Abstract
Neonatal uterine bleeding (NUB) has been hypothesized as a relatively common early-life event potentially linked to the later development of endometriosis through retrograde menstruation. However, its true prevalence and biological nature remain uncertain. This study aimed to determine the prevalence, biological features, and maternal–neonatal correlates of reddish diaper staining in newborns, and to evaluate its purported association with endometriosis. A prospective cross-sectional study was conducted between September 2023 and March 2024 at a tertiary obstetric center with postpartum outpatient follow-up. A total of 638 pregnant women with singleton pregnancies (530 female and 108 male fetuses) were enrolled. Participants completed prenatal questionnaires, and neonatal outcomes, including diaper staining, were assessed during follow-up. Diapers with reddish staining were collected and analyzed using light microscopy and biochemical assays to identify the presence and origin of blood. Umbilical cord blood samples from female newborns were analyzed for steroid hormone concentrations. Reddish diaper staining was observed in 22% of female and 13% of male neonates. Only two female samples (2.5%) tested positive for menstrual blood. Most of the reddish diapers contained urate crystals rather than blood. Maternal characteristics, exposure to endocrine-disrupting factors, and cord blood steroid hormone levels did not differ significantly between female neonates with and without reddish diaper staining. These findings indicate that reddish diaper staining in neonates is predominantly due to urate crystal excretion rather than true NUB. By identifying a major and previously unrecognized confounder, this study calls into question a long-standing biological assumption and reshapes current understanding of NUB and its supposed link to endometriosis. Our results provide a substantial paradigm shift in the interpretation of this phenomenon, redefining its clinical and biological significance and opening new perspectives for research in early-life determinants of reproductive health.
Keywords: Neonatal uterine bleeding, Endometriosis, Umbilical cord blood, Steroid hormones, Urate crystals, Genital crisis
Subject terms: Diseases, Endocrinology, Health care, Medical research, Physiology
Introduction
Neonatal uterine bleeding (NUB) has been described as menstrual-like bleeding that occurs in some female newborns a few days after birth. This phenomenon has always been regarded as ‘physiological’ and attributed to the abrupt decline in steroid hormone levels that follows the detachment of the placenta in the newborn female1.
As part of Sampson’s theory of retrograde menstruation in endometriosis onset, NUB has also been associated with the reflux of uterine blood into the peritoneal cavity through the tubes carrying endometrial cells, stromal fibroblasts and possibly also stem cells2,3. These cells could contribute to the formation of ectopic implants that may remain dormant until puberty, when increased estrogen levels may stimulate their reactivation and contribute to the formation of endometriotic lesions4. The anatomy of the neonatal uterus, with a longer cervix and abundant cervical mucus and stem cells, might facilitate menstrual reflux by reducing the outflow of menstrual blood through the vagina2,5. This interesting theory could eventually also explain the pathogenesis of rare cases of early-onset endometriosis described before menarche, during childhood or adolescence6–9 which are not well explained by the more widely accepted theories of retrograde menstruation and coelomic metaplasia10. Moreover, it aligns with the idea that endometriosis may also depend on factors influencing the earliest stages of life, such as the intrauterine environment11.
Although the NUB theory for the onset of endometriosis is appealing, solid supporting data are very limited12,13. The current literature describes NUB occurring in female neonates 3–5 days after delivery, lasting 3–4 days, with a reported prevalence ranging from 3 to 53.8%1,14. Its incidence seems to vary according to certain obstetric outcomes, such as post-maturity, pre-eclampsia, fetal growth restriction, low birth weight, and feto-maternal blood incompatibilit15–18. However, most of the studies are outdated and highly heterogeneous. It remains unclear why, although the physiological postnatal drop in steroid hormones occurs in all female neonates, NUB is observed in only a small percentage of cases.
Of further note, important confounding factors, such as the excretion of urate crystals in newborn urine, have not been considered in any of the articles available in the literature on this topic. The high concentration of urate in maternal and neonatal blood characteristics of the last trimester of pregnancy promotes its precipitation in the urine of newborns, typically presenting as red–orange spots19,20. This often raises concern among mothers who may mistake them for blood. Uric acid excretion is observed in 22% of newborns, and it is more frequent in breastfed infants in the first days of life, lasting 3–4 days21,22. Manifestations of NUB and uric acid excretion are similar and share many clinical characteristics.
