To investigate whether the reproductive outcomes of oocytes with smooth endoplasmic reticulum aggregates (SERa) are impaired.A total of 2893 intracytoplasmic sperm injection (ICSI) cycles were performed between January 2010 and December 2019 in our center. In 43 transfer cycles, transferred embryos were totally derived from SERa+ oocytes. Each of the 43 cycles was matched with a separate control subject from SERa- patient of the same age ( ± 1 year), embryo condition, main causes of infertility, type of protocols used for fresh or frozen embryo transfer cycles. The clinical pregnancy, implantation, ectopic pregnancy and live birth rate were compared between the two groups.vs. 65.11%, p=0.5081), implantation rate  and live birth rate  between the SERa+ oocyte group and the matched group. No congenital birth defects were found in the two groups.43 embryo transfer cycles from SERa- patient were matched to the 43 transferred cycles with pure SERa+ oocytes derived embryos. No significant difference was observed in clinical pregnancy rate (55.81% Our results suggest that the implantation, clinical pregnancy, live birth and birth defects rate of embryos derived from oocytes with SERa are not impaired. Aggregates of smooth endoplasmic reticulum (SERa) in the ooplasm is one of the cytoplasmic dysmorphisms of oocytes. These aggregations appear as round flat disks in the ooplasm corresponding to large tubular SER clusters surrounded by mitochondria .Otsuki et al. firstly reported significantly lower pregnancy rates in SERa+ cycles and the transfer of embryos derived from SERa+ cycles resulted in Beckwith-Wiedmann syndrome . SubsequAfter the consensus was published, more research focused on this field. However, the conclusions are inconsistent. The clinical pregnancy rate was comparable between SERa+ and SERa− cycles in the most of the published studies , 3, 6–13However, most of the published literatures compared the clinical pregnancy outcomes between SERa+ and SERa− cycles , 16, 17.Therefore, in order to clarify whether SERa have adverse effects on reproductive outcomes, direct comparison should be made between embryos derived from SERa+ oocytes in SERa+ cycles and embryos from SERa- cycles. Furthermore, it should be acknowledged that these studies are fraught with potential biases and confounders. Factors, such as maternal age, embryo conditions, main causes of infertility, type of protocol etc., were not matched in these studies, which could lead to inaccurate conclusions.In this report, we conducted a matched, case-control study to determine whether the embryos derived from SERa+ oocytes were associated with negative reproductive outcomes.All women undergoing intracytoplasmic sperm injection (ICSI) cycles that did not involve pre-implantation genetic testing (PGT) in the Centre for Reproductive Medicine, Guangzhou Women and Children’s Hospital between January 2010 and December 2019 were included in the study. Since the presence of SERa cannot be observed on the day of insemination, patients undergoing conventional IVF and split IVF-ICSI were excluded from the study. The study was approved by the Independent Ethics Committee of Guangzhou Women and Children’s Hospital.SERa+ oocytes were defined as those oocytes where one or more SERa were visible with an inverted microscope after denudation just prior to ICSI. A SERa+ cycle indicates that at least one SERa+ oocyte is observed among the cohort. The SERa- cycles had morphologically normal oocytes. When all the cycles are SERa-, the patient is defined as a SERa- patient.Both fresh and frozen embryo transfer (ET) cycles were included in the study. The reproductive outcomes were only analyzed in transfer cycles where transferred embryos were totally derived from SERa+ oocytes.Standard controlled ovarian stimulation protocols were used. Briefly, the patients underwent either GnRH agonist or GnRH antagonist protocol for ovulation induction. The ovarian stimulation was performed using recombinant FSH  in combination with GnRH antagonist  or agonist . The initial doses were based on antral follicle counts, female age, and basal FSH. The subsequent doses were adjusted according to follicle growth and serum estradiol levels. HCG or GnRH agonist was administered when at least three leading follicles reached a mean diameter ≥ 18 mm. Transvaginal oocyte retrieval was scheduled 34–38 h later.2, 37°C until days three. Embryos with extended culture were transferred on day three to G2  media and cultured individually under the same conditions until day five or six. Cleavage stage embryos were assessed for cell stage, percent fragmentation, multinucleation, and blastomere symmetry according to ESHRE/ALPHA consensus . The embryo transfers were done with ultrasound guidance by using a standard embryo transfer catheter .In 43 transfer cycles, including 13 fresh transfer cycles and 30 frozen transfer cycles, the transferred embryos were totally derived from SERa+ oocytes. The matched group was drawn from all ICSI cycles from SERa- patients. Matching was accomplished by the following criteria: [1] age( ± 1 year); [2] embryo condition  [3] main causes of infertility ; [4] type of protocol used for controlled ovarian hyperstimulation (COH) in fresh embryo transfer or endometrium preparation protocol in frozen embryo transfer; [5] infertile years ( ± 2 year); [6] number of transfer cycles; [7] embryo transfer date ( ± 1 year); [8] embryo transfer physician. We required exact matching for criteria 1–4; for criteria 5–8, we attempted matching as closely as possible and in most cases were able to match at least two of these criteria. To reduce the introduction of potential bias, researchers were blinded to reproductive outcomes during the matching process. If multiple patients fit the criteria, one was chosen at random.The implantation rate was calculated as the number of gestational sacs divided by the number of transferred embryos. Clinical pregnancy was defined as the presence of an intrauterine gestational sac with a yolk sac, a fetal pole, and fetal heart pulsations. Ectopic pregnancies were diagnosed by ultrasound or by laparoscopic visualization of an extrauterine gestational sac or by the absence of an intrauterine gestational sac and increasing β-hCG levels following the failure of suction D&C to reveal products of conception. Live birth was defined as the birth of a live infant at ≥28 weeks of gestation.P value <0.05 was considered statistically significant.Data are presented as the mean ± standard deviation. Statistical comparisons of 2 experimental groups were evaluated by 2-tailed Student t test if data distribution passed the normality test. The Mann-Whitney U test was used for comparison if data distribution failed the normality test. A chi-square test or Fisher’s exact test to compare fertilization, pregnancy and live birth rate etc. vs. 21.6 ± 3.0), stimulation protocols and total gonadotrophins dose (2090.7 ± 918.7 vs. 2043.8 ± 943.9) between the two groups. Compared with SERa-cycles, significant younger age (33.5 ± 5.4 vs. 34.5 ± 5.6), more stimulation days (9.3 ± 2.5 vs. 8.9 ± 2.8), higher estradiol levels at hCG-trigger (9973.3 ± 5403.3 vs. 8662.2 ± 5477.4), and more mean number of retrieved oocytes (11.5 ± 7.9 vs. 8.8 ± 7.1) and MII oocytes (9.9 ± 6.7 vs. 7.4 ± 5.8) were found in SERa+ cycles.A total of 2893 ICSI cycles were performed between January 2010 and December 2019 in our center. Among them, 482 were SERa+ cycles, the incidence rate was 16.7%. The baseline characteristics, stimulation protocols and outcomes of SERa+ cycles and SERa- cycles are shown in In the 482 SERa+ cycles, 43 embryo transfer cycles were exclusively derived from SERa+ oocytes, including 13 fresh transfer cycles and 30 frozen transfer cycles. Totally 71 embryos, including 39 good quality embryos, were transferred. 43 embryo transfer cycles from SERa- patients were matched to the pure SERa+ oocyte group (1:1).vs. 86.05%), cleavage rate (99.09% vs. 97.80%) and D3 good embryo quality rate (33.84% vs. 32.69%) were all comparable between SER+ oocyte group and the matched group. vs. 65.11%), implantation rate (47.89% vs. 50.70%), ectopic pregnancy rate (4.17% vs. 0%), miscarriage rate (8.33% vs. 14.29%), live birth rate (48.84% vs. 55.81%), gestational week at birth (37.9 ± 1.7 vs. 37.8 ± 1.8), or birth weight (2.9 ± 0.6 vs. 2.8 ± 0.6) between the SERa+ oocyte group and the matched group. From these 43 transfers in SERa+ oocyte group, 21 live births were obtained and resulted in the birth of 28 healthy infants (7 twin pregnancy). No congenital anomalies were found in the two groups.The cycle characteristics of pure SERa+ oocyte group and matched group are presented in The occurrence rates of SERa reported in previous researches are different, ranging from 5.4% to 23.1% . ConsistSince Otsuki et al. firstly reported significantly lower pregnancy rate and fetal anomalies derived from SERa+ oocytes , more anTransfer of embryos originating from SERa+ oocytes was not avoided in our center. Previous investigation also showed that only 14% of centers discarded SERa+ oocytes and 43% of centers that did not discard oocytes followed up neonatal data  according to our embryo scoring criteria. This may explain the high pregnancy rate (55.81%) and live birth rate (48.84%) in our SERa+ oocyte group. 21 out of the 43 transfer cycles had alternative embryos, but embryo scoring was better in the SERa+ oocytes derived embryos. In the left 22 cycles in which no alternative embryos existed, the mean age (34.9 ± 7.0) was older, mean number of retrieved oocytes (7.0 ± 5.8) was relatively small. The acceptable clinical pregnancy rate (45.5%) and live birth rate (36.4%) in the 22 aged cycles also indicated that the clinical outcomes of SERa+ embryos are not impacted when there is no other alternative in embryo selection.To minimize the impact of the clinical heterogeneity observed between patients, we used multiple matching criteria. We attempted in particular to match for age, embryo condition, main causes of infertility, type of protocol used for COH in fresh embryo transfer or endometrium prepare protocol in frozen embryo transfer, which are known to affect IVF success. In our study, 43 transfer cycles with pure SERa+ oocyte derived embryos were included and compared with matched controls. This suggests that SERa+ oocytes are not associated with both decreased clinical pregnancy rate, implantation rate, gestational week, birth weight, live birth rate, and increased miscarriage rate and birth defect rate.As we mention above, if the presence of SERa in the cytoplasm of oocytes has a negative impact on clinical and neonatal outcome, the reproductive outcomes of the SERa+ embryo transfer cycle are most likely to be affected. After finding that the reproductive outcomes of SERa+ embryos were totally normal, we decided not to continue other subgroup analysis.Gurunath et al. reported that a gradual reduction in live birth rates was observed when the percentage of oocytes containing SERa increased and the group containing >50% of oocytes with SERa demonstrated no live births . HoweverWhether SERa increase birth defects has always attracted extensive attention. Based on previously reported cases of Beckwith-Wiedemann syndrome , diaphraSome limitations of our research should be noted. The nature of SERa makes it a retrospective study, which by nature cannot exclude selection bias. Furthermore, SERa can be classified on the basis of its size: large, medium and small aggregates . In mostThe implantation, clinical pregnancy, live birth and birth defects rate of embryos derived from oocytes with SERa are not impaired.The datasets generated for this study are available on request to the corresponding author. Requests to access the datasets should be directed to LS, sunling6299@163.com.The studies involving human participants were reviewed and approved by Independent Ethics Committee of Guangzhou Women and Children’s Hospital. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.LS was responsible for the conception and design of the study, interpretation of data, revised the article critically for important intellectual content, and approved the final draft for publication. JX and LY contributed to collect the data, analysis and interpretation of data; draft and revise the whole article. Z-HC, M-NY, and JC contributed to collecting the data, drafting and revising the article for important intellectual content. All authors contributed to the article and approved the submitted version.This study is supported by the National Natural Science Foundation of China (81800110).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Moreover, the expression of five candidate genes was increased to more than twofold by pretreatment with lipopolysaccharide in HTR-8/SVneo cells. Changes in gene expression between preterm birth and term birth is strongly correlated with oxidative stress and infection during pregnancy. Specific expression patterns of genes could be used as potential markers for the early identification of women at risk of having a spontaneous preterm birth.Preterm birth affects approximately 5% to 7% of live births worldwide and is the leading cause of neonatal morbidity and mortality. Amniotic fluid supernatant (AFS) contains abundant cell-free nucleic acids (cfNAs) that can provide genetic information associated with pregnancy complications. In the current study, cfNAs of AFS in the early second-trimester before the onset of symptoms of preterm birth were analyzed, and we compared gene expression levels between spontaneous preterm birth (n = 5) and term birth (n = 5) groups using sequencing analysis. Differential expression analyses detected 24 genes with increased and 6 genes with decreased expression in the preterm birth group compared to term birth. Upregulated expressions of RDH14, ZNF572, VOPP1, SERPINA12, and TCF15 were validated in an extended AFS sample by quantitative PCR . Five candidate genes displayed a significant increase in mRNA expression in immortalized trophoblast HTR-8/SVneo cell with H Spontaneous preterm birth constitutes a complex and multi-factorial phenotype accompanied by numerous gestational tissues to induce parturition and is the result of an interaction of both genetic and environmental factors5. However, the genetic and molecular characteristics of preterm birth remain elusive and preventive strategies have not yet been established. Amniotic fluid (AF) contains nutrients and growth factors for fetal development and is a source of cells for the prenatal diagnosis of chromosomal abnormalities and fetal infections, as well as for the determination of fetal lung maturity7. While the cells present in the AF are utilized in these diagnostic methods, the remaining AF supernatant (AFS) is usually discarded. However, the AFS contains abundant cell-free nucleic acids (cfNAs), a uniquely dispersed form of genetic material. CfNAs may be derived from active cellular secretions via exosomes and shedding vesicles or microparticles through apoptosis or necroptosis9.Preterm birth, defined as birth before the completion of 37 weeks of gestation, is the leading cause of neonatal morbidity and mortality. Despite recent advances in neonatal care, the burden of disease in preterm birth remains significant, increasing the risk of developmental and medical disabilities14. Moreover, the AF directly contacts the placenta and may also include cfNAs originating from placental tissues15. CfNAs can serve as intermediate messengers to convey genetic information and cellular signals, which affect various cellular responses such as oxidative stress and immune response9. Recently, molecular studies using next-generation sequencing (NGS) have suggested that cfNAs obtained from AFS could serve as a good diagnostic tool for the detection of abnormal fetal conditions such as fetal aneuploidies19, fetal growth restriction21, twin to twin transfusion syndrome22, and altered molecular pathways24. However, despite substantial research on cfNAs in fetal development, the correlation between AFS-derived cfNAs and preterm birth is still elusive.AF-derived cfNA is of relatively fetal origin and is less likely to be contaminated with maternal-derived nucleic acidsThe aim of this study is to characterize the gene expression signatures related to a spontaneous preterm birth from AFS during second-trimester pregnancy and to reveal the biological information about genes involved in the pathogenesis of preterm birth for early diagnosis and appropriate treatment. We hypothesized that the cfNAs from AFS could provide real-time information on spontaneous preterm birth along with nucleic acid from amniocytes.2), or gestational age at amniocentesis  between preterm birth and term birth groups, whereas a significant difference was observed between both groups for gestational age at delivery .We obtained 74 AF samples from pregnant women at 16 to 19 weeks of gestation. As a result of amniocentesis in all study populations, no patients were diagnosed with chromosomal abnormalities, including Down syndrome. Among them, 24 women had a preterm delivery, and 50 delivered at term. The average amounts of total nucleic acid in the spontaneous preterm birth and term birth groups were 219.2 ± 102.1 ng and 151.3 ± 69.27 ng, respectively, which were significantly different (P = 0.0012). To pursue sequencing analysis, we paired cases and controls based on the amount of total nucleic acid and selected 5 of the 21 preterm birth and 5 of the 40 term birth samples. The demographic and clinical characteristics of the study population are presented in Supplementary Table To examine the gene expression signatures in women destined to have a preterm birth, we performed nucleic acid sequencing on second-trimester AF samples and determined the differentially expressed genes (DEGs) between the preterm and term birth groups. The body fluid samples constantly change components such as RNA/DNA/protein according to intrinsic and extrinsic environmental conditions. Although various normalization approaches have been proposed, there is no standardized manner. Therefore, we applied three normalization methods, i.e., Quantile, reads per million (RPM), and reads per kilobase million (RPKM) normalization, in addition towhil the non-normalization method. Figure The candidate markers were miRNAs, SNORA, and uncharacterized genes LOC105374432), including the ordinary genes. To overview statistical significance along with differential gene expression, volcano plots were drawn, in which the magnitude of gene expression ratios and the significance of the difference in gene expression between pools were displayed on the x-axis and y-axis, respectively. Red dots indicate the six upregulated DEGs  were chosen from the ranked gene lists. For qRT-PCR based validation of the candidate genes, 21 s-trimester AFS samples from the preterm birth group and 40 from the term birth group, with an RNA quantity of more than 10 ng/ul were chosen. Consistent with the sequencing results, all 5 DEGs  were found to be differentially expressed in the preterm birth samples with the same trends , and the inhibitory effect of H2O2 on both cells was dose-dependent. HTR-8 cells were more sensitive than JEG-3 cell lines to H2O2 cytotoxicity. The IC50 values of HTR-8/SVneo and JEG-3 cells were 34.50 and 78.66 μM after 24 h and 32.73 and 71.54 μM after 48 h of treatment, respectively. Next, we performed expression analyses of RDH14, ZNF572, VOPP1, SERPINA12, and TCF15 using qRT-PCR analysis in the RNA samples extracted from two cell lines with H2O2 treatment to validate the candidate genes. Five candidate genes displayed a significant increase in mRNA expression in the HTR-8/SVneo cells after H2O2 treatment, but no change in the JEG-3 cells due to resistance to H2O2 treatment -induced oxidative stress is associated with preterm labor2O2-induced cell damage, we performed a cell viability assay following the treatment of 0 μM and 100 μM H2O2 with or without ritodrine for 24 h. As shown in Fig. 2O2 was similar to that of the control group. Ritodrine significantly reversed the damaging effects of H2O2 on HTR-8/SVneo cells (P < 0.001), which indicated that ritodrine may exhibit potent protective potential against H2O2-induced oxidative damage. Furthermore, ritodrine significantly decreased the mRNA expression levels of RDH14, TCF15, ZNF572, and SERPINA12, which are normally increased by H2O2 in HTR-8/SVneo cells  generation.To evaluate the effect of ritodrine on HThis is the study comparing gene expression in the AFS between spontaneous preterm and term births using NGS. In this study, we analyzed the differences in global gene expression in the AF-derived cfNAs before the onset of preterm birth to identify the pathogenesis of spontaneous preterm birth. It is well known that AF is a complex and dynamic solution that can provide valuable information about fetal development and health status, and many studies have therefore reported the presence of cfNAs in AF. Although numerous studies have reported the use of AF cfNAs for discovering novel biomarkers for pregnancy complications, there have been no studies using AF-derived cfNAs to explore the pathogenesis of preterm birth. While other researchers have reported gene expression via microarray in AFS, few studies have performed gene expression analyses using sequencing analysis from AFS.30. The knock-down of VOPP1 overexpressed in squamous carcinoma cells induced cell death by increasing ROS and mitochondrial dysfunction, and N-acetyl cysteine as a ROS scavenger abolished the induction of apoptosis by VOPP1 knock-down31. In addition, SERPINA12 (Vaspin) inhibited apoptosis in human hepatocellular carcinoma cells, associated with reduced nitric oxide and superoxide anion33. Therefore, these results suggested that our candidate genes can be correlated with oxidative stress stimuli induced preterm birth.In this study, 30 DEGs were identified in the preterm birth cases compared with the term birth using nucleic acid sequencing. In particular, the expression patterns of 5 DEGs were verified by qRT-PCR in the second-trimester AFS of the preterm birth group and were highly associated with oxidative stress in the pathophysiology of preterm birth. Most DEGs were not deduced a valid ontology for each gene and particularly oxidative stress-related ontology because they were rarely studied on their functions. However, previous studies have shown that our candidate genes have already been correlated with oxidative stress in various experimental conditions and diseases. For example, the cardiotoxin-mediated transient acute mouse model of muscle degeneration lowered glutathione's reduction/oxidation rate as an oxidative stress marker and changed the protein expression level of RDH1434. They reported that changes in selected gene expression related to oxidative stress and inflammation occur before the onset of preterm birth, which may facilitate early detection of pregnancies at higher risk of preterm birth.Oxidative stress, characterized by imbalances in the redox system in the maternal–fetal intrauterine compartments, has been reported to play a critical role in the pathogenesis of spontaneous preterm birth. The present study showed that even before the onset of symptoms of preterm birth, there is a possibility of