHEAR) laboratories, including Emory University’s Laboratory of Exposure Assessment and Development for Environmental Research (LEADER), New York University’s Laboratory Hub (NYU), and the Wadsworth Center Laboratory in New York. The samples were centrifuged  to demonstrate data quality assurance in serum PFAS analysis.The Human Health Exposure Analysis Resource (HHEAR) replaced CHEAR in 2019 and requires adherence to strict quality control procedures so services at any of the laboratories are harmonized and comparable to globally recognized, external laboratories114. In accordance with the American College of Obstetricians and Gynecologists (ACOG) and the Society for Maternal Fetal Medicine (SMFM), gestational age at birth was categorized as follows: gestational age 22– < 37 weeks was considered PTB; gestational age 37–38 weeks was considered ETB; full-term births had a gestational age ≥39 weeks100. Information about the presence or absence of pregnancy complications and the labor and delivery course was abstracted from the medical record and used to further categorize the early births (those that were PTB or ETB) as spontaneous  or medically indicated ; in addition, full-term births were further categorized as healthy full-term births if the pregnancy was without complications  and these healthy full-term births were used as the referent category in analyses.Gestational age at birth (weeks) was calculated from the estimated date of conception, based on the mother’s self-reported last menstrual period and/or early pregnancy ultrasound, to the date of delivery118. In comparison to whole blood samples containing serum and plasma fractions, newborn DBS samples are also reliable, reproducible, and representative biospecimens of the circulating metabolome in humans, plus carry the advantage of novel insights into the intrauterine environment, which are largely uncharacterized118.Newborn DBS samples are routinely collected at the time of birth for medical screening and public health surveillance then archived for future biomonitoring purposes. Metabolomics analysis of newborn DBS samples has recently emerged as a powerful method to phenotype exposures and health outcomes, such as those in the present study, at the population level119. On the day of collection, card specimens were transported to the Georgia Department of Public Health Laboratory for storage in a walk-in refrigerator (2–8 °C) without a desiccant for up to three months and then transported to an Emory University Laboratory for storage in freezer (−80 °C) within gas-impermeable bags with dessicant until assay119. For this study, we obtained single 15-mm punches  that were collected from 279 children between 2016 and 2020116. An additional set of 15-mm punches was obtained from adjacent portions of filter paper from the same Guthrie cards to use as blanks.DBS samples were collected within 24–48 h after birth from a heel stick performed by trained hospital nursery personnel using a standardized protocol that involves cleaning the skin of the heel with 75% isopropanol , using a sterile 2.5 mm lancet to obtain the sample, and collecting the sample onto a standard Guthrie card by saturating each circle with ~75 uL of bloodUntargeted high-resolution metabolomics profiling was conducted in a single run by the North Carolina HHEAR Hub (NC HHEAR Hub) in UNC Nutrition Research Institute, North Carolina Research Campus  using established methods. Blood spot samples were extracted with 1 mL ice-cold methanol containing 500 ng/mL L-tryptophan-d5, vortexed at 5,000 rpm for 10 min, and then sonicated for another 20 min. After centrifugation at 16,000 rcf for 10 min at 4 °C, 600 µL of the extracted supernatant was transferred into a 2.0 mL low-bind microfuge tube. An additional 70 µL of the extracted supernatant from each of the study samples was pooled together and then redistributed into new set of tubes with 600 µL per tube to generate total study pool samples. The empty blood spot card (without blood) matched with each of the study samples in terms of size and material was used as a blank and processed using the same procedures as the study sample. Supernatant (70 µL) from each of the blanks were mixed together and then distributed into multiple low-bind 2.0 mL tubes with a volume of 600 µL per tube. All supernatant aliquots (600 µL) including study samples, study pools, and blanks were dried by vacuum concentrator overnight and reconstituted with 100 µL water-methanol  for the ultra-high performance (UHP) LC-HR-MS analysis. Aliquots of 50 µL of NIST reference plasma (SRM 1950) were also prepared as external quality control samples. Briefly, 400 µL of methanol containing 500 ng/mL L-tryptophan-d5 was vortexed at 5,000 