Accordingly, this study investigated the prevalence and underlying causes of reddish diaper staining in neonates, distinguishing between menstrual-like bleeding and urate crystal excretion. We assessed the clinical and laboratory characteristics of participating mothers and neonates to explore factors associated with reported reddish staining, traditionally interpreted as NUB in the literature. By clarifying the extent to which urate crystal excretion may account for this finding, our results contribute to refining the estimation of true neonatal uterine bleeding and to reassessing its hypothesized link with endometriosis.
Materials and methods
Study design and participants
We conducted a prospective cross-sectional study involving n = 638 women carrying a singleton fetus before delivery (n = 530 women carrying a female and n = 108 a male fetus) who chose to give birth at Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy. Eligible participants were pregnant women aged between 18 and 42 years, with a single, uncomplicated pregnancy progressing normally, with no indication for delivery before 37 weeks of gestation. Informed consent was obtained from all the participants. Between September 2023 and March 2024, participants were asked to complete a comprehensive questionnaire to collect detailed information on their general and gynecological clinical characteristics, current pregnancy status, and personal lifestyle habits. Specifically, the questionnaire included medical history, previous surgeries, menstrual and reproductive health, prior gynecological conditions or treatments, and family history of gynecological diseases. Additional questions about the pregnancy such as type of conception (natural or assisted), weight gain, use of medications, history of hospitalizations and comorbidities, smoking habits, and the use of dietary supplements. Furthermore, the questionnaire assessed lifestyle factors such as diet, physical activity, and working schedule, aiming to provide a comprehensive overview of potentially influencing factors.
In the case of a female fetus, a cord blood sample was collected from the placental side of the umbilical cord at the time of delivery. Subsequently, mothers were contacted by telephone 10–15 days postpartum to complete a follow-up questionnaire addressing obstetric outcomes and the presence of any episodes of reddish staining observed in diapers. The underlying rationale was that, if meaningful differences were detected between maternal or neonatal characteristics and the occurrence of red diaper staining, such differences could help identify factors associated with this phenomenon.
Diaper sample collection and analysis
In addition, a total of 146 diapers (n = 78 from female infants and n = 68 from male infants) in which mothers had identified reddish stains were collected, representing a subset of the original cohort. During daily neonatal ward rounds, whenever neonatologists identified a diaper showing reddish discoloration, the diaper was immediately collected and transferred to the hospital laboratory for analysis. After collection of a stained diaper from a given neonate, no additional diapers from the same infant were included in the analysis, even if the phenomenon recurred, in order to avoid multiple sampling from the same subject.
Samples were placed in sterile sealed containers and transported promptly under controlled conditions. Laboratory analysis was initiated as soon as possible after receipt to reduce pre-analytical variability. When immediate processing was not feasible, diapers were stored and transported at 4 °C to minimize degradation of blood components and preserve crystalline structures. In the laboratory, for microscopic evaluation, non-dried biological material was recovered with a sample collection spatula and resuspended in 50 ml TRIS buffer. Microscopic evaluation was conducted under light microscopy to identify erythrocytes, epithelial cells, and crystalline structures, including urate crystals.
Biochemical testing using the SERATEC PMB Test was performed according to the manufacturer’s instructions to distinguish between menstrual blood and peripheral blood. The visibly stained portion was carefully excised from the absorbent core using sterile instruments. The material was suspended in the extraction buffer provided with the kit. An extraction time of 10 min. was used. The assay is based on the detection of hemoglobin and D-dimer, as D-dimer concentrations tend to be higher in menstrual blood than in peripheral blood. The SERATEC PMB Test can detect human hemoglobin at concentrations as low as 20 ng/mL and human D-dimer at 400 ng/mL21,22. The test did not show any cross-reactivity with other proteins in blood.
Evaluation of umbilical cord levels of steroid hormones
At the time of delivery, umbilical cord blood samples were collected from the placental side, and a subset was selected for hormone analysis. Samples were matched in a 1:3 ratio between mothers of the same age whose infants exhibited reddish diaper stains and those who did not. The samples were centrifuged at 3000 rpm and stored at − 20 °C. Steroid hormones levels, including aldosterone, androstenedione, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEA-S), dihydrotestosterone, estradiol, 17-OH hydroxyprogesterone, progesterone and testosterone were measured by liquid chromatography-mass spectrometry (LC–MS/MS) using IVD-MS kits (MassChrom, Steroids in Serum/Plasma, Chromsystems, Gräfelfing, Germany) as previously described23.A relative standard deviation of up to 10% was considered acceptable.