a redox imbalance in the maternal–fetal intrauterine compartments. Vora et al. examined the differences in gene expression in the cfRNA from AFS in women who delivered preterm and those who delivered at term using a customized nanostring panel containing genes related to oxidative stress35. Recent studies have demonstrated that oxidative stress plays an important role in infection-induced apoptosis and in the pathogenesis of preterm labor37. LPS triggers inflammatory responses in human gestational tissues by increasing pro-inflammatory cytokines and phospholipid metabolites mediated by activation of the NF-κB pathway. In addition, glucose deprivation results in oxidative stress, and the alteration of the redox status of cells triggers stress-activated or other signal transduction pathways leading to cell death. In the present study, the expression of candidate genes was increased under conditions of oxidative stress and ROS production, which was induced by H2O2, LPS treatment, and glucose deprivation.The role of inflammation related to the pathophysiology of preterm birth has been well studied, and several investigators have reported various inflammatory biomarkers associated with preterm birth. Oxidative stress, characterized by the generation of ROS, is an integral factor in the inflammatory process40. The balance between ROS and antioxidants can maintain good maternal health and safe childbirth42. Redox imbalance is closely related to the fundamental pathogenesis of many pregnancy complications. The overproduction of ROS in inflammation and cell damage can generate pathways leading to preterm birth. The risk factors for preterm birth can lead to redox imbalance caused by high levels of ROS, such as hydroxyl radicals, hydrogen peroxide, superoxide anion, and nitric oxide. As a result, ROS-mediated oxidative stress contributes to collagen degradation and consume antioxidant defenses. Collagen damage in human gestational tissue can result in uterine contractions and tears in the chorioamniotic sac, leading to spontaneous preterm birth.The production of ROS is necessary for the regulation of aerobic energy production. Redox balance is maintained through an intricate equilibrium between ROS production and subsequent elimination, and an interactive network of enzymatic and non-enzymatic antioxidant systems. Cells can protect themselves against oxidative stress by increasing the levels of endogenous and exogenous antioxidants or decreasing the production of ROS2-adrenoreceptor agonist used as a treatment for preterm labor, decreased the expression of candidate genes, thereby inhibiting H2O2-induced cell damage. β2-adrenoreceptor agonists have been considered to have anti-inflammatory and antioxidant properties, which can be of great interest in treating several diseases44. However, the exact mechanism for decreasing oxidant production of inflammatory cells remains unknown. In previous studies, low doses of β2-adrenoreceptor agonist significantly inhibited LPS-induced activation of NF-kB and production of proinflammatory cytokines normally mediated by the NF-kB and MAPK signaling pathway43. Also, these studies showed reduced ROS generation and decreased apoptosis after β-adrenergic receptor stimulation, which support our findings that demonstrate that β2-adrenergic receptor stimulation increases the cell viability. Our results suggest that β2-adrenergic receptors stimulation inhibits the activation of candidate genes, supporting reduced apoptosis and low level of ROS after β2-adrenergic receptor stimulation.This study showed that ritodrine, a type of β45. Cff NAs in maternal plasma are now established as molecular diagnostic materials for noninvasive prenatal testing and have been routinely used in clinical care. Through advancements in sequencing technology and large scale genomic profiling in both clinical and research fields, simple methods to generate comprehensive and accurate whole-genome and transcriptome sequencing data will become increasingly valuable. Until now, RNA-sequencing analysis provides detailed information of the transcriptome while enabling a novel RNA transcript variation to be detected. Recently, the integration of DNA and RNA analysis has shown promising results in the detection of fusion transcripts and alternative transcripts. These sequencing data can provide useful information to explore genetic variation underlying human disease and normal phenotypic variability46.Cell-free fetal nucleic acids (cff NAs) have become the subject of intense interest in prenatal diagnosis since Lo et al. found their presence in maternal blood in 199749, and most of these are focused on the transcriptome of preeclampsia, not on preterm birth. In addition, published studies on human labor using NGS have been confined to the placenta or blood. Until now, many studies have focused on maternal factors, such as inflammation, infection, and maternal gestational tissues, in studying the causes of preterm birth; however, little is known about the fetal contribution to preterm birth52. Although maternal factors are known to contribute to preterm birth risk, there are emerging data that variations in the fetal gene expression, not the maternal, may induce preterm labor53. If sequencing technologies can analyze the DEGs in preterm birth to identify unique gene signatures at the fetal molecular level, it will help to understand more about the fetal contribution to pregnancy-specific diseases such as preterm birth. Further, it will lay the groundwork for preventive, diagnostic, and therapeutic strategies in obstetrics and pediatrics. Overall, both maternal and fetal genomes can simultaneously or separately contribute to the occurrence of spontaneous preterm birth, influenced by the environmental factors.Nucleic acid sequencing is an advanced technology for genome analysis that enables a comprehensive characterization of gene expression. Most gene expression data regarding human pregnancy are based on microarrays54. Second, this study may have annotation biases because of inequality in gene annotations of bioinformatics analysis. Third, it lacks sufficient power to demonstrate the mechanisms leading to early delivery due to the lack of early preterm births in our study. The preterm birth group in our study included only late preterm births (34–36 weeks), and there was only a difference of about 5 weeks compared to the mean gestational age at delivery of the term birth group. The absence of an early primordial group in sample collection resulted in a limitation, but it is still another significance of this study that gene expression differences occurred in second-trimester AF prior to 20–25 weeks before delivery.We aimed to investigate gene expression signatures that contribute to preterm birth prior to the onset of symptoms of preterm birth. Based on these results, we identified the difference in gene expression associated with redox imbalance as a contributing factor to preterm birth. This led to further investigations to define high-risk populations and characterize gene expression in the AFS. However, there are several limitations in this study. First, we performed a comparative study with sequencing data obtained from five samples each of preterm and term births. This relatively small sample size makes it difficult to draw definitive conclusions. While it is widely acknowledged that increasing the number of replicates in sequencing experiments usually results in more reliable results, the exact relationship between the number of replicates and the possibility of identifying DEGs has not been thoroughly investigated. However, Conesa et al. recommended that sequencing experiments have at least three biological replicates if sample availability is not limited to allow all sequencing analysis to utilize reproducibility between replicatesDespite these limitations, this study has many strengths. This study investigated differences in gene expression levels using cfNAs isolated from AFS in the early second-trimester of gestation for the prediction of spontaneous preterm birth. This information regarding the variations occurring at the genome or transcriptome level will be help in improved antenatal recognition, thereby reducing the morbidity and mortality associated with preterm birth. Identification of differences in gene expression between preterm birth and term birth before the onset of clinical symptoms will lay the groundwork for future prospective studies to delineate mechanisms leading to preterm birth.In conclusion, specific expression patterns of genes associated with high oxidative stress in pregnant women may indicate placental tissue damage predisposing to spontaneous preterm birth. Expression patterns of candidate genes shown in our study may be a criterion for assigning therapeutic targets so as to regulate their function at the earliest, thereby modulating the expression of proteins and their pathogenic pathways in spontaneous preterm birth. These DEGs may elucidate the underlying mechanisms of preterm birth and be used to predict preterm birth in early pregnancy.AF samples were obtained from 74 pregnant women who underwent amniocentesis for fetal karyotyping and genetic diagnosis at 16 to 19 weeks of gestation in the Department of Obstetrics and Gynecology, Gangnam Severance Hospital  between March 2011 and May 2017. This study was reviewed and approved by the Institutional Review Board of Gangnam Severance Hospital (Ethics ref.: 3-2011-0147). All participants provided written informed consent to participate in the study before AF was obtained. All experiments were performed in accordance with the relevant guidelines and regulations. For all subjects, gestational age was assessed during early gestation using the crown-rump length measurement by transvaginal ultrasonography. We included only cases of spontaneous preterm birth and excluded all pregnancies complicated by preeclampsia, major fetal anomalies, abnormal karyotypes, gestational diabetes mellitus and multiple pregnancy. We defined control subjects as those who had normal term births, defined as a live birth from 37 to 42 weeks of gestation.g for 10 min at 4 °C. The supernatant samples were spun at 1500 rpm for 10 min at 10 °C to remove residual vernix and then stored at -80 °C. CfNAs were extracted from 1 mL of AFS using the QIAamp Circulating Nucleic Acid kit  according to the manufacturer's protocol and eluted with RNase-free water. The concentration of cfNAs in each sample was measured using a NanoDrop ND-1000 spectrophotometer . Samples with an A260/A280 ratio greater than 2.0 were stored at −70 °C until further analyses.AF samples were centrifuged at 350 × The quantity and quality of the extracted nucleic acids were assessed using an Agilent 2100 Bioanalyzer RNA chip  according to the manufacturer’s instructions. Sequencing libraries were prepared using the SMARTer Stranded Total RNA-Seq kit-v2 Pico Input Mammalian  and validated using a DNA 1000 chip on an Agilent 2100 Bioanalyzer (Agilent Technologies) according to the manufacturer’s instructions. Nucleic acid sequencing was performed using the Illumina NextSeq 500 platform  with a 75-nucleotide paired-end indexed run.55. Significant differences in gene expression were determined via Student's t-test. DEGs were retained if they were detected by at least two of the methods used. All analyses were performed using DNASTAR Lasergene 15 software. Common markers were identified using a heat map with hierarchical clustering and Venn Diagram analysis.Nucleic acid sequencing reads were mapped to the human genome reference . The read counts for each gene were calculated and analyzed using three different