rpm for 2 min. Afterwards, extracts were centrifuged at 16,000 rcf for 10 min at 4 °C, and 350 µL of the supernatant was dried by vacuum concentrator overnight and reconstituted with 100 µL water-methanol  prior to analysis. Study samples were randomized before sample preparation and data acquisition. Quality control study pools, NIST plasma references, and blanks were interspersed at a rate of 10% amongst the study samples during the analysis sequence. Samples were run across three batches.The untargeted data was acquired on a Vanquish UHPLC system coupled with a Q Exactive™ HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer . A volume of 5 µl was injected for analysis. Metabolites were separated via an HSS T3 C18 column  at 50 °C with mobile phases of water (A) and methanol (B), each containing 0.1% formic acid (v/v). The UHPLC linear gradient began with 2% B, and increased to 100% B in 16 min, then held for 4 min, with a flow rate at 400 µL/min. The untargeted metabolomics data was acquired in a range of 70-1050 m/z using the data dependent acquisition mode for the MS/MS spectra data.120. The remaining peaks were normalized by Progenesis QI using the “normalize to all” feature. Signals that significantly differed amongst the three running batches  correction q < 0.05) were excluded for further analysis. Finally, signals were filtered to only include those detected in 95% of serum samples and the metabolomics data was log2-transformed to normalize positive skewness.The UHPLC-HR-MS data were processed by Progenesis QI  for peak identification and alignment. Background signals were excluded if the mean intensity across blanks were higher than that across the quality control study pools based on the unnormalized data121. The serum PFAS concentrations were positively skewed and log2-transformed to normalize their distribution. For every pair of log2-transformed PFAS in this study, Pearson correlations were examined. Descriptive statistics were also performed for the clinical, sociodemographic, and anthropometric variables. Continuous variables are presented as the mean ± standard deviation (SD) and categorical variables are presented as the count and relative frequency.All statistical analyses were conducted in R . Serum PFAS concentrations were first analyzed with descriptive statistics, including their detection frequency, distribution percentile, and geometric mean (GM) and standard deviation (GSD). For serum samples with nondetectable PFAS concentrations (<5%), the exposure values were imputed with the limit of detection (LOD) divided by We tested for collinearity among variables by examining bivariate associations. Other assumptions to proceed with regression models were satisfied by visually inspecting distributions of the variables. Multivariable regression models were used to investigate individual maternal PFAS concentrations in relation to individual birth outcomes. All of the models were adjusted for the same set of covariates: maternal age at enrollment, maternal prenatal BMI, maternal prenatal tobacco and marijuana use, parity, maternal level of education, neonatal sex, and gestational week at sample collection. Linear regressions were fitted to the continuous birth outcome, gestational weeks at birth, while logistic regressions were fitted to the binary birth outcomes . For logistic regression models, the reference group was healthy, full-term newborns ). We also modeled dose-response relationships between quartiles of serum PFAS concentrations and birth outcomes.Intensity is the signal for every metabolomic signal detected in the DBS after data filtering, Intensity of every metabolomic signal. The same set of covariates was included in the models fitted for binary birth outcomes as shown below:Nine MWAS were carried out to determine the newborn DBS signals associated with the prenatal PFAS concentrations and birth outcomes. First, the exposure-metabolomic signal relationships were investigated for each PFAS species by fitting a linear model with the following form:p-values obtained from the MWAS were corrected for FDR using the Benjamini-Hochberg and Bonferroni procedures since multiple comparisons were made123.The Mummichog (Version 1.0.10), which predicts biological networks, pathways, and metabolites based on significant signals with tentative chemical identities124. We serially reduced the significance level at which signals were eligible for inclusion in the pathway enrichment analyses, ranging from a raw p-value < 0.05 to Bonferroni corrected q-value < 0.01 as long as there were ≥100 signals available. Additionally, to minimize the chance of false positive discoveries, we only retained pathways with Mummichog’s p-value < 0.05 after 