Statistical analysis
Data were analyzed using the Statistical Package for Social Sciences (SPSS 26.0, IL, USA). Results for continuous variables were expressed as mean ± standard deviation (for normally distributed data) or median and interquartile range (for non-normally distributed data). In the first case they were compared by Student’s t-test, in the second case by Mann–Whitney test. Categorical data were reported as numbers (%) and compared by means of chi-square test or Fisher Exact test. A binomial distribution model was applied to estimate the 95% confidence interval (CI) for the frequency of the genital crisis.
To identify predictors of red staining in diapers, a multivariate analysis was performed including variables with a p < 0.10 in univariate analysis. Values of p < 0.05 were considered statistically significant.
The sample size was calculated for the first aim of quantifying the frequency of NUB in female neonates, and then adapted for the other investigated items based on the emerging findings. Specifically, we initially planned about a sample size of 450 women delivering a female fetus, assuming a 5% reported NUB rate and targeting a 95%CI of ± 2%. The recruitment was extended to 530 participants to account for the weakness of the basic assumption (5% rate of bleeding that could be as low as 3%) and to ensure an appropriate number of reported cases or red staining in diapers to allow the second aim, i.e., the comparison of umbilical cord steroid hormones levels between those who did and did not show red staining in diapers. Even with a 1:5 ratio between cases and controls (no staining in diapers), at least 16 cases of reddish stains (corresponding to 3% of 530) would have been required for a reliable analysis (Type I and II errors at 0.05 and 0.2 and objective to demonstrate a 4 folds increase frequency of cases above the 90th percentile of the hormones among cases).
Ethical approval
The study was conducted in accordance with ICH/GCP guidelines (International Conference on Harmonization/Good Clinical Practice) and all applicable laws, including the Declaration of Helsinki of June 1964, as amended by the World Medical Association General Assembly in Seoul, 2008. The study protocol was reviewed and approved by the local Independent Ethics Committee (CE Milano Area 2) prior to study initiation. The Ethics Committee also reviewed and approved the informed consent form and all written materials provided to participants before enrollment in the study (Comitato Etico Milano Area 2: 980_2021, ENDO-2021-12,371,933).
Results
Prevalence of red staining in diapers of female newborns
We initially investigated the prevalence of reddish staining in the diapers, perceived by mothers as suspected NUB with a specific questionnaire (Fig. 1A–B). The phenomenon was reported in n = 115 female neonates out of a total of n = 530 women included in the study, corresponding to a frequency of 22% (95%CI 18–25%). The median duration of discharge was 2 days [interquartile range: 1–4], while the median onset was recorded at 2 days after birth1–4.
Fig. 1.
Representative images of diapers with reddish stains from neonates. (A) Diaper from a female neonate who tested positive for menstrual blood using the SERATEC PMB test. Abundant red blood cells were observed under optical microscopy (400x). (B) Diaper from a female neonate who tested negative for both menstrual and peripheral blood using the SERATEC PMB test but was positive for uric acid crystals under light microscopy (400x). (C) Diaper from a male neonate showing uric acid crystals under light microscopy (400x).
Red staining in diapers and maternal obstetric and gynecological characteristics
Baseline characteristics of the mothers of female neonates, collected through the questionnaire, are presented in Table 1. As shown in the table, no significant differences were observed between mothers of female neonates with and without reddish diaper staining in terms of age, gravidity, parity, menstrual cycle duration and regularity, age at menarche, use of combined oral contraceptives, presence of polycystic ovary syndrome (PCOS) as well as a previous diagnosis of endometriosis. Among the variables assessed, mothers of female newborns with reddish diaper staining showed a slightly but significantly higher median pre-pregnancy body mass index (BMI) compared to those without red stains: 22.8 (20.5–25.9) kg/m2vs21.2 [19.4–23.5] kg/m2, p < 0.001. Similarly, BMI at the end of pregnancy was also higher in the former group than in the latter (27.5 [25.1–30.4] kg/m2 vs 25.8 [23.6–28.3] kg/m2, p < 0.001). Another variable that emerged as different between the two groups was the duration of time to conception, which was slightly longer in the first group than in the second (3 months1–11 vs. 2 months [0–6] , p = 0.02). No differences were found regarding pregnancy-associated hypertensive disorders, such as chronic hypertension, gestational hypertension and preeclampsia (Table 1).
Table 1.