normalizations, that is, quantile, RPM, and RPKM, and non-normalization methods for differential expression detection. Gene transcript numbers comparing preterm birth versus term birth were considered to be significantly different by using a combination of FC > 1.5 and P-value 4 cells and 1.0 × 105 cells per well, respectively, in 0.5 ml of growth media. After 24 h, the medium was replaced with DMEM containing 10% FBS. Meanwhile, H2O2  was added to the corresponding wells and incubated at 37 °C for 24 h or 48 h. For the crystal violet staining assay, cells were fixed with fixation solution ,stained with 0.5% crystal violet staining solution with 20% methanol for 1 h, photographed, and extracted using 1% SDS solution. The absorbance was measured at 595 nm using a VersaMax microplate reader . All experiments were performed in triplicates. We investigated the effect of ritodrine , a type of β2-adrenoreceptor agonist used as a treatment for preterm labor, on H2O2-induced cell damage. HTR-8/SVneo cells were seeded onto 24-well plates at a density of approximately 2.0 × 105 cells per well in 0.5 ml of growth media. After 24 h, the medium was replaced with DMEM containing 10% FBS. Meanwhile, cells were treated with 0.1 mg/ml of ritodrine; 30 min later, 0 μM or 100 μM H2O2 was added to the plate and incubated at 37 °C for 24 h.JEG-3 and HTR-8/SVneo cell lines were plated onto 24-well plates at densities of approximately 8.0 × 105 cells in six well plates and treated with 10 ng/ml and 100 ng/ml LPS  for 24 h. For glucose deprivation, the complete DMEM medium in culture plates was replaced with glucose-free DMEM medium  and cells were cultured for 24 h.HTR-8/SVneo cells were seeded at 4.0 × 10Total RNA (50 ng) from each sample was reverse-transcribed into cDNA using Maxima First Strand cDNA Synthesis Kit  according to the manufacturer’s protocol. Real-time polymerase chain reaction (PCR) was performed to quantify mRNA expression using SYBR Green PCR Master Mix  and an ABI PRISM 7300 real-time PCR system  according to the manufacturer’s instructions. Relative mRNA expression was quantified using the comparative Ct (ΔCt) method and expressed as 2-ΔΔCt, where ΔΔCt = ΔE–ΔC, ΔE = CtE target–CtE GAPDH, and ΔC = CtC target–CtC GAPDH . Each assay was performed in triplicate and expressed as the mean ± standard error (SE). Serial dilutions were prepared from a stock solution of total RNA to generate a standard curve and determine reaction efficiencies. The primers used for PCR are listed in Supplementary Table Experimental results were statistically evaluated with a two-tailed paired Student’s t-test using GraphPad Prism 9. All tests of significance were set at P < 0.05.Supplementary Tables.Supplementary Figure 1.


Differences in low birth weight rate depending on maternal socioeconomic characteristics have not yet been demonstrated using the Vital Statistics in Japan; therefore, this study aimed to investigate these differences according to maternal occupations. “Report of Vital Statistics: Occupational and Industrial Aspects” and the Vital Statistics in Japan were used every five years from 1995 to 2015. Nine types of occupations were compared. The low birth weight rate was calculated according to maternal occupations and year. Also, the standardized low birth weight ratio was obtained by dividing the number of low-birth-weight infants for each maternal occupation by an expected number of low birth weight infants. The standardized low birth weight ratio for manual workers was the highest among all occupations from 2000 to 2015, and it was significantly higher than one throughout the years. The ratio for clerical workers was also significantly higher than one from 1995 to 2010. Whereas, the ratio for farmers was significantly lower than one in most of the years. It was suggested that health guidance and prenatal care are particularly needed for manual workers, and a study investigating the differences in prenatal characteristics among maternal occupations is necessary for finding a reason for disparity. Low birth weight is one of the major adverse birth outcomes in perinatal health, and is an important public health indicator ,2. AlthoLow birth weight is known to be associated with the socioeconomic characteristics of women in the world ,9,10. InIn this study, we investigated the association between low birth weight and maternal occupations using Vital Statistics by occupation and industry in Japan.The data of “Report of Vital Statistics: Occupational and Industrial Aspects” in Japan from 1995 to 2015 were used . In JapaData on the number of births by maternal occupations, maternal age groups, and years and the number of low birth weight infants by maternal occupations and years were publicly available and used in the analysis. Low birth weight infants are infants whose birth weight is less than 2500 g. As the occupation classifications, professional and engineering workers, administrative and managerial workers, clerical workers, sales workers, service workers, security workers, agriculture, forestry and fishery workers, and unemployed persons are available. Regarding manual workers, occupational classifications changed in the analyzed periods, and transport and communication workers, and craft, mining, manufacturing, construction, and labor workers were available in 1995. In 2000 and 2005, a classification of production processes and related workers became available instead of the classification of craft, mining, manufacturing, construction, and labor workers. In 2010 and 2015, these classifications were removed, and manufacturing process workers, transport and machine operation workers, construction and mining workers, and carrying, cleaning, packaging, and related workers, were available. Therefore, we classified these types of workers into one group as manual workers as was done in a previous study [In addition, the Vital Statistics data for every five years from 1995 to 2015 were also used , and speFirst, we summarized the number of births and number of low birth weight infants by maternal age group, maternal occupation, and year, using the data of “Report of Vital Statistics: Occupational and Industrial Aspects” and the census data. In addition, low birth weight rate by the maternal age group, maternal occupation, and year were calculated from the result. We also summarized maternal age-specific births by maternal occupation for each year. Then, we calculated the percentage of births for each maternal age group and occupation per year, in order to study the differences in maternal age among occupations.https://www.r-project.org/ accessed on 29 July 2021).The low birth weight rate is known to be largely affected by maternal age; as such, it is meaningful to compare the low birth weight rate standardized by maternal age among maternal occupations. However, we could not obtain the data on the number of low birth weight infants by maternal occupations and age groups and could not calculate the low birth weight rate standardized by maternal ages. Therefore, we calculated the standardized low birth weight ratio just like the standardized mortality ratio. To calculate the standardized low birth weight ratio, we calculated the low birth weight rate in all of Japan for each year and maternal age group using the Vital Statistics at first. Then, we calculated an expected number of low birth weight infants for each maternal occupation and year by multiplying the number of births for each maternal age by the maternal age-specific low birth weight rate in all of Japan. The standardized low birth weight ratio was calculated by dividing the number of low birth weight infants for each maternal occupation by the expected number for every maternal occupation and year. All statistical analyses were conducted using R 3.6.3  is a risk factor for low birth weight . An incrThe use of antenatal care is another factor associated with low birth weight ,26. In JIn contrast to manual workers, the standardized low birth weight ratio of agriculture, forestry, and fishery workers remained low throughout the analyzed periods. Although the educational level of agriculture, forestry, and fishery workers is not high in Japan, their smoking prevalence is relatively low among occupations in women . AccordiIt was shown that manual workers had a high standardized low birth weight ratio as compared to the other maternal occupations. The disparity in low birth weight, depending on maternal occupations was revealed. Although it might be difficult to ameliorate the physical differences between mothers depending on maternal occupations, we might be able to change the health behaviors of pregnant manual workers. For example, guidance for smoking cessation is effective not only in prenatal care but also in workplaces. Workplaces are important places for creating awareness that smoking cessation is particularly needed for pregnant women against manual workers. The recommendation for consultation of antenatal care against manual workers in workplaces is also needed. It is considered that establishing a medical staff that workers can consult easily in workplaces is effective for ameliorating these prenatal behaviors in workers. Additionally, antenatal care is not completely free of charge, as previously noted, and making the utilization of antenatal care completely free of charge against low-income households may be effective for easing the disparity among maternal occupations. Moreover, grasping percentage of maternity leave by maternal occupations is also important because maternity leave might be difficult depending on the occupation. If taking maternity leave is difficult for manual workers, we need to create systems for increasing childbearing support in workplaces.There are some limitations in the current study. First, we could not obtain data on other characteristics of mothers than maternal occupations. As other factors, Vital Statistics collects data on parity, number of pregnancies, legitimacy of child, nationality, gestational ages, birth height, sex, place of residence, and paternal occupation, while individual data containing these factors are not publicly available. Data on these factors can be obtained when using individual data, and an analysis using these factors will be important in the future. In addition, we could not obtain data on the number of low birth weight infants by age group for each maternal occupation, and we could not calculate the low birth weight rate standardized by maternal age. Furthermore, the standardized low birth weight ratio tended to be higher than the average level in clerical workers; however, reason for the phenomenon is uncertain. An accurate reason for the association between maternal occupation and low birth weight is uncertain from this study, and an epidemiological study investigating differences in social and physical characteristics of mothers will be necessary to determine the reason for the disparity.We revealed differences in the standardized low birthweight ratio between maternal occupations using Vital Statistics. As a result, the standardized low birth weight ratio of manual workers was the highest in most of the years, and it was significantly higher than one throughout the years. On the other hand, the standardized low birth weight ratio of agriculture, forestry, and fishery workers remained low throughout the analyzed periods. It was suggested that more attention and medical care aimed at preventing low birth weight is needed, particularly in women who are manual workers, and a further study investigating reasons for the association between mothers being manual workers and low birth weight is necessary.