1000 permutations were performed and pathways enriched with ≥10% overlapping signals relative to the pathway size.Pathway enrichment analyses were performed using the bioinformatics software 127. Metabolites that matched to the IESL by RT (± 0.5 min), MS ( <5 ppm), and MS/MS (similarity score >30) are labeled OL1, whereas those that match by RT and MS are labeled OL2a. Metabolites with OL1 and OL2a were identified with the highest level of confidence in this study. An OL2b label was provided for signals matched by MS and MS/MS to the IESL but were outside the RT window (±0.5 min). Metabolites annotated with OL2b are the conjugates/isomers that share similar or the same moieties with the matched compound in IESL. A public database (PDa) label was provided for signals matched by MS (<5 ppm) and experimental MS/MS (similarity score >30) to public databases. Annotated signals with a PDa label could be the listed compound, isomer, or derivative of the listed compound. OL2b and PDa were considered confident annotations in this study. Metabolites annotated with less confidence were not retained in subsequent analyses.Signals were identified by matching against the NC HHEAR Hub in-house experimental standards library (IESL) generated from acquired data for over 2400 compounds, including endogenous metabolites associated with host metabolism and exogenous metabolites related to lifetime exposures including environmentally relevant compounds, ingested food components, drugs and medications, as well as derivatives/conjugates formed in vivo after exposure. All standard reference compounds in the IESL were run under identical conditions to study samples. The same set of signals were also matched to public databases, including NIST and METLIN. These signals were matched with metabolites and labeled with ontology levels (OL) to indicate the evidence that supported the identification or annotation, including RT, exact mass (MS), MS/MS fragmentation pattern, and/or isotopic ion pattern39. Specifically, the pathways and metabolites significantly enriched in both the PFAS MWAS and birth outcomes MWAS were identified as intermediate biological mechanisms and biomarkers. These results informed our construction of a hypothesized diagram relating the effect of prenatal PFAS exposure on birth outcomes in our study population.We analyzed the neonatal metabolome for molecular signatures underlying the relationship between maternal PFAS concentrations and gestational age at birth outcomes with the MITM framework129. To assess the influence of this hypothesized confounder on the enriched signals and pathways associated with PFAS exposure, we conducted sensitivity analyses without adjusting for the week when the maternal serum sample was collected and compared the results to those obtained in the main analyses. We also performed sensitivity analyses to identify similarities across the enriched pathways associated with PFAS and birth outcomes at the serially reduced significance thresholds, as described above.The PFAS body burden in fetal tissues has been shown to increase with gestational age and is believed to coincide with trans-placental transferThe content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.Further information on research design is available in the Supplementary Information FileReporting Summary


Weight, length/height and head circumference (HC) were collected at birth, discharge and at 2-year CA, and Δ z-scores were calculated. The MOM group had significantly larger negative change in weight and length z-scores between birth and discharge, and smaller positive change in HC z-score (–0·47 (sd 0·41) v. −0·25 (sd 0·36), P = 0·01; −0·63 (sd 0·75) v. −0·27 (sd 0·75), P = 0·03; 0·13 (sd 0·67) v. 0·41 (sd 0·55), P = 0·04, respectively). Almost half of the MOM-fed infants experienced poor length growth by discharge compared with 22 % of PF-fed infants (P = 0·03). By 2-year CA, both groups had similar positive change in weight and HC z-scores, but MOM-fed infants had a slower increase in height z-score (0·64 (sd 1·30) v. 1·33 (sd 1·33), P = 0·02), and only 40 % had achieved catch-up height compared with 68 % of the PF group (P = 0·02). Our study indicates that fortified MOM-fed SGA preterm infants may need extra nutritional support in the first 2 years of life to achieve height growth potential.Small for gestational age (SGA) is typically defined as birth weight < 10th percentile for age. Limited data are available regarding the growth of SGA preterm infants in relation to feeding type. We aimed to study SGA preterm infants fed fortified mother’s own milk (MOM) or preterm formula (PF) on growth patterns and catch-up growth at discharge and 2-year corrected age (CA). Our