Maternal and perinatal characteristics according to the presence or absence of red staining in diapers of female newborns.
| Red staining (n = 115) | Absence of red staining (n = 415) | p-value | ||
|---|---|---|---|---|
| Maternal age | 35 [32–38] | 35 [32–38] | 0.32 | |
| Age at menarche | 12 [11–13] | 12 [12–14] | 0.26 | |
| Endometriosis | 8 (7%) | 26 (6%) | 0.83 | |
| PCOS | 24 (21%) | 87 (21%) | 1.00 | |
| Menstrual cycle duration | 0.78 | |||
| < 21 days | 1 (1%) | 5 (1%) | ||
| 21 35 days | 104 (90%) | 366 (88%) | ||
| > 35 days | 10 (9%) | 41 (10%) | ||
| Amenorrhea | 0 (0%) | 3 (1%) | ||
| Plurigravity | 53 (46%) | 202 (49%) | 0.67 | |
| Pluriparity | 26 (23%) | 120 (29%) | 0.20 | |
| Pre-pregnancy BMI (Kg/m2) | 22.8 [20.5–25.9] | 21.2 [19.4–23.5] | < 0.001 | |
| BMI at the end of pregnancy (Kg/m2) | 27.5 [25.1–30.4] | 25.8 [23.6–28.3] | < 0.001 | |
| Time to conception (months) | 3 [1–11] | 2 [0–6] | 0.02 | |
| Pregnancy onset | 1.00 | |||
| Spontaneous | 101 (88%) | 365 (88%) | ||
| ART | 14 (12%) | 50 (12%) | ||
| Smoking at conception | 13 (11%) | 43 (11%) | 0.74 | |
| Smoking during pregnancy | 15 (13%) | 30 (7%) | 0.06 | |
| Gestational diabetes | 12 (11%) | 52 (13%) | 0.63 | |
| Hospitalization for preterm labor | 5 (4%) | 21 (5%) | 1.00 | |
| Steroids for lung maturity | 3 (3%) | 25 (6%) | 0.24 | |
| Hypertensive disorders during pregnancy | 14 (12%) | 34 (8%) | 0.20 | |
| Physical activity during pregnancy (hours/week) | 1 [0–4] | 1 [1–3] | 0.86 | |
| Delivery | 0.65 | |||
| Vaginal | 63 (55%) | 217 (52%) | ||
| Vacuum-assisted | 7 (6%) | 19 (5%) | ||
| Cesarean section | 45 (39%) | 179 (43%) | ||
| Fever during labor | 9 (8%) | 31 (8%) | 0.84 | |
| Newborn birth weight (grams) | 3250 [3000–3520] | 3150 [2850–3445] | 0.007 | |
| Infant feeding | 0.08 | |||
| Formula milk | 47 (42%) | 211 (52%) | ||
| Breastfeeding | 13 (11%) | 52 (13%) | ||
| Both | 53 (47%) | 144 (35%) |
PCOS Polycystic Ovary Syndrome, BMI Body Mass Index, ART Assisted Reproductive Technology.
Continuous variables are presented as median [interquartile range] and were compared using the Mann–Whitney U test. Categorical variables are expressed as number (percentage) and were compared using the Chi-square test or Fisher’s exact test, as appropriate. A two-sided p-value < 0.05 was considered statistically significant.
Red staining in diapers and neonatal outcomes
Regarding the characteristics of female newborns (Table 1), although low birth weight has been described as a risk factor for the onset of genital crisis17, in our study, birth weight was found to be higher in female newborns with reddish diaper staining than those without it (3.250 kg [3.000–3.520] vs 3.150 kg [2.850–3.445], p = 0.007). On the other hand, breastfeeding showed no significant differences between the two groups.
Red staining in diapers and exposure to EDCs and lifestyle factors in pregnant women
To assess whether exposure to EDCs such as phthalates and phytoestrogens in the lifestyle and diet of pregnant women may have an impact on red diaper staining, questionnaires were distributed to analyze the frequency of consumption of foods like soy (which contains genistein and daidzein, substances structurally similar to estradiol) and the consumption of canned foods, an important source of phthalates (Table 2). Soy consumption, canned food intake, use of plastic containers, hairsprays, and perfumes during pregnancy did not differ between mothers of female newborns with or without reddish staining in diapers.
Table 2.