Although the hormonal intrauterine system has limited availability in low- and middle-income countries, this highly effective long-acting reversible contraceptive method has the potential to be an important addition to the method mix. Introduction of the method in the public sector under “real-world” conditions in Kenya and Zambia shows promise to increase contraception use and continuation. Making the hormonal intrauterine system (IUS) available in public facilities in low-income countries could increase uptake of long-acting contraception because it appeals to some women who would not otherwise choose a long-acting method; 30% of hormonal IUS adopters would have chosen a short-acting method if the hormonal IUS had not been available.Satisfaction and continuation rates were high among interviewed hormonal IUS adopters. Providers also reported that most hormonal IUS adopters were satisfied and rarely returned with complaints that could not be addressed with additional counseling.Providers reported that many women were not willing to try a method they were hearing about for the first time.When taking steps to increase the availability of the hormonal IUS, donors and policy makers must fund demand-creation efforts to increase awareness of a new long-acting contraceptive option that has characteristics that are distinctly different from other long-acting and short-acting methods.Program managers should develop introduction and scale-up plans that allow for ongoing support and mentorship of providers in offering a new long-acting contraceptive method. Few women in low- and middle-income countries have access to the hormonal intrauterine system (IUS). Past research from a small number of facilities and the private sector suggest the IUS could be an important addition to the contraceptive method mix because it is the only long-acting method some women will adopt and users report high satisfaction and continuation. We aimed to determine whether these promising results were applicable in public facilities in Kenya and Zambia.We used a mixed-methods approach with program monitoring data, interviews with women who received an IUS, and qualitative focus group discussions with providers. Data were collected in 2017–2019.Facilities in Kenya and Zambia reported 1,985 and 428 IUS insertions, respectively. If the IUS had not been available, 30% of adopters would have chosen a short-acting method. Women and providers gave diverse reasons for adopting the IUS, with the desire for fewer side effects being frequently mentioned in focus group discussions. Many IUS adopters first heard of the method on the day it was inserted , yet providers reported that many women were unwilling to try a method they were just hearing about for the first time. Satisfaction and continuation were high: 86% of adopters in Kenya were still using the method 3–6 months after insertion and 78% were in Zambia (average 10 months post insertion). Providers also reported that most IUS adopters were satisfied; they rarely returned with complaints that could not be addressed with additional counseling.Expanding IUS access through the public sector shows promise to increase contraception use and continuation in low- and middle-income countries. Efforts to strengthen availability should consider demand and engage directly with various communities, including youth, around availability of a new long-acting option. Generally, increasing the number of available methods increases contraceptive use, although adding a method that is a variation of existing methods may have a greater effect on continuation than uptake.–,–,,Past research on the hormonal IUS in LMICs suggests women will choose it when it is offered as part of a mix of methods.The U.S. Agency for International Development (USAID) flagship Maternal and Child Survival Program (MCSP) aimed to fill this evidence gap by introducing the hormonal IUS into public facilities in 2 counties in Kenya and 4 provinces in Zambia. MCSP used donated products from the ICA Foundation with implementation activities executed in partnership with the USAID-funded Afya Halisi projectThis article explores characteristics of women adopting the hormonal IUS at public facilities with comparison to IUD adopters, reasons women chose the hormonal IUS, sources of information about the method, user satisfaction, and continuation rates. Data collected from women were triangulated with provider perspectives. MCSP's intent was to learn whether promising results from earlier studies on the hormonal IUS in LMICs hold true when the method is introduced in the public sector under “real-world” conditions. This evidence contributes to the learning agenda developed by a global Hormonal IUS Consultative Group of donors, implementers, and suppliers.This study investigated whether promising results from earlier studies hold true when the hormonal IUS is introduced in the public sector under “real-world” conditions.,In Kenya and Zambia, MCSP consulted with Ministry of Health (MOH) stakeholders to plan and design the program, including tools for on-the-job training with ongoing mentorship, an approach shown to be cost efficient for building provider skills.BOX,Traditional training through extended, off-site, group-based workshops often fails to improve health care provider performance.In light of this evidence, and to ensure hormonal intrauterine system (IUS)/long-acting reversible contraceptive (LARC) training translated into performance, the Maternal and Child Survival Program (MCSP) used an on-the-job, modular (no session longer than 3 hours) approach to help build and strengthen the competency and confidence of providers to learn and perform essential job skills with minimal disruption to services. Training was interactive and held at health care facilities with special attention given throughout to learning and practicing skills through role plays and simulations  with family planning clients. Training content was tailored at each site based on identified learning needs of providers, using the appropriate modules from MCSP's LARC Learning Resource Package as needed. For example, in Kenya, the copper intrauterine device (IUD) module was used in addition to the hormonal IUS and postpartum modules when LARC providers were not sufficiently proficient in IUD counseling and insertion, so providers could confidently and competently provide both the IUD and hormonal IUS. The LARC Learning Resource Package contains 5 clinical modules plus modules on family planning counseling and assessing medical eligibility (with activities designed to help providers strengthen their communication skills to enable clients to make well informed and voluntary decisions), infection prevention, and quality care.Mentoring complements on-the-job-training by supporting skill transfer and service implementation after training, ensuring that remaining skill gaps of learners are addressed during post-training mentorship sessions. MCSP defined mentoring as “the process through which an experienced and empathetic person who is proficient in her/his content area (a mentor) teaches and coaches another individual (mentee) or group of individuals (mentees), in person and/or virtually, to ensure competent workplace performance and provide ongoing professional development.”To implement cascade training, MCSP trained providers (mainly nurses and midwives) who had been identified as MOH mentors to support other providers in building their clinical skills. The first step of the process was to train the mentors in hormonal IUS counseling and insertion and removal techniques and to review the process for training and mentoring other providers. After mentors conducted 10 insertions in Kenya (as many as possible under observation during training and mentorship and the remainder done independently and logged) and 4 insertions in Zambia (2 while being coached and 2 under observation during training or mentorship), they underwent assessment to become certified hormonal IUS providers. After becoming certified, mentors were then expected to train and provide ongoing support for other providers (mentees) in facilities where they worked and/or nearby facilities, as well as continue to conduct insertions. Mentees were other providers  selected collaboratively by MOH coordinators, the project team, and facility in-charges. On-site training of mentees was spread over 4 consecutive days and lasted no more than 3 hours per day. Mentors then developed a follow-up mentoring plan based on the performance of the mentees. The criteria for becoming a certified hormonal IUS provider were the same for mentees as for mentors .In Kenya, the mentors had been previously trained by MCSP in other LARCs (implants and IUDs) in 2016 but were inexperienced in hormonal IUS. With hormonal IUS introduction, MCSP led additional training for mentors on hormonal IUS plus postpartum insertion (for the IUS and IUD) in December 2016, using the appropriate modules from the Learning Resource Package. Additional mentors were trained in 2017 on all LARCS under the Afya Halisi project. As of mid-2019, 65 mentors were trained in hormonal IUS with 48 certified plus 190 mentees trained by mentors, although none were certified due to delays in rolling out the assessments. In Zambia, the SM360+ project trained maternity care providers to become LARC providers and mentors from February to August 2017, with MCSP contributing materials and trainers to incorporate the hormonal IUS into the training. The mentors had previous training in maternal and newborn health and mentorship skills, but were often inexperienced with LARCs. They were expected to start integrating LARC counseling and provision into their maternity clinical work and mentorship of other providers of maternity services.  As of mid-2019, 68 mentors were trained in all LARCs including hormonal IUS  with 49 certified plus 134 mentees trained by mentors with 91 certified. In both countries, the number of trained and certified providers continues to rise as cascade training is ongoing.Mentors and mentees worked in facilities that offered short-acting contraceptive methods as well as IUDs and implants , although temporary stock-outs or staffing disruptions sometimes limited availability of certain methods. Hormonal IUS was offered free of charge, as are all contraceptive methods in the public sector in these countries. MCSP distributed donated ICA Foundation commodities directly to facilities. Providers in both countries gave information on the hormonal IUS during group talks at facilities and one-on-one counseling. Information on the hormonal IUS was incorporated into discussions or counseling on family planning and the various methods available, and not done separately. Providers had been trained to give a woman more information on the characteristics of a method once she made her choice, including expected side effects, bleeding changes, and noncontraceptive benefits. Providers also explained to the woman that she could have it removed at any time and what she could expect before, during, and after insertion. Information about the hormonal IUS was also shared through community outreach. In Kenya, community health volunteers work with facilities to promote health behaviors and care-seeking; facilities where the hormonal IUS was available were responsible for orienting community health volunteers on the method so they could share information in communities as part of their general efforts to promote family planning. In Zambia, SM360+ oriented existing Safe Motherhood Action Groups on LARCs including the hormonal IUS, although orientations happened late in the study period. MCSP did not track data on these demand-creation activities.We used a mixed-methods approach that included analysis of program monitoring data, interviews with women who received a hormonal IUS or an IUD, and qualitative focus group discussions (FGDs) with providers. IUD adopters were interviewed for comparison since adopters of hormonal IUS and IUD were both presumed to desire long-acting contraception and to be undeterred by having a device in the