retrospective cohort study included data from medical records and follow-up questionnaires about SGA preterm infants born at < 37 weeks fed on MOM ( Globally, 15 million babies are born preterm annually, a preterm birth rate of ∼11 %.. SGA is typically defined as birth weight < 10th percentile for age. Others define SGA as birth weight below the 3rd or 5th percentile, or less than –2 sd of expected birth weight for gestational age (GA)–4. Fetal growth restriction occurs due to several factors, including placental insufficiency, genetics, inadequate nutrition, deprived social-environmental states and maternal health issues. SGA preterm infants have a two to fourfold elevated risk of mortality compared with non-SGA preterm infants. Moreover, they are prone to multisystem complications, such as anaemia, respiratory failure, necrotising enterocolitis, retinopathy of prematurity, bronchopulmonary dysplasia and patent ductus arteriosus,5.Of these infants, ∼20 % are born small for gestational age (SGA). Mother’s own milk (MOM) is the first choice to feed preterm infants–6, the advantages include improved immune defence and gastrointestinal function, reduction in the incidence of necrotising enterocolitis and sepsis, and improved long-term neurodevelopment outcomes compared to feeding with preterm formula (PF),8. However, preterm infants’ nutritional needs are beyond what is provided in human milk. Therefore, fortifying human milk with a human milk fortifier is recommended for preterm infants with a birth weight < 1800 g. When MOM and donor milk are not available, the recommendation is to feed preterm infants with PF that supply has a higher nutrient density than standard infant formula. PF appear to match the nutritional needs of preterm infants, and they support good growth; however, limited data are available regarding growth patterns of SGA preterm infants.Nutrition plays an essential role in the first 1000 d of a preterm infant’s life, with critical health consequences on growth, morbidity and cognitive developmentOur aim was to study growth patterns of SGA preterm infants fed fortified MOM or PF at two time points: discharge and 2-year corrected age (CA) follow-up.This is a retrospective cohort study conducted during the year 2020, using data collected from medical records of SGA preterm infants born at Kaplan Medical Center’s (KMC) Neonatal Intensive Care Unit (NICU) between the years 2012–2018.Anthropometric data at 2-year CA were extracted from KMC’s neonatal follow-up clinic medical records and from family healthcare centres, subject to parental consent. In addition, a follow-up telephone questionnaire was conducted with parents in 2020(average age of the children in 2020 was 4·8 years). The questionnaire included standard questions that are also asked at the Israeli follow-up health clinics, such as information regarding duration of breast-feeding, type of formula and timing of introduction of complementary food.This study was conducted according to the Declaration of Helsinki guidelines, and all procedures involving human were approved by the KMC’s Ethics Committee (0130-19-KMC). Verbal informed consent was obtained from all parents.. Exclusion criteria included mixed feeding (MOM ≤ 80 % or PF ≤ 80 % of daily volume) (n 89), lack of matching (n 10), mortality (n 5), congenital malformations (n 4) and genetic syndrome (n 3). After applying the exclusion and inclusion criteria, we included eighty SGA preterm infants and excluded 111 SGA preterm infants. Using MedCalc software, the sample size was calculated to be at least seventy SGA preterm infants, thirty-five in each group with 80 % power and a significance level of P-value < 0·05 to detect 50 % difference between groups.A total of 191 SGA preterm infants were born at KMC between January 2012 and December 2018 with GA < 37 + 0 weeks. Inclusion criteria included SGA preterm infants fed MOM or PF. SGA was defined as per Fenton’s growth chart. We divided the data into two groups: the MOM group included forty preterm infants primarily fed with MOM (volume of feeds > 80 %) and the PF group included forty preterm infants primarily fed with PF (volume of feeds > 80 %). Each preterm infant from the MOM group was matched to a preterm infant from the PF group. The criteria were birth week (sd1 week), birth weight (sd100 g) and birth year. Follow-up data, including anthropometric measures at 2-year CA and eligible questionnaires, were available for seventy-five infants .Anthropometric measures, including infant weight, length/height and head circumference (HC), were collected at birth, discharge and 2-year CA according to StRoNNGhttps://www.who.int/tools/child-growth-standards/standards).Anthropometric growth data were converted to z-scores according to standardised age at assessment and infant sex. Changes in z-scores (Δ z-score) were calculated from birth to discharge using Fenton’s growth chart (12), and from birth to the 2-year follow-up, using the WHO growth chart /([(W2 + W1)/2]/1000)/number of days]. Clinical and demographic data such as maternal age, delivery mode, maternal morbidities, ethnicity, intra-uterine growth restriction, day of full enteral feeding, postnatal weight loss, day of birth weight regain, days of hospitalisation and neonatal morbidities were also extracted from medical records.According to standardised procedures, weight was measured to the nearest 5 g using a calibrated electronic scale , and length was measured with a recumbent length board measured to the nearest 1 mm  and HC with a non-stretchable tape measure. All measurements were done with standardised equipment and techniques by professionals that were trained in measuring preterm infants and toddlers. Expected growth was defined as the change in z-score on the Fenton growth chart between −0·8 and 0·8 from birth to discharge–27. Trophic feeding (< 24 ml/kg per d) was initiated within the first 24 h of life and no later than 48 h, with advancements of 20–35 ml/kg per d until full enteral feed was reached. To meet the nutritional needs, MOM was fortified by bovine milk-based human milk fortifier powder , 0·25 g protein; Similac Abbott Nutrition). Fortification was introduced when MOM feeding exceeded 50–80 ml/kg/d, starting with two packets of human milk fortifier/100 ml  and advanced to four packets of human milk fortifier/100 ml  when reaching 100 ml/kg per d. PF preterm infants were fed . Decisions regarding feeding volume and fortification were made by the physician and dietitian, based on the feeding guideline. The goals for enteral nutrition were 140–160 ml/kg per d, 120–130 kcal/kg per d and 3·5–4 g protein/kg per d. All the SGA preterm infants in the study received 150–160 ml/kg per d and achieved the nutritional goals as they reached full enteral feeding.The KMC’s NICU follows a feeding protocol based on the European Society for Paediatric Gastroenterology, Hepatology and Nutrition’s (ESPHAGAN) recommendations of parenteral and enteral + nutritionP values < 0·05 were considered statistically significant, 95 % confidence interval, with 80% power. Data were analysed using the Statistical Package for the Social Sciences .Data are presented as mean values and standard deviations or numbers and percentages. Continuous variables between the study groups were tested for normality by a Shapiro–Wilk test. Since normal distributions were found, and T-tests were performed for continuous variables. Fisher’s exact tests were used to test the relationship between two categorical variables. A Pearson correlation two-tailed was preformed between day of regain and Δ height z-score at 2-year CA. Eighty SGA preterm infants were included in this study (forty MOM fed and forty PF fed). The baseline characteristics of the SGA preterm infants and their respective mothers are presented in v. 45 % in the PF group (P = 0·17). GA in the MOM group was 33·8 (sd 2·4) compared with 34·7 (sd 1·9) in the PF group (P = 0·06), and birth weight was 1409 (sd 319) in the MOM group compared with 1490 (sd 310) in the PF group (P = 0·25). Significant difference was found between the feeding groups in multiple births . We anticipated that the PF group would have more multiple births since mothers find it difficult to express milk for twins or triplets. Furthermore, the MOM group had significantly fewer episodes of regurgitation during hospitalisation compared with the PF group (2 (sd 2·5) v. 5·9 (sd 8·5), respectively, P = 0·007).In the SGA preterm infants MOM group, 62 % were boys sd 0·41) v. PF: −0·25 (sd 0·36), P = 0·01) and Δ length z-score (MOM: −0·63 (sd 0·75) v. PF: −0·27 (sd 0·75), P = 0·03). The MOM group had a significant smaller positive ΔHC z-score compared with the PF group (0·13 (sd 0·67) v. 0·41 (sd 0·55), respectively, P = 0·04). Absolute weight, length, HC and their z-scores were similar in both groups at discharge. The MOM group had weight gain (g/kg per d) equal to the PF group at discharge (P = 0·56).The growth outcomes at discharge are shown in sd in length-for-age z-score, compared with 22 % in the PF group (P = 0·03). No significant difference was found between groups for poor weight and HC growth (Forty seven percentage of SGA preterm infants in the MOM group experienced poor length growth with a significant decline of > 0·8 C growth .sd 1·3) v. 0·64 (sd 1·3), respectively, P = 0·02). However, regarding Δ weight z-score and Δ HC z-score, the MOM group accomplished similar increase to the PF group . Weight-for-height z-score was similar in both groups (P = 0·79)  .P = 0·01); the longer it took SGA preterm infants to regain weight back to birth weight, the more it impacted their height growth over time. No correlation was found in the PF group (P = 0·29)  . After dsd 1·4) v. 7·8 (sd 3·2), P = 0·04). The mean age of the children at the time the parents answered the questionnaire was 5·1 (sd 1·6) years in the MOM group and 4·5 (sd 1·6) years in the PF group (P = 0·07).No difference was found between formula type and the infants’ growth outcome. The MOM group was introduced to complementary foods significantly earlier than the PF group (6·1 (P = 0·10). A significant difference was found between feeding type and infants’ height outcome; 40 % of the MOM group achieved height catch-up growth compared with 68 % in the PF group (P = 0·02). The PF group also had a higher proportion of infants achieving HC catch-up growth   .Our retrospective cohort study findings demonstrate that feeding SGA preterm infants with fortified MOM from birth is associated with significant twofold loss in weight and length z-scores at discharge compared with SGA preterm infants fed with PF. Furthermore, PF feeding is associated with a fourfold increase in HC z-scores compared to feeding with MOM at discharge.n 32) on fortified MOM-fed non-SGA very low birth weight (VLBW) preterm infants found a significant decrease in weight z-score from birth to discharge compared with PF-fed non-SGA VLBW preterm infants. No differences were detected between the groups for Δ length or Δ HC z-scores. Similarly, fortified-MOM or donor milk-fed non-SGA VLBW preterm infants  were found to have significant reduction in weight z-score from birth to discharge compared with PF-fed preterm infants. Length and HC were not examined. Contrary to these studies, a retrospective cohort study  showed that low birth weight preterm infants fed with fortified breast milk (MOM and donor milk) had a higher weight z-score and lower decrease in HC z-score at discharge compared with PF-fed infants. No differences in weight z-scores were found in VLBW preterm infants fed the different feeding types. The differences in z-scores outcomes between the studies could be due to several reasons, including studied populations , differences in the breast milk type (MOM or donor) and the amount of breast milk (exclusive or any). Our research demonstrates that SGA preterm infants have a unique growth pattern that is affected by feeding type, and expressed by length and not only by weight and HC.SGA preterm feeding and growth consequences are challenging issue with complicated outcomes and scarcely studied. However, previously published findings regarding non-SGA preterm infants, type of feeding and growth parameters at discharge are partially consistent with our results. A small prospective observational study  SGA preterm infants experienced an increase in weight and height over a longer period of time. A recent large observational study on SGA and non-SGA VLBW preterm infants demonstrated slower growth in Δ weight and HC z-scores at discharge among MOM fed compared with PF fed. However, these differences were not observed at age 10. Also, in non-SGA preterm infants, an observational cohort study on two large cohorts (LIFT and EPIPAGE) showed greater loss in weight z-score at discharge in MOM-fed preterm infants compared with PF-fed preterm infants. However, at 2-year CA, weight, height and HC z-scores were significantly higher in MOM fed compared with PF fed. The EPIPAGE study also found that breast-feeding at discharge was associated with a lower incidence of short stature at 5 years of age. Taken together, although there are differences in methodology between the various studies, it can be concluded that MOM-fed SGA preterm infants achieve weight, HC and height goals over longer period than FP-fed SGA preterm infants. However, the question related to the time frame taken to achieve this catch-up is still not entirely clear and depends on feeding type and birth status.At 2-year CA, both groups exhibited similar increases in weight and HC z-scores. However, the change in height z-score was different between the two groups, possibly suggesting that the improvement in weight and HC growth of the MOM-fed group was at the expense of height growth. This indicates possible prolonged effects of feeding type on height growth potential. Similarly, Toftlund . Possible explanation for the difference in height catch-up growth is the amount of protein in the diet. It has been reported that protein content in human milk decreases throughout lactation and varies between mothers,40. Also, human milk contains less protein compared with formula and might contribute to slower growth rate. According to the ‘early protein hypothesis’, the higher protein content in infant formula compared with human milk could stimulate the production of Insulin growth factor (IGF)-I and insulin which promotes growth. In later infancy (∼ 9 months of age), term infants who were still breastfed showed lower levels of IGF-I, IGFBP-3 and insulin than infants no longer breastfed,43. Nevertheless, it is important to mention that MOM is the gold standard for feeding preterm infants. Therefore, the question is whether it is right to refer growth on formula as the optimal growth since preterm infants have achieved a greater improvement in height z-score and catch-up growth or rather to refer growth on MOM as the preferred growth in which height catch-up growth occurs over a longer period of time.In our study, at 2-year CA the height difference still remained, whereas the PF group exhibited significant linear catch-up growth compared with the MOM group. Poor catch-up growth is affected by prematurity, SGA status, genetics and nutrition and is associated with low IQ and short stature,45. Although the increase was slower in MOM group, still 98 % of the SGA preterm infants in the study were in the normal range, which may predict positive neurodevelopmental outcomes.In addition, our study demonstrates that feeding SGA preterm infants with fortified MOM from birth was significantly associated with slow increase of HC z-score growth compared with PF feeding at discharge. Studies have shown that poor HC growth in SGA preterm infants may lead to neurodevelopmental impairments outcomeAt 2-year CA, the increase in HC z-score was similar in both groups, with PF-fed group showing non-significant trend to higher rate of HC z-score catch-up. Thus, feeding type probably has no effect on HC growth at 2-year CA.. Even though there were more episodes of regurgitation among the PF group, our findings indicate that it did not affect their growth compared with the MOM group.Regarding the nutritional gastrointestinal feeding acceptability, during hospitalisation, significantly less episodes of regurgitation were observed in the MOM group than in the PF group, presumably due to the unique MOM composition which includes digestive enzymes, growth factors, hormones and prebiotic factors which benefit the immature gastrointestinal functionsOur study has several limitations: the feeding information during hospitalisation and post-discharge until the age of 3 months was controlled and thus is detailed and extensive. However, other un-recognised nutritional factors in the feeding post-discharge could have influenced growth. Additionally, parents have fulfilled the questionnaires several years after discharge. Our study has several advantages: we studied a unique population that is hardly investigated, the study population data were fully documented and we studied all the anthropometric parameters. Also, KMC’s NICU follows a standardised and controlled feeding protocol with the same medical staff and dietitian over the years which reduce bias.In summary, our study points to a link between the feeding type and the rate of linear catch-up growth at discharge and at 2-year CA. SGA preterm infants warrant special attention and closer monitoring of their nutrition before and after discharge to support growth, particularly in MOM-fed infants. Furthermore, supporting earlier birth weight regain should be the focus. This might assist in increasing the height z-score at 2-year CA in MOM-fed SGA preterm infants. Further research is needed to determine the best approach to fortify MOM or to set specific standards to this population in order to achieve optimal growth of SGA preterm infants during the NICU period and at post-discharge period.The early growth of the SGA preterm infants is regulated by complex interplay between genetic, environmental, nutritional and endocrine factors, coupled with therapeutic interventions. Our findings indicate that feeding type in SGA preterm infants has an effect on growth in all anthropometric parameters at discharge, whereas at 2-year CA the effect remains only on height growth. Feeding with fortified MOM during hospitalisation compared with PF leads to significant larger decrease in weight and length z-scores and smaller increase in HC z-score at discharge. Feeding SGA preterm infants with unfortified MOM for at least 3 months post-discharge is associated with slower increase in height z-score and delayed height catch-up growth at 2-year CA, suggesting possible prolonged effects on growth potential influenced by feeding type.Due to differences found in this study, close nutritional monitoring of SGA preterm infants beyond the age of 2 years is warranted.