Exposure to potential endocrine-disrupting factors according to the presence or absence of red staining in diapers of female newborns.
| Red staining (n = 110) | Absence of red staining (n = 401) | p-value | ||
|---|---|---|---|---|
| Consumption of canned food | 0.16 | |||
| Never | 10 (9%) | 65 (16%) | ||
| Daily | 4 (4%) | 7 (2%) | ||
| Weekly | 52 (47%) | 191 (48%) | ||
| Monthly | 44 (40%) | 138 (34%) | ||
| Soy consumption | 0.17 | |||
| Never | 62 (56%) | 179 (45%) | ||
| Daily | 9 (8%) | 37 (9%) | ||
| Weekly | 15 (14%) | 65 (16%) | ||
| Monthly | 24 (22%) | 120 (30%) | ||
| Use of plastic containers for food storage | 0.63 | |||
| Never | 20 (18%) | 96 (24%) | ||
| Daily | 37 (34%) | 131 (33%) | ||
| Weekly | 36 (33%) | 118 (29%) | ||
| Monthly | 17 (15%) | 56 (14%) | ||
| Use of perfumes | 0.39 | |||
| Never | 20 (18%) | 91 (23%) | ||
| Daily | 58 (53%) | 187 (47%) | ||
| Weekly | 24 (22%) | 78 (10%) | ||
| Monthly | 8 (7%) | 45 (11%) | ||
| Use of nail polish | 0.75 | |||
| Never | 31 (28%) | 107 (27%) | ||
| Daily | 23 (21%) | 75 (19%) | ||
| Weekly | 18 (16%) | 58 (14%) | ||
| Monthly | 38 (35%) | 161 (40%) | ||
| Use of hair sprays | 0.55 | |||
| Never | 82 (75%) | 322 (80%) | ||
| Daily | 10 (9%) | 28 (7%) | ||
| Weekly | 6 (5%) | 21 (5%) | ||
| Monthly | 12 (11%) | 30 (8%) | ||
| Use of chemical substances | 3 (3%) | 29 (7%) | 0.11 | |
| High-risk occupational activity | 12 (11%) | 32 (8%) | 0.34 |
Categorical variables are expressed as number (percentage) and were compared using the Chi-square test or Fisher’s exact test, as appropriate. A two-sided p-value < 0.05 was considered statistically significant.
Similarly, occupational exposure was also surveyed. The proportion of women reporting being engaged in potentially hazardous work environments (such as shift work or strenuous work) was similar across both groups.
Red staining in diapers and steroid levels in fetal circulation
Because potential differences in hormone levels between neonates with and without reddish-stained diapers might help clarify factors associated with suspected NUB, we analysed the steroid hormone cascade in 104 umbilical vein blood samples collected from female fetuses at birth. Owing to a higher-than-expected prevalence of NUB, a 1:3 case–control matching ratio was applied instead of the initially planned 1:5 ratio. The differences between women who reported reddish stains in their newborn daughters’ diapers and those who did not are summarized in Table 3. For all steroids evaluated, no significant differences were observed between the two groups.
Table 3.
Umbilical cord steroid hormone levels according to the presence or absence of red staining in diapers of female newborns.
| Red staining (n = 24) | Absence of red staining (n = 80) | p-value | |
|---|---|---|---|
| Aldosterone | 0.34 [0.22–0.48] | 0.34 [0.22–0.59] | 0.87 |
| Cortisol | 31.9 [25.2–47.0] | 34.4 [22.7–45.6] | 0.89 |
| Cortisone | 134.5 [89.0–173.5] | 114.2 [74.4–150.3] | 0.33 |
| 11-Deoxycortisol | 9.2 [7.4–16.9] | 9.5 [7.1–14.3] | 0.99 |
| Corticosterone | 1.8 [1.0–3.9] | 2.3 [1.3–3.9] | 0.40 |
| 11-Deoxycorticosterone | 1.0 [0.6–1.4] | 1.0 [0.7–1.6] | 0.90 |
| DHEAS | 1613 [1143–2307] | 1891 [1161–2618] | 0.54 |
| DHEA | 2.0 [1.4–3.4] | 2.2 [1.5–3.5] | 0.49 |
| Estradiol | 7.1 [5.3–18.7] | 9.3 [5.5–13.8] | 0.72 |
| Androstenedione | 0.7 [0.5–1.0] | 0.2 [0.5–0.9] | 0.61 |
| Testosterone | 0.06 [0.03–0.09] | 0.06 [0.04–0.10] | 0.55 |
| 17-OHProgesterone | 29.1 [23.6–47.2] | 27.7 [20.2–37.8] | 0.22 |
| Progesterone | 614.9 [522.5–806.2] | 615.5 [504.5–752.9] | 0.67 |
DHEA Dehydroepiandrosterone, DHEAS Dehydroepiandrosterone sulfate;
Continuous variables are presented as median [interquartile range] and were compared using the Mann–Whitney U test. A two-sided p-value < 0.05 was considered statistically significant.