uterus, so we could compare other factors that influence women's decision making and experiences.Ethical approval was received from the ethics review committees at the Johns Hopkins University School of Public Health (USA), Maseno University (Kenya), and ERES Converge (Zambia).In Kenya, women were enrolled in the study immediately after hormonal IUS or IUD insertion beginning in April 2017 . All women receiving a hormonal IUS or an IUD at participating facilities were eligible for the study. Providers received training on research ethics and were given informed consent scripts to read to women after insertion. After providers obtained oral consent, they completed a short paper-based questionnaire collecting sociodemographic characteristics and contact information for follow-up. This questionnaire also collected reasons for choosing the method, what method they would have chosen if the hormonal IUS or IUD was not available, and when and how they heard about the hormonal IUS, which are questions implementers in the global hormonal IUS consultative group all agreed to collect.In Zambia, providers completed a paper-based questionnaire immediately after insertion as part of program monitoring and study recruitment. Data were collected on all women who received a hormonal IUS or IUD in participating facilities and were willing to answer the questions. Data collection started during the initial training for mentors. The questionnaire collected information similar to the one used in Kenya, but contact information was documented only for women that expressed interest in learning about the research study. The study consent process and interview were done sequentially via phone by MCSP staff. In Zambia, phone interviews were limited to women who received the hormonal IUS or IUD within 1 year after giving birth or after receiving postabortion care. The population was restricted due to the programmatic focus on maternity providers and because Society for Family Health was simultaneously introducing the hormonal IUS and collecting similar data in other public facilities in Zambia without a postpartum focus.We present results based on data collected by providers at 42 facilities in Kenya from April 2017 to March 2019 and 41 facilities in Zambia from February 2017 to September 2019, although program expansion continued into additional sites after these dates. Follow-up phone interviews were conducted over 2 time periods—September 2017 and March 2019 in Kenya, and March–December 2018 and April–July 2019 in Zambia. These periods were selected based on availability of consultants and staff conducting interviews. To meet reporting requirements from the ICA Foundation, MCSP also collected the number of hormonal IUS commodities distributed to facilities and contacted facilities semiannually to obtain the total number of hormonal IUS insertions according to facility records. Data were extracted from these reports for the purpose of assessing overall uptake of hormonal IUS in program-supported facilities.FGDs were held with providers, with separate groups for mentors and mentees to allow different questions about the training and mentorship approach. Participants were selected by MOH staff. Each participant worked in a different facility to increase the representativeness of the sample. FGDs were conducted in English, recorded, and transcribed. In all, 2 FGDs were done with mentors (23 participants) and 4 with mentees (36 participants) in Kenya and 4 with mentors (23 participants) and 3 with mentees (15 participants) in Zambia. FGDs were held in February 2019 in Kenya and August 2018 in Zambia. Providers did not receive compensation for participation.Quantitative analysis was done using Stata v14. Using data collected on the day of insertion, we ran cross-tabulations to compare hormonal IUS and IUD adopters in terms of sociodemographic characteristics, timing of insertion relative to last birth, and the method nonpostpartum adopters were switching from. We used the Pearson chi-squared test with Rao-Scott correction to adjust for clustering by facility. We also explored reasons hormonal IUS adopters chose the method; what method they would have chosen if hormonal IUS were unavailable; and where they first heard about the hormonal IUS. Using follow-up phone interviews, we cross-tabulated side effects or physical changes that hormonal IUS and IUD adopters reported providers mentioned to them on the day of insertion. In Kenya, we also examined whether the time lapse from insertion to interview seemed to affect the side effects women mentioned by restricting the analysis to women who received the hormonal IUS or IUD within 6 months before the phone call. We could not do the same in Zambia due to the small number of women participating in phone interviews. As markers of satisfaction, we looked at whether hormonal IUS adopters would recommend or have recommended the method (the question was asked differently in Kenya and Zambia) and what benefits they would mention to other women. Finally, we examined continuation rates for hormonal IUS adopters. Since there was a wide range in the time lapse from insertion to phone interviews, we restricted analysis to women interviewed 3–6 months (92–183 days) after insertion in Kenya to make the sample more homogenous. Due to the small sample size in Zambia, we were unable to restrict the sample.FGDs were explored for information to enhance the quantitative findings, namely provider perspectives on the reasons women chose the hormonal IUS, information women received on the method, challenges to providing counseling, and client satisfaction with the method. Transcripts were coded using these themes; codes were then analyzed using principles from the One Sheet of Paper method.MCSP Kenya and Zambia received 2,930 and 1,205 hormonal IUSs from ICA Foundation, respectively, with 1,985 and 428 insertions reported by facilities in Kenya and Zambia, respectively, by mid-2019. In Kenya, 289 adopters of the hormonal IUS were interviewed on the day of insertion , of which 182 (63%) participated in phone interviews. Additionally, 143 copper IUD adopters were interviewed on the day of insertion, with 87 (61%) participating in follow-up phone interviews. In Zambia, 395 adopters of the hormonal IUS were interviewed on the day of insertion ; 246 were postpartum or postabortion clients, of which 40 (16%) participated in phone interviews. Additionally, 359 IUD adopters were interviewed on the day of insertion; 183 were postpartum or postabortion clients, of which 42 (23%) participated in follow-up phone interviews. Day-of-insertion interviews ranged from 1 to 47 per facility in Kenya  and from 1 to 121 in Zambia . The average time lapse between insertion to follow-up interview was 5 months (range 43–668 days) in Kenya and 10.4 months (range 52–773 days) in Zambia.Totals of 1,985 and 428 insertions of the hormonal IUS were reported by facilities in Kenya and Zambia, respectively, by mid-2019.In Kenya, no statistically significant differences were observed in the characteristics examined between hormonal IUS and IUD adopters , althougAmong nulliparous and interval adopters (women whose last birth was more than 1 year prior), around half in Kenya and two-thirds in Zambia were switching from a short-acting method. In both countries, around 10% of hormonal IUS and IUD adopters were not switching from another method .Women were allowed to give multiple reasons for choosing the hormonal IUS; answers were unprompted. Women had diverse reasons for adopting the hormonal IUS. The most commonly mentioned reasons in Kenya were each mentioned by less than 40% of adopters and included the desire for fewer side effects (37%) and the facts that it is reversible (31%), can be used for spacing (30%), and is long-lasting (29%). In Zambia, the ability to use the hormonal IUS for spacing was the only reason mentioned by over half of women (55%); other top reasons included it is long-lasting (36%) and reduces bleeding (36%) . The abiIn Kenya, 37% of women chose the hormonal IUS because they desired fewer side effects. In Zambia, 55% of women chose the method because of the ability to space pregnancies.If the hormonal IUS had not been available in Kenya, 48% of hormonal IUS adopters would have opted for an implant, 15% an IUD, 30% a short-acting method, and 4.5% a traditional method or no contraception. In Zambia, 37% would have chosen an implant, 15% an IUD, 30% a short-acting method, and 3% a traditional method or no contraception .When asked why clients chose the hormonal IUS, providers mentioned many of the same reasons as the interviewed women. The desire for fewer side effects came out strongly in the focus groups. Providers explained that women's desire for fewer side effects were, at times, tied to negative experiences using other hormonal methods, and some hormonal IUS adopters had come to the facility with the intent of switching to a method with fewer side effects. Although there was not extensive conversation about the specific side effects women sought to avoid, providers mentioned weight changes, reduced sexual desire, and cardiac effects . Providers reported that clients found it appealing that the hormonal IUS releases less hormone than other hormonal methods and that the hormone is localized to the uterus. These features eased women's concerns about side effects and using a hormonal method. Providers also reported women desired less bleeding to avoid discomfort, inconvenience, and disruption to their lives. The appeal of bleeding reductions came up more frequently in the Kenya interviews. There was no mention of negative reactions to reduced bleeding in any focus group.Maybe the hormonal level in hormonal [IUS] where the hormonal is less and it is concentrated in the uterus most of the time. Personally, as a user of hormonal I have gone into amenorrhea and that is an advantage. The hormone level is okay with me, I don't feel any palpitations, I don't have any other problem. That is the reason why I choose hormonal. So if you counsel, because with many women they tend to fear hormones, so even when you counsel and tell them that it is a 5-year and it is hormonal, unless you elaborate further that it is a hormone that concentrates mostly in the uterus and very little can be released into the system which will not affect [them], they will go for it. —Kenya, Migori County mentorProviders mentioned that some women like that the hormonal IUS is long-lasting but not as long-lasting as the IUD . This preference implies women are unaware that IUDs can be removed by choice before their full effective period. This lack of awareness may be due to inadequate counseling, misinterpreted marketing, or even difficulty experienced in accessing removals (providers in an FGD in Zambia said women reported resistance from providers when attempting to have an IUD removed before full duration). Whatever the reason, providers shared it can be challenging to overcome the perception that the IUD is only appropriate for women who want 10 years of protection, and some women still feel more comfortable with a method intended for removal within 5 years . The 5-year duration may be especially appealing to adolescents because it aligns well with their reproductive intentions—that is, to finish school and start childbearing soon thereafter.The other reason is because it takes a shorter period, some women would want to have an [intrauterine device] that does not go up to 12 years like the copper T, so during the counseling when you discuss with them then they tell you, I would rather have this one because previously with the one that takes long, the copper IUD, I realized that because of ineffective counseling sometimes women are not told that whenever you want to have it removed they can just come back, that part sometimes misses that you find clients coming back to you with IUDs that were inserted 20 years ago, so for them if you talk about this, most of them say I would rather have the one with a shorter period. —Kenya, Kisumu County, mentorProviders also mention that the hormonal IUS can appeal to women who desire fewer visits to a facility, for convenience, cost saving, or, as 1 provider mentioned, to hide the use of contraception from her partner. Few women mentioned the ability to use the method discretely as a reason for choosing it and this reason was likewise only mentioned by a few providers. Still, it could be an important reason for a minority of women.I have a certain group of clients who don't want their spouses to know that they are using family planning methods yet they want the method. I have a mother who [had 10 previous births]. She just delivered yesterday and the husband is very cruel that he doesn't allow the mother to take any method. She has twelve children because she had a twin delivery previously. And all these children are alive and the spacing between them is not that good. So we counseled this mother at [antenatal care] so when she was coming for delivery, she just opted up for that method so that the husband will not know but will not take a long time as copper T. —Kenya, Migori County, menteeProviders mentioned additional attractive qualities that were not recorded in interviews with women, although discussion was not extensive and these may not be driving reasons for many women. These factors included a bias some women have in favor of a new method, believing it would be superior to methods that have been around a long time; the hormonal IUS does not require an incision; and removal is less painful compared with subcutaneous implants.Most hormonal IUS adopters in Kenya heard of the method for the first time on the day it was inserted (70%), and 22.5% had heard about it from a provider during a previous visit to the facility. In Zambia, 47% heard about the method for the first time on the day of insertion, and 36% had heard about it from a provider previously .Among adopters of the hormonal IUS, 70% in Kenya and 47% in Zambia heard of the method for the first time on the day it was inserted.During follow-up interviews, women were asked, without prompting, what physical changes or side effects the provider mentioned at the time of insertion, and all answers were recorded. In Kenya, most women recalled the provider telling them about potential physical changes or side effects; only 3% of hormonal IUS adopters and 10% of IUD adopters reported the provider did not mention any or could not recall if the provider had mentioned any. The majority of hormonal IUS (76%) and IUD adopters (60%) said the provider mentioned changes in menstrual bleeding and 38% of hormonal IUS adopters and 39% of IUD adopters mentioned abdominal discomfort or pain. Other side effects were mentioned by a small number of women. The majority of women were told what to do if they experienced side effects . When restricting the analysis to women interviewed within 6 months after insertion, results did not materially change (data not shown). In Zambia, a larger proportion of women reported the provider did not mention any side effects or could not recall if the provider mentioned side effects than in Kenya . The difference between IUS and IUD adopters within Zambia was not statistically significant in this small sample. Similar to the results in Kenya, the most common side effects women recalled providers discussing were changes in bleeding  and the majority were told what to do if they experienced side effects , although the levels were not as high as in Kenya .Although a large proportion of hormonal IUS adopters had not previously heard of the method, a common theme across FGDs with providers was that many women are not willing to try a method they are hearing about for the first time.Okay, for me, the challenge is just lack of information. The clients are not aware of the method, so when you try to tell them about the method, they will just tell you let me go and think about it, then I will come later. —Kenya, Kisumu County, menteeProviders shared that they found it difficult to counsel women who have no baseline awareness of the method and to overcome pervasive rumors about IUDs and contraception, including that IUDs do not stay in place and that contraception can cause cancer or infertility. The providers also shared that some women appeared interested in the method, but were unwilling to start the method that day, needing time to think and to consult their husbands. And a few providers shared stories of women who did receive the method, but then returned requesting a removal because their husbands did not like the method or did not approve of them using it. Because of these experiences, providers across most of the focus groups appealed for investments in spreading accurate information on the hormonal IUS in the community through mass media, community health workers or volunteers, or community groups so women come to facilities with awareness of the hormonal IUS, openness to using long-acting contraception, and support from their partners.Providers appealed for investments in spreading accurate information on the hormonal IUS in the community so women come to facilities with greater awareness of it.Although the content of counseling was infrequently discussed, in a couple of instances, providers made a direct link between counseling on side effects and women's willingness to initiate or continue using contraception. They explained that truthful conversations about side effects meant women were better prepared and were more trusting of providers if they experienced unwelcomed effects.Just to add-on to what she has said, like in the counseling there are times when you discover that when people are given counseling at first, the side effects never used to come out. So now we would concur with people as they are mentoring them, you come in the audience without people noticing you and listen to the on-going counseling. They will talk about the benefits as she put it but side effects are not talked about. So mothers would start shunning out family planning, but when you tell the mothers the truth of what they are going to face or to experience the first 3 months of the hormonal IUS and other IUCDs they will understand that this is what I was told and afterwards the side effects go. At least this time it's coming up bit by bit. —Zambia, Central Province, mentorThese FGDs cannot be used to assess the quality of counseling, but it should be noted that providers often discussed feeling overworked and complained that facilities were understaffed. A few times during the discussions, providers mentioned that this situation contributed to inadequate counseling.Not adequately because of lack of personnel so a client will come but I'll feel I am not giving them enough time for counseling.—Kenya, Kisumu County, mentorIn Kenya, 79% of hormonal IUS users had recommended the method to other women and 95% in Zambia would recommend the method. In terms of willingness to recommend their method, no difference was found between IUS and IUD adopters. The top benefits that IUS users in Kenya would mention to other women were reduced bleeding (49%), fewer side effects (33%), and the fact that it is long-lasting (29%). The top benefits IUS users in Zambia would mention to other women were convenience (63%), fewer side effects (34%), and high effectiveness (29%) .In Kenya, 79% of hormonal IUS users had recommended the method to other women and 95% in Zambia would recommend the method.Among hormonal IUS adopters in Kenya interviewed 3–6 months after insertion, 128 women (86%) were still using the method, 7 (5%) experienced an expulsion, and 11 (7%) had the method removed, and 2 responded “I don't know” to the question “Is the same IUD still in place, as far as you know?” (plus 1 with missing data). In Zambia, where the average time lapse from insertion to interview was over 10 months with a wide range for a small sample size, 31 women (79%) reported still using the method, 4 (10%) experienced an expulsion, and 4 (10%) had the method removed (plus 1 with missing data). In both countries, continuation rates were slightly higher among IUD users  and there were fewer expulsions among IUD users, but the difference between IUS and IUD users was not statistically significant.Continuation was very high among women who did not report experiencing a “major” problem with the hormonal IUS  and was lower among women who did (60% in Kenya and 56% in Zambia). Among the 11 women who had the hormonal IUS removed in Kenya, 4 did not report having a major problem, and the remaining 7 women reported the following problems: cramping or pain (4), too much bleeding (2), husband disapproval (2), strings were too long (2), and irregular bleeding (1). Among 4 removals in Zambia, women reported the following problems: too much bleeding (4), dizziness (2), and cramping or pain (1). For comparison, 15 women still using the IUS in Kenya (12%) reported cramping or pain (8), too much bleeding (2), discharge (2), reduced bleeding (1), irregular bleeding (1), or husband disapproval (1). Ten women still using the IUS in Zambia (32%) reported cramping or pain (6), too much bleeding (3), too little bleeding (2), fever (2), or irregular bleeding (1).Continuation was very high among women who did not report experiencing a “major” problem with the hormonal IUS.Women were asked about amenorrhea separately. In Kenya, 40 women still using the IUS (31%) reported experiencing amenorrhea, with 7 saying it was a negative change. None who had the IUS removed experienced amenorrhea. In Zambia, 5 women still using the IUS (16%) reported amenorrhea, with 3 saying it was a negative change, and 1 of the 4 who had the IUS removed experienced amenorrhea saying that was a positive change (she reported dizziness as a problem).In the focus groups, providers frequently expressed confidence that most hormonal IUS adopters were satisfied with the method because they did not return with complaints. Occasionally, providers contrasted the absence of complaints among hormonal IUS adopters with grievances heard from users of other methods.I feel they are satisfied because so far no one has approached me with any complaint. I have removed copper T, I have removed Implanon and Jadelle, but not hormonal [IUS]. Even those that I meet, when I ask them about the method they tell you they are comfortable. —Zambia, Southern Province, mentorProviders also reported hearing positive feedback from hormonal IUS users, who said they did not have any problems or were pleased to have fewer side effects than previously experienced with other methods.We have had interactions with them through other services that they come to seek. Maybe they have come for under-5, maybe they are sick with other ailments then you tend to interact with them and say how are you finding the hormonal [IUS] that you had received from here? They say it's just okay, I thought it would give me problems but there is nothing. Such things, so some of the women are coming out openly to say that there is no problem. And we are just encouraging them also to help us by sensitizing the women in the community because the community believes more their fellow community members than us the health practitioners. —Zambia, Eastern Province, menteeProviders talked about the powerful influence satisfied users can have within their circle of friends, family, and neighbors when they share their experiences using the method. Some providers reported that satisfied users in their communities led to increased interest among other women. And some providers said they had invited satisfied users to talk to other women with the intention of garnering more demand for the method.Providers talked about the powerful influence satisfied users can have within their circle of friends, family, and neighbors.Providers also told stories of women returning with complaints and how they were able to successfully address them with counseling and occasionally through treatment of side effects such as bleeding. With additional counseling to assuage concerns, providers reported that many women opted to continue using the method and found that the side effects eventually faded. However, a few providers gave examples of women who had bad experiences with the hormonal IUS and actively discouraged other women from using the method.Providers also recounted cases of hormonal IUS removals. In many of the accounts mentioned, providers believed the male partners were driving the decision to have the method removed and the woman was not necessarily unhappy with the method. In 1 case, the woman even had the provider secretly reinsert the method. Providers also shared examples of women wanting or needing to have the method removed, each for a different reason, including undesired bleeding changes (spotting or excessive bleeding were mentioned), discharge, infection, and diagnosis of hypertension.This study provides information on characteristics of women who adopted the hormonal IUS in public facilities in 2 low-income countries. Generally, these characteristics are likely correlated with the profile of the typical family planning clientele of these facilities, which probably accounts for many differences in hormonal IUS adopters across countries and studies. A large proportion of Kenyan adopters in this study and a prior study of postpartum adopters at a public facility in KenyaComparisons of hormonal IUS to IUD adopters at the same facilities allows additional insight into whether hormonal IUS appeals to a different set of women. The results from this study suggest that hormonal IUS may appeal more to young women than the IUD since a greater proportion of hormonal IUS adopters were under age 25 in both countries . More adopters of the hormonal IUS in Zambia were never married and had lower educational attainment, which may be attributable to the differences in the age profile. Provider testimonies that shorter duration of effectiveness is a favorable attribute of the hormonal IUS could explain why the method may be more appealing to younger women than IUDs, as they may want to become pregnant in the near to intermediate term.A substantial proportion of women choosing the hormonal IUS (35% in Kenya and 33% in Zambia) would have opted for a short-acting or traditional method or none at all, if the hormonal IUS had not been an option. This finding is similar to what Hubacher et al.Many adopters would have opted for a short-acting or traditional method or none at all, if the hormonal IUS had not been an option.,,,As in other studies, women gave a range of reasons for choosing the hormonal IUS. A desire for fewer side effects emerged at the top. Among adopters we interviewed, 37% in Kenya mentioned hormonal IUS having fewer side effects than other methods and 23% specifically mentioned reduced bleeding. In Zambia, 22% mentioned fewer side effects and 36% specifically mentioning reduced bleeding. Users of the hormonal IUS also reported that fewer side effects and reduced bleeding were benefits they would mention to other women when recommending the method. A desire for fewer side effects was also a popular reason for women in past studies.Women gave diverse reasons for choosing the method, suggesting that it can appeal to women wishing to adopt contraception for a broad range of reasons.,Many hormonal IUS adopters had never previously heard of the method. Providers reported that lack of awareness of the method among their clients handicapped uptake, but they saw potential for further growth in interest in hormonal IUS if information on the method could be disseminated through community channels. Although few adopters reported hearing about the method from family or friends, providers believed that satisfied users can be powerful assets to encourage other women to try the method. The critical role of satisfied users to increasing demand for IUDs has been shown in past research.Most women reported being told what to do if they experienced side effects, and many recalled being told about bleeding changes and abdominal pain, although other side effects were rarely recalled and may not have been emphasized or mentioned at all. Providing clients with information about contraceptive methods in general and their chosen method in particular is associated with improved continuation.,Despite some indications that the quality of counseling before insertion was not always optimal, user satisfaction levels were high, as evidenced by the high proportion of users who would recommend the hormonal IUS to other women. We also found a high level of continuation among women in Kenya 3–6 months after insertion (86%), on par with the rate seen in a previous study in Kenya at 12 months (89%).User satisfaction levels were high, as evidenced by the high proportion of users who would recommend the hormonal IUS to other women.Uptake of the hormonal IUS was higher in Kenya than Zambia. Two prolonged health care worker strikes (December 2016 to March 2017 and June–November 2017) disrupted services in Kenya during the study period, but demand quickly recovered after each strike ended. The difference between countries may be partially explained by general trends in contraceptive use in each country. The modern contraceptive prevalence rate (mCPR) was 34.1% in Zambia in 2018.For context, we compared the number of IUS insertions in project-supported facilities to MOH data from the same facilities over the same period. In Kenya, we reported 1,985 IUS insertions over a period when approximately 8,000 total intrauterine insertions (IUS + IUDs) and 37,000 implant insertions were reported by the same facilities. In Zambia, we reported 428 IUS insertions over a period when approximately 2,700 total intrauterine insertions (IUS + IUDs) and 16,000 implant insertions were reported by the same facilities. We found wide variation in uptake across facilities. In some participating facilities, no IUS insertions were reported. We know from project reports and focus groups that some trained providers were transferred to different facilities and some did not yet feel comfortable in their skills. As mentioned above, providers reported challenges in offering a new method that women were not familiar with. From the MOH data, we also see a wide range across participating facilities in terms of total LARC insertions. Adoption of new practices is often uneven, although it could be improved with careful tracking and by quickly responding when providers are transferred or are found to not be adequately skilled. Investments in widespread demand creation could also support more even adoption.Study limitations include that findings may not be representative of all women receiving a hormonal IUS; in Kenya, only around 15% of women receiving the IUS were interviewed at the time of insertion, either because women refused to participate or providers did not take the time to enroll all eligible women. We do not know if interviewed adopters were different from those not interviewed in ways that might affect the results presented in this article. In both countries, the study conducted follow-up phone interviews with a minority of adopters because many women were unable or unwilling to share phone numbers or did not answer their phones after multiple attempts to reach them. Data from follow-up interviews are thus likely biased toward women who have greater phone access, including women with a higher income. We did not track how many insertions were done by providers who were still in training, but it is likely to be many because there was a long training and mentorship process before certification. It is possible that clients' experience of care, satisfaction, and continuation may shift over time as the number of more experienced providers increases. In Kenya, we conducted fewer interviews with IUD adopters than IUS adopters, which affected our ability to detect statistically significant differences between the groups. Results from follow-up interviews are subject to recall bias. In addition, women were interviewed at very different time points after insertion, so data on continuation have to be interpreted cautiously. Qualitative interviews were conducted with providers only, and there may have been a social desirability bias, especially since program staff were in attendance to assist the qualitative interviews and interviews were conducted on project premises. Also, MOH may have identified providers that were high performing or held more positive views on the training and mentorship experience to participate in the interviews.Expanding access to the hormonal IUS through the public sector as part of a range of contraceptive choices shows promise to increase overall use of contraception and continuation in both Kenya and Zambia. Supporting providers to acquire skills in both counseling and insertion is essential and efficiencies can be gained if integrated within efforts to fill LARC competency gaps, rather than offering stand-alone hormonal IUS training, although proficient LARC providers only need tailored support to add this method to their skillset. Ensuring that training includes skills for insertion in interval, postabortion, and immediate postpartum services can improve the diversity of clients who benefit from this method and requires only a marginal additional investment. Efforts to strengthen availability of the services should not ignore demand-related efforts and engaging directly with various communities around the availability of a new LARC option with its own method characteristics. These communities could include youth groups and channels for unmarried women as well as men. With global donors and multilateral agencies planning to introduce hormonal IUS as part of offerings for commodity donations to countries, there will be more opportunities for women in LMICs to access and voluntarily opt for this method.


Although hymenal tags are not uncommon in newborn girls, there is a paucity of literature on this condition. Few photo images of hymenal tags have been published. We report the case of a four-month-old girl with a large hymenal tag noted at birth. Approximately 30% of hymenal tags may persist at three years of age. As the number of prepubertal girls who require evaluation for sexual abuse increases, physicians should familiarize themselves with the appearance of a hymenal tag to differentiate it from signs of sexual abuse, especially in older girls whose hymenal tags have not resolved. Hymenal tags, also known as hymenal polyps, are common findings in infant girls. Despite its prevalence, there is a paucity of literature on hymenal tags. A PubMed search of the English literature conducted in April 2021 using the key terms “hymenal tag” OR “hymenal polyp” revealed only seven publications . The majA four-month-old Chinese infant girl presented with a protruding vaginal mass which was first noted at birth. The infant was born to a gravida 2 para 1 27-year-old mother at 39 weeks of gestation, following an uncomplicated pregnancy and normal vaginal delivery. The mother was not on any medications during the pregnancy. The Apgar scores were 6 and 9 at one and five minutes, respectively. Her birth weight was 3.02 kg (between 25th and 50th centile), length was 51.1 cm (between 50th and 75th centile), and head circumference was 35.4 cm (75th centile). The labor was smooth and there was no history of birth trauma or vaginal bleeding. The infant was exclusively breasted and thriving. The mass was asymptomatic and there was no history of bleeding from the mass or of sexual abuse. The infant came from a loving and supportive family. Her mother was the sole care provider.On examination, the infant looked alert and was not in distress. Her weight was 6.8 kg, length was 64 cm, and head circumference was 41.7 cm. A smooth, pink mass was seen extending from the hymenal ring  and inferior  positions of the hymen and, less commonly, in the lateral position ,2. HymenHymenal tags tend to regress with time . Some auRoutine physical examination of children should include a careful examination of the external genitalia. Physicians should be familiar with hymenal tags to diagnose them accurately so that they will not be mistaken for sexual abuse, especially in an older girl with the persistence of a hymenal tag of considerable size.