Microscopic analysis of red stains in diapers of male and female neonates
To assess whether the excretion of urate crystals could represent a confounding event, during the study, our analysis was expanded to include male infants. The prevalence of reddish stains in the diapers was reported in n = 110 male neonates, yielding a frequency of 13% (95% CI: 8–20%). A representative sample of reddish stains in a male neonate is shown in Fig. 1C.
The collection of diapers containing reddish stains allowed us to apply specific analyses to detect the presence of blood and determine its origin, as well as to perform cell characterization. Using microscopic evaluation analysis and the SERATEC PMB Test, peripheral blood was detected in 8/78 (10.3%) female and 7/68 (10.3%) male samples (Fig. 1). Among the female samples, two were positive for menstrual blood at the SERATEC PMB Test (Fig. 1A), suggesting that 2.5% of the examined reddish stains could be attributed to uterine bleeding.
In blood-positive cases at SERATEC PMB test, light microscopic analysis showed that no cells other than erythrocytes or desquamated epithelial cells were present in any diapers of female newborns. The presence of urate crystals was observed microscopically in red-coloured diapers of both males and female when blood was not detected by SERATEC PMB test (Fig. 1B–C, respectively).
Discussion
The issue of NUB prevalence
The NUB has been a topic of interest to writers, physicians, midwives, and even philosophers for over a millennium and has long been misinterpreted24. Initially, there was confusion between neonatal bleeding, early menstruation due to early puberty, and bleeding caused by disease or even malignant influences. The first scientific publication on this subject was in 1822, and since then, scientific descriptions of what is now called “neonatal menstruation”, “neonatal uterine bleeding” or “genital crisis” began to appear.
Interest in the phenomenon was recently renewed when a new theory suggesting a link between NUB, accelerated endometrial maturation, and endometriosis1,14. In this regard, a report from a workshop held by the World Endometriosis Society (WES) and the World Endometriosis Research Foundation (WERF) recommended that medical practitioners record the presence of the NUB, identifying it as a potential early-life biomarker for endometriosis25.
In our study, the presence of red colored discharge in the diapers, perceived by mothers as NUB, was found in 22% of cases (95% CI 18–25).
Screening methods used in previous studies to evaluated NUB included postmortem histological examinations of the uterus16,26, hemoglobin detection tests27, genital examinations, colposcopic examinations, vaginal smears and real-time ultrasonography1. It is highly plausible that part of these suspected cases of NUB in previous and in this study were actually due to the excretion of reddish uric acid crystals in urine, which are difficult to distinguish from blood (Fig. 1 versus 2–3). A surprising limitation of previous studies is the failure to considered the potential confounding effect of red uric acid crystals in urine. This is the reason why to elucidate the origin of stains in diapers reported by mothers, we subsequently expanded our analysis to include male infants. To the best of our knowledge, this represents the first study to put into question the true nature of the red discharge found in diapers. Notably, 13% (95% CI 8–20%) of mothers of male infants contacted a few days after delivery described red spots in their son’s diaper. According to the literature, uric acid crystals can be observed in the urine of 22% of all infants (males and females) in their first week of life, especially if they are breastfed21,22. This data is fully consistent with the result of our observed of 22% rate of red staining in female neonates.
Moreover, light microscopy analysis conducted on diapers in our study confirmed the presence of urate crystals in diapers from both male and female newborns (Fig. 1). This finding, together with the microscopic absence of endometrial cells in female diapers, supports the hypothesis that many cases of reddish staining in female diapers have been misinterpreted as NUB, both in the past and present studies.
The use of SERATEC PMB Tests showed negative results in the large majority of the studied population, which is noteworthy considering the high analytical sensitivity of the assay. Among the few positive cases, the test revealed the presence of peripheral blood in equal proportions among red-stained diapers from both male and female newborns. The detection of peripheral blood in both female and male neonates warrants cautious interpretation. The presence of blood in male infants clearly indicates that not all reddish stains originate from the genital tract. Possible sources include minor perineal or urethral irritation, superficial skin lesions, microtrauma related to diaper friction, or irritation secondary to urate crystal passage. Residual maternal blood contamination or minor umbilical stump bleeding cannot be entirely excluded28
Interestingly, Ogawa and coworkers recently considered alternative sources of NUB28. They analyzed 15 postmortem neonatal endometria for several biological markers, including endometrial stem cells (eMSCs), showing an absence of epithelial/stromal cells in the endometrium and eMSCs in the NUB. They even suggested that the possibility that some NUB cases could originate from the neonatal vaginal wall. On the other hand, they did not consider the possibility that the bleeding could originate from the newborn urinary tract.
Predictive factors of red staining in diapers
According to the current literature, NUB appears to be more common in post-term neonates, possibly due to postulated greater exposure to maternal hormones and more mature endometrial development. In contrast, preterm infants seem to have a lower prevalence of NUB, likely due to limited hormone exposure and immaturity of the endometrium. In addition, obstetric complications such as preeclampsia, hypertensive disorders, and feto-maternal blood group incompatibility, as well as neonatal factors such as low birth weight, have been associated with a higher prevalence of NUB1,29. These data indicate that hormonal influences, developmental factors and pathological conditions may play a crucial role in the occurrence of NUB12.
In our study, there were no significant differences between mothers who reported red staining in diapers and those who did not in terms of maternal characteristics or obstetric and newborn outcomes with the exception of pre-pregnancy BMI, post-pregnancy BMI, newborn birth weight, and time to conceive. The longer time to conceive in the group that reported red staining in diapers could represent a type I error as the difference between the two groups is minimal (therefore devoid of clinical significance). Additionally, the isolated finding of a higher maternal BMI, although statistically significant, is not useful evidence: this is a factor that could be associated with both NUB (different estrogen concentrations in pregnancy) and urate crystals exertion (different nutrition). To note, while BMI was higher, the increase in BMI during pregnancy did not differ between groups.
Although the literature suggests low birth weight as a risk factor for the onset of NUB, in our study newborns with reddish spots in diapers showed higher birth weight than those without such spots. Interestingly, low birth weight appears to be associated with a higher incidence of uric acid crystals in urine21,22, supporting the hypothesis that some cases interpreted as NUB by mothers might be due to the presence of uric acid in urine rather than true menstrual-like bleeding.
Steroid hormone levels in the umbilical cord blood and their impact on red staining in diapers
The phenomenon of NUB is thought to be caused by the sudden drop in steroid hormone levels occurring after the detachment of the placenta18. Although this is a common physiological process in all female neonates, this hormonal shift is thought to trigger NUB in only a small subset of them. After delivery, steroid hormone production transitions from the placenta to the neonate’s ovaries and subsequently to adrenal glands, resulting in a massive reduction of hormones thought to provoke the shedding of the endometrial mucosa and to cause menstrual-like bleeding.
Our analysis performed by LC–MS/MS allowed the precise evaluation of ovarian steroid hormones in cord blood of the female neonates. Even for the hormonal assessment, our analysis found no significant difference in umbilical cord blood steroid hormone levels in women who reported reddish leaks in newborn diapers and those who did not. This result reinforces the conclusion that the reddish spots in diapers traditionally interpreted as NUB may not be a consequence to a uterine bleeding.
Strengths and limitations of the study
Our study has several strengths compared to previous literature. First, its prospective design in nature is ideal for evaluating a phenomenon in close temporal proximity to recruitment. In addition, recruitment with follow-up was performed by the same trained operators throughout the duration of the study, ensuring more consistent and accurate data collection. The same operators also personally assessed the diapers under observation in the postpartum ward. Second, the present study is the first to consider the presence of urate crystals in urine as a potential confounding factor in the interpretation of NUB. Finally, it should be noted that the recruitment of male infants with reddish diapers staining during follow-up represents a unique methodological addition, reinforcing the hypothesis that genital crisis is highly overestimated.
The study also has some limitations. First, the follow-up was conducted mainly through telephone interviews in a single center. Women reported the presence of any blood leaks on diapers without these being systematically verified by the operators. As a result, the assessment of staining was subjective, potentially contributing to overestimation of genital crisis in the early phase of the study. However, the microscopic analysis together with the use of SERATEC PMB Test to detect menstrual blood on newborn diapers allowed an accurate characterization of reddish diaper stains. Moreover, diaper collection was not systematic across all cases of reported staining, which may have affected representativeness.
Second, the recruitment method was modified during the course of the study when we realized the potential impact of the urate crystals in urine as a potential confounder, thus not allowing for the same statistical power with regard to male infants and the diaper study. In addition, the SERATEC PMB Test, which is used in forensics to distinguish menstrual blood from peripheral blood was a particularly useful tool in the study of stains origin in diapers, however, its diagnostic validity in neonates remains unconfirmed. Therefore, some degree of misclassification cannot be entirely excluded, including the possibility of false-positive results among the two cases classified as menstrual blood. It should also be emphasized that the number of SERATEC-positive cases was extremely small, which further limits the strength of any inference regarding true prevalence of NUB. Nevertheless, it is noteworthy that all cases with negative SERATEC results showed concordant findings on optical microscopy, with no erythrocytes detected. This concordance, together with the high analytical sensitivity of the test, further supports the reliability of the negative results observed in the present cohort. Moreover, the high absorbency of diapers and the potential for dehydration prior to analysis may have influenced crystal morphology and the preservation of blood components, possibly affecting microscopic interpretation and biochemical detection. Nonetheless, the concordance between biochemical testing and microscopic evaluation supports the internal consistency of blood detection in our samples.
Moreover, the cellular characterization of the diapers’ content was performed exclusively through optical microscope analysis, without the use of more specific methods. Nevertheless, epithelial cells were found to be very rare in the analyzed slides.
Conclusions
The theory that NUB may represent the first retrograde menstruation episode, contributing to the pathogenesis of endometriosis before menarche is intriguing but presents significant limitations. Our study contributes to an extremely heterogeneous body of literature, highlighting that defining the real prevalence of NUB remains one of the most challenging aspects. Throughout the project, difficulties characterizing the staining in diapers, combined with the presence of another phenomenon virtually indistinguishable from NUB (the presence of urate crystals in urine which has not been previously considered in the literature), along with the lack of differences in the physical characteristics of the enrolled pregnant women, obstetric and neonatal outcomes, and steroid concentrations in the umbilical cord of the newborns, raise serious concerns regarding the true prevalence of NUB. Our findings suggest that confirmed menstrual blood may be considerably less frequent than previously reported. To the best of our knowledge, this is the first study to systematically consider uric acid crystals as an important confounder of neonatal uterine bleeding, potentially challenging previous literature and then, the theory linking NUB to endometriosis. This underscores the need for a novel investigative approach to accurately determine the true nature and clinical significance of reddish spots in diapers of both male and female infants. Such an approach could clarify the actual existence of NUB and, if so, its potential association to the development of endometriosis.
Author contributions
G.F., P.Vi, E.S. and L.B. conceived the study and drafted the initial manuscript. G.F., I.M., G.D.S., C.F., S.F., E.P., E.I., M.O., I.P. and L.C. contributed in data collection. G.F., I.M., G.D.S., M.C., C.F. P.Vi, P.Ve, E.S. and L.B. contributed to data interpretation and manuscript writing. G.F., P.Vi, P.Ve, E.S. and L.B. critically revised the final version of the manuscript. All authors read and approved the final manuscript.
Funding
This research was funded by the Italian Ministry of Health (ENDO-2021-12371933) following peer review in the competitive grant “Bando Endometriosi 2021”.
Data availability
The datasets generated and/or analysed during the current study are available from the corresponding author upon reasonable request. Individual participant data are not publicly available in order to protect participant confidentiality. The anonymized dataset supporting the findings of this study, together with the analytical code used for statistical analysis, can be shared with qualified researchers for non-commercial academic purposes upon approval by the Ethics Committee of the Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan.
Declarations
Competing interests
P.Ve. is a member of the Editorial Board of Human Reproduction Open, the Journal of Obstetrics and Gynaecology Canada, and of the International Editorial Board of Acta Obstetricia et Gynecologica Scandinavica; has received royalties from Wolters Kluwer for chapters on endometriosis management in the clinical decision support resource UpToDate; and maintains both a public and private gynaecological practice. E.S. is Editor-in-Chief of Human Reproduction Open; discloses payments from Ferring for research grants and honoraria from Merck-Serono for lectures; and maintains both a public and private gynaecological practice. P.Vi. is Co-Editor-in-Chief of Journal of Endometriosis and Uterine Disorders. M.O. became a member of the Editorial Board of Scientific Reports after the submission of this manuscript. All other authors declare they have no conflict of interest.
Footnotes
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The datasets generated and/or analysed during the current study are available from the corresponding author upon reasonable request. Individual participant data are not publicly available in order to protect participant confidentiality. The anonymized dataset supporting the findings of this study, together with the analytical code used for statistical analysis, can be shared with qualified researchers for non-commercial academic purposes upon approval by the Ethics Committee of the Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan.