Microbiota transplantation in restoring cesarean-related infant dysbiosis: a new frontier

ABSTRACT

C-section is crucial in reducing maternal and neonatal mortality when medically indicated, but one of its side effects could be the disruption of vertical transmission of maternal-infant microbiota during delivery, potentially leading to gut dysbiosis and increased disease risks in C-section infants. To address such dysbiosis, it seems reasonable to supplement “what is missing” during C-section procedure. This idea has prompted several clinical trials, including proof-of-concept, investigating interventions like vaginal microbial seeding, oral administration of maternal vaginal microbes and even oral administration of maternal fecal materials. Hereby, we have summarized these trials to help understand the current state of these researches, highlighting the predominantly pilot nature of most of these studies and emphasizing the need for well-designed studies with larger sample to guide evidence-based medicine in the future.

GRAPHICAL ABSTRACT

PUBLIC SUMMARY

• C-section is associated with gut dysbiosis in CS infants and increased disease risks from childhood to adulthood.

• Apart from using traditional probiotics to restore CS-related dysbiosis, a new research direction is to investigate the potential of mimicking natural inoculation process would alleviate infant gut dysbiosis.

• Several small-scale studies have shown that transplanting maternal vaginal or even fecal microbiota might restore CS-related infant dysbiosis. Controversy remains regarding the clinical applicability, safety, efficacy and mechanisms of these approaches.

KEYWORDS:

• Microbiota transplantation
• Vaginal microbial seeding
• Gut microbiota
• C-section
• Infant

Introduction

The global incidence of cesarean sections (CS) has reached 21.1% in 2015 and continues to rise unabated. Projections indicate that by 2030, the global CS rate will approach a staggering 30%, surpassing the World Health Organization’s recommended range of 10–20% for CS.¹ While CS undoubtedly provides an effective approach to reduce maternal and neonatal mortality, as well as mitigate complications associated with pregnancy and childbirth, such as pre-eclampsia, placental abnormalities, birth defects, and dystocia, it is important to acknowledge that CS has the potential to disrupt the natural colonization of the neonatal microbiota originating from the maternal birth canal. Consequently, this disruption can lead to early gut dysbiosis in CS infants.²,³

Furthermore, compared with their counterparts born via vaginal delivery (VD), infants delivered via CS is associated with short-term and long-term effects, including elevated risk of developing neurological disorders,⁴ chronic immune diseases,^5,6 obesity ,^7–9 allergic disease,^10,11 type 1 diabetes¹² and inflammatory bowel disease.^13,14 Although certain postnatal interventions, such as breastfeeding¹⁵ and probiotics,¹⁶ have shown some efficacy in mitigating the disparities arising from the mode of delivery, it remains evident that CS infants are deprived of the crucial microbial contact inherent in the passage through the birth canal. Therefore, in 2012, Dominguez-Bello put forth the concept of ecological approaches, including “microbial bathing,” as a means to enable CS infants to mimic the experience of vaginal delivery by facilitating contact with maternal vaginal fluids. Subsequently, a series of studies utilizing microbiota transplantation methods have been published, with the aim of restoring the early perturbed gut microbiota in CS infants and ultimately enhancing their overall well-being. Hereby, we offer a comprehensive overview of these ecological interventions designed to rectify infant gut dysbiosis, thereby fostering a more holistic understanding of their current status.

Swabbing the C-section infants using vaginal fluid

Infants delivered vaginally are exposed to maternal vaginal fluid, while infants born via C-section may miss out on this initial colonization process. Therefore, the concept of simulating a natural delivery process through vaginal microbiota seeding (VMS) has emerged as a method to restore the disordered gut microbiota in CS infants. The pioneering study by Dominguez-Bello in 2016 was the first to propose VMS as an intervention.¹⁷ This study included 18 infants and their mothers, consisting of 11 VD infants and 7 planned CS infants, of which 3 CS infants received no intervention and 4 infants received VMS. During the VMS procedure, a sterile gauze was inserted into the mother’s vagina prior to CS delivery and then used to swab the baby’s mouth, face, and rest of the body. In comparison to CS infants, the gut microbial composition of VMS infants was more similar to that of VD infants, particularly in samples taken from the anal and skin. For example, in anal samples from VMS and VD infants, the study observed an early enrichment of Lactobacillus followed by a bloom of Bacteroides from week 2. Furthermore, the study also analyzed the bacterial diversity and relative abundance of the vaginal microbiota in mothers who received antibiotics and those who did not. The results showed no significant differences, suggesting that residual antibiotics on the swabs were unlikely to have caused the differences in infant microbiota between the intervention and control groups. Overall, this seminal study provides the first scientific report on the VMS technique and explores its potential for partially restoring the gut microbiota of CS infants, garnering considerable attention within the academic community.

In 2021, Dominguez-Bello’s group reported a larger-scale study to assess the long-term effects of VMS following CS delivery on the naturalization of microbial developmental trajectory in CS infants within the first year of life.¹⁸ The study included a total of 177 infants, divided into three groups: 98 infants in the VD group, 30 infants in the VMS group, and 49 infants in the CS group. Samples were collected from multiple body sites within hours to one year after birth. The results showed that the microbial developmental trajectory of VMS infants exhibited closer resemblance to that of VD infants, especially in fecal and skin. Importantly, VMS effectively supplemented the bacterial taxa missing in CS infants, notably Bacteroides. Furthermore, the study observed an interesting phenomenon that vaginal microbiota diversity increased on the day of delivery along with the presence of bacteria from other body sites, which was not observed in non-pregnant women. Although the underlying mechanisms for this phenomenon remain unclear, it suggests that VMS may involve not only vaginal microbiota but also microbiota from other body sites, such as gut microbiota. This intervention study expands on the findings of previous smaller studies and further demonstrates that the microbial differences associated with delivery mode can be reduced through VMS.

In 2023, Zhou et al. reported the first blind randomized controlled trial to evaluate the effects of VMS on neurodevelopment, as well as the gut microbiome and metabolome.¹⁹ The study enrolled a total of 76 infants, who were randomly assigned to three group: the VMS group (n = 35), the CS group (n = 41), and an additional reference control group of 33 VD infants. Vaginal samples were collected from all mothers and stool samples were obtained from the infants at four time points (days 3, 7, 30 and 42). Neurodevelopmental assessments were conducted using the babies’ Ages and Stages Questionnaire, Third Edition (ASQ-3), with total score and subdomain scores at 3 and 6 months. The safety of VMS was evaluated as the primary outcome, and it was found that the incidence of adverse events were not significantly different between the VMS and control group. Interestingly, infants in the VMS group showed significantly higher total ASQ-3 scores at 3–6 months compared to the CS group, representing an approximate 10% increase. Furthermore, VMS was found to promote the maturation of the gut microbiota in CS infants within the 42 days of life, bringing it significantly closer to that of VD infants. The effect was characterized by an increase in beneficial bacteria such as Lactobacillus, Bifidobacterium, and Escherichia, as well as a reduction in opportunistic pathogens including Klebsiella. Additionally, VMS increased the levels of 15 gut metabolites, including L-lactic acid, homovanillic acid, and indolelactic acid, which were reported to associate with mental disorders.²⁰ These results suggest that VMS might affect neurodevelopment in CS infants through the modulation of the gut microbiota and related metabolites. Importantly, this study is the first to employ a blinded design to investigate the effects of VMS and has identified phenotypic differences. However, it also highlights the need for future research with hard clinical endpoints and expanded sample sizes studies to validate these findings.

Notably, the trials on VMS do not always show beneficial effects on the infants. In 2022, Liu et al. reported a randomized controlled trial to investigate the effects of VMS on gut microbiota, growth, and allergy risk in CS infants.²¹ The study included 117 mother-infant pairs, with 57 assigned to the VMS group and 60 to the control group. Meconium samples were collected from all infant, and measurement of their length and weight were taken. Fecal samples were collected at 6, 12, 18, and 24 months and allergen-specific IgE levels were assessed in infant serum at 18 months. The results revealed no statistically significant differences between the two groups in terms of BMI, BMI z-scores, total allergy risk, experienced allergic symptoms, or diagnosed with allergic diseases at any of the four time points assessed(6, 12, 18, and 24 months). Furthermore, the relative abundance of dominant phyla (Proteobacteria, Firmicutes, Bacteroidota, Actinobacteriota) in both meconium and fecal samples were similar between the two groups at all four time points, and there were no significant differences observed in microbial community richness or diversity. However, at 6 months, a slightly higher relative abundance of genera Lactobacillus and Bacteroides was observed in the VMS group. The researchers suggested that there may be differences during the early critical window that they did not include in their study design. Additionally, factors such as the preparation and testing of maternal vaginal fluids gauze, infant feeding mode, and the impact of COVID-19 pandemic might also affect their results.

Oral administration of maternal vaginal microbes

During the process of vaginal delivery, infants might also ingest their mother’s vaginal fluid. Based on this rationale, Wilson et al. reported in 2021, that using a single-blind, randomized, placebo-controlled trial to assess whether oral administration of maternal vaginal fluids could alter the gut microbiota of CS infants.²² After birth, CS infants were randomly assigned to receive either 3 ml of processed vaginal solution (CS-seeded, N = 12) or sterile water (CS-placebo, N = 13), while VD infants served as the reference group (VD, N = 22). To prepare the vaginal solution, sterile gauze was inserted into the mother’s vagina for 30 minutes prior to the CS. Upon removal, half of the gauze was placed into a 10 ml sterile syringe, and five ml of sterile water was aspirated and passed through the gauze 20 times. Subsequently, three ml of the solution was administered into the newborn’s mouth to trigger the swallowing reflex. Meconium samples were collected after birth, and anthropometric measurements and fecal sample collection were conducted at one month and three months. Body growth and composition were measured at three months. Even though the trial did not report any adverse events, it did not reveal any significant differences in the fecal microbiota composition between the intervention and control groups. Furthermore, the study found limited colonization of the infant’s gut by maternal vaginal microbiota, with only four CS-seeded infants having maternal strains present in their gut at one month, and only one CS-seeded infant still having maternal strains (Bifidobacterium breve) at three months. Regarding growth outcomes, no differences in anthropometric measurements or body composition were observed between the three groups of infants at one month or three months. And the author claimed that the small sample size and dilution factor applied to the vaginal fluid might have limited the ability to interpret their results. Even though in a mouse model, Jasarevic et al. reported in 2021 that administering different maternal vaginal microbiota to newborn mice via gavage resulted in long-lasting effects on growth, circulating immunity, and hypothalamic transcriptional profiles.²³

Oral administration of maternal fecal microbiota

In fact, most women experience some degree of fecal elimination during delivery,²⁴ which may also be a potential source of microbial vertical transmission. In 2020, Korpela et al. published a proof-of-concept study to evaluate whether postnatal oral administration of fecal microbiota transplantation(FMT) could restore gut dysbiosis in CS infants.²⁵ The study included seven mother-infant pairs who received the intervention, which consisted of mixing one ml of pre-processed fecal material with 10 ml of breast milk and administering a dose of five ml(except for one infant who received 10 ml) as the first feeding within two hours of birth. Meconium samples and subsequent fecal samples were collected weekly until three months after birth. Apart from three neonates reporting mild gastrointestinal symptoms, no other treatment-related symptoms, illnesses, or need for medical appointments were reported, suggesting that the FMT procedure may be safe. A highly promising finding was that following this intervention, the infants’ gut microbiota rapidly became dominated by Bacteroides and Bifidobacterium (two days after birth), making it more similar to VD infants than non-intervened CS infants. Furthermore, to further evaluate the effects of FMT compared to VMS, the study incorporated publicly available microbiota datasets, including 163 VD infants, 38 CS infants and six infants who received VMS. The results showed that the microbiota of FMT infants clustered together with that of VD infants, but non-intervened CS infants or VMS infants. Based on these findings, the authors concluded that FMT could be a safe and more effective approach than VMS in restoring the gut microbiota development in CS infants. However, in this study, breastmilk was used to dilute the samples, whereas other studies utilized saline solution. Therefore, the observed restoration of microbiota in the intervention group may be attributed to the combined effects of the fecal samples and breastmilk. In a mouse model, Cerdó et al. reported in 2023 that fecal transplants from infants with higher composite cognitive scores into GF mice resulted in improved memory functions. ²⁶ Additionally, similar to the findings observed in human infants in this study, the gut microbiota was found to modulate the histidine metabolome in mice, indicating that the regulation of histidine metabolome levels could potentially be a mechanism through which the gut microbiota influences neurodevelopmental function.

Controversies

Firstly, although some short-term studies show promise in addressing gut dysbiosis in infants delivered via C-section (Table 1), the long-term effects remain uncertain due to a lack of comprehensive follow-up data. Secondly, there is ongoing debate regarding the long-term safety of these intervention methods. The primary focus is on potential risks such as infection transmission, immune reactions, and other unknown risk factors. Despite the existing research having rigorous inclusion criteria (Table 2), both fecal and vaginal fluids can potentially carry pathogens, necessitating rigorous pathogen screening prior to the implementation of these interventions. More importantly, even though using the same intervention method, there can be variations in the operational methods and procedures across different studies, making it difficult to compare and replicate research findings. Standardizing the operational methods and procedures of these methods is crucial to ensure the reliability and reproducibility of results. Additionally, geography and race have been identified as baseline determinants of microbial community composition in both the vagina²⁷ and gut.²⁸ Therefore, the widespread application of these standardized interventions in different regions and populations remains a significant challenge. Currently, the mechanisms underlying the effects of these interventions are still unclear, and the key microbes and metabolic pathways involved are yet to be identified. Furthermore, the success of these interventions is influenced by multiple factors, including recipient factors, administration route, timing, dosage and frequency of administration, as well as post-intervention diet and medication.²⁹

Table 1. Safety and efficacy of the included studies.

Study	Country	Intervention method	Sample sizes	Major adverse events n/N(%)				Efficacy
				Adverse events	Serious adverse events	Adverse events	related to the intervention
Dominguez-Bello et al.,^Citation17 2016	USA	VMS	1) VD group:11 2) CS group:3 3) Intervention group:4	Not reported	Not reported	Not reported	Not reported	1) The proportions of different maternal microbiome in the anal, oral, and skin samples of VMS infants are more similar to those of VD infants compared to CS infants. 2) The microbiome composition of VMS infants is more similar to that of VD infants compared to CS infants.
Song et al.,^Citation18 2021	USA, Chile, Bolivia, Spain	VMS	1) VD group:98 2) CS group:49 3) Intervention group:30	Not reported	Not reported	Not reported	Not reported	1) The VMS infants led to a developmental trajectory that more closely resembled that of VD infants most prominently in feces and skin. 2) The VMS procedure was able to establish microbes missing in CS born babies. 3) In the initial few days, a majority of the microbiota in the infant’s body is shared with corresponding maternal sites, regardless of birth mode or seeding status.
Zhou et al.,^Citation19 2023	China	VMS	1) VD group:33 2) CS group:41 3) Intervention group:35	None	VMS:4/32(12.5) CS:5/36(13.9)	Papules, erythema, pustule, cough and nasal congestion, fever	No	1) Compared to the infants in the CS group, the infants in the VMS group showed a significant improvement in ASQ-3 scores. 2) VMS procedure can accelerate the maturation of the gut microbiota in cesarean-born infants, making it more similar to that of VD infants, and this rate of maturation is correlated with higher ASQ-3 scores. 3) VMS may affect neurodevelopment through gut microbiota species and metabolites.
Liu et al.,^Citation21 2021	China	VMS	1) CS group:60 2) Intervention group:57	Not reported	VMS:4/53(7.5) CS:6/64(9.4)	Mild asphyxia, wet lung, jaundice, renal pelvis separation	No	1) There were no significant differences in infants BMI and BMI z-score between groups. 2) There were no significant differences in median total allergy risk, experienced allergic symptoms, or diagnosed with allergic diseases between two groups. 3) The gut microbiota community structures and relative abundance of dominant phyla were similar between the two groups of infants, and there were no significant differences in microbiota richness.
Wilson et al.,^Citation22 2021	New Zealand	Oral administration of maternal vaginal microbes	1)VD group:22 2)CS group:13 3)Intervention group:12	None	CS-seeded:7/12 CS:6/13	Neonatal hypoglycemia, neonatal care unit/hospital admission, colic, reflux	No	1) The group did not observe any difference in the microbial composition of stools collected from CS infants who received vaginal seeding compared to those receiving the placebo at either 1 month or 3 months of age and Microbial alpha diversity and gene richness were similar between the three infant groups. 2) In total, 20 microbial pathways were more abundant in VB infants when compared to CS infants at either 1 month or 3 months of age and for the majority of differentially abundant pathways (14/20), Bacteroides spp. were responsible for the increase in pathway potential. 3) The colonization of infant gut by maternal vaginal microbiota is limited, By 3 months of age, only one of these maternally- derived strains was still present in the gut of a CS-seeded infant.
Korpela et al.,^Citation24 2020	Finland	FMT	1)VD group:163 2)CS group:38 3)VMS group:6 4)Intervention group:7	Not reported	Not reported	Not reported	No	1) The microbiota in FMT-treated infants’ samples was found to be very different from the microbiota of the mothers who donated the transplant, indicating highly selective outgrowth upon transplantation. 2) FMT treatment changed the microbiota of CS- born infants in such a way that the temporal development of all relevant groups resembled that of vaginally delivered infants. 3) Maternal FMT but not Vaginal Swabbing Restored the Microbiota in CS-Born Infants.

Table 2. Exclusion and inclusion of the included studies.

		Study
		Dominguez-Bello et al,^Citation17 2016	Song et al,^Citation18 2021	Zhou et al,^Citation19 2023	Liu et al,^Citation21 2021	Wilson et al,^Citation22 2021	Korpela et al,^Citation24 2020
Exclusion criteria	HIV	√	√	√	√	√	√
	Chlamydia	√	√	√	√	√
	Group B Streptococcus	√	√	√	√	√	√
	Genital herpes			√	√
	Treponema pallidum			√		√	√
	Gonorrhea			√		√
	Human papillomavirus			√		√
	Trichomonas			√		√
	Bacterial vaginosis			√		√
	Vulvovaginal candidiasis			√
	Hepatitis A					√	√
	Hepatitis B			√	√	√	√
	Hepatitis C			√		√	√
	Hepatitis E						√
	Vaginitis				√
	Herpes simplex viruses					√
	Candida albicans					√
	Human T-cell lymphotrophic virus						√
	Severe obstetric complications			√
	Serious physical diseases			√
	Infectious diseases			√
	Communicable diseases			√
	SARS-CoV-2			√
	Used drugs/abused alcohol			√
	Were assisted in becoming pregnant			√
	Serious mental illness/psychological disorders			√
	Apgar scores were less than seven at 1, 5 and 10 minutes after birth evaluated by neonatologist			√
	Any congenital anomalies or intrauterine infections at birth			√
	Spontaneous rupture of membranes					√
	Maternal antibiotic treatment within 3 months of delivery						√
	Any travel outside the EU within the last 3 months						√
	CS after the onset of labor						√
	Protozoa						√
	Helminths						√
	Entamoeba histolytica						√
	Clostridium difficile						√
	Enteric pathogens						√
	Helicobacter pylori						√
	Norovirus						√
	Giardia lamblia						√
	Cryptosporidium parvum						√
	Methicillin-resistant Staphylococcus aureus						√
	Gram-negative multidrug-resistant bacteria						√
	Vancomycin-resistant enterococci						√
Inclusion criteria:	Vaginal pH < 4.5 at 1–2 h preceding the procedure	√	√		√
	Healthy pregnancy	√	√				√
	Non-obese mothers with no pregnancy complications		√
	Had intact amniotic membranes at the time of delivery		√
	No signs of vaginosis or viral infections as determined by their obstetrician	√	√
	Aged 18 to 45 years			√
	Singleton term			√	√		√
	Gestational weeks ≥ 37			√	√
	Had no evidence of bloody appearance or premature rupture of the membranes			√
	Women or their spouses had severe allergic diseases and conditions not suitable for participation				√
	Healthy, term-born, normal-weight breastfed infants with no antibiotic exposure, except for those associated with cesarean section, were selected as controls.						√

Perspectives

Several small-scale population studies suggest that microbial community reconstruction through interventions such as VMS, oral administration of maternal vaginal microbes, and FMT may potentially improve the gut microbiota, gut metabolism, and even phenotypic outcomes in CS infants. The results of several VMS interventions have shown inconsistent findings, with the predominant bacteria in the infant gut not being derived from the vaginal microbiota. This suggests that the influence of vaginal microbiota on newborns may be indirect. In contrast, administrating mother’s feces showed more direct effects in restoring the gut dysbiosis in CS infants. However, it is unclear if the adult microbiota is compatible for their infants regarding early-life development. Whether using the gut microbiota from VD infants as donors to restore CS infants’ dysbiosis remains an interesting question. Overall, based on the positive findings from the existing studies, it is warranted for further research with expanded sample sizes in human populations, as well as investigations into the underlying mechanisms involved.

Currently, several large-scale clinical trials are underway. For example, one clinical trial in the United States (NCT03298334) is investigating the effects of swabbing vaginal fluid on health outcomes in CS infants over a three-year period, including microbial development, immune development, metabolic outcomes, and any adverse events. A French clinical trial (NCT02567071) is evaluating the effect of a seeding method, in which newborns suck on gauze soaked in maternal perineal and vaginal secretions after cesarean delivery, on the infant gut microbiota diversity and their health status during the first year after birth. Additionally, a Finnish clinical trial (NCT04173208) is assessing the influence of FMT on the trajectory of gut microbiota development during the first year after birth, as well as evaluating markers of atopic-related diseases and changes in immunomarkers. These larger-scale studies will provide stronger evidence to help guide potentially implementation of these techniques in clinical usage. Mechanism studies are also warranted to understand the microbiota succession, development and their impact on infant phenotypes to help develop more precise interventions.

Author contributions

Y.H and R.H.X provided direction and guidance throughout the preparation of this manuscript. S.N.L collected and interpreted studies and was a major contributor to the writing and editing of the manuscript. X.X.L reviewed and made significant revisions to the manuscript. L.P.Z collected studies. All authors read and approved the final manuscript.

Acknowledgments

We express our gratitude to the participants and their families who contributed to this study. We also extend our appreciation to the funding agencies whose support made this research possible. Specifically, we acknowledge the National Key R&D Program of China (2019YFA0802301 [to Y.H.]) and the National Natural Science Foundation of China (NSFC82022044 [to Y.H.] and NSFC82272391 [to Y.H.]). Their contributions were instrumental in advancing our research endeavors.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The work was supported by the National Key Research and Development Program of China [2019YFA0802301 [to Y.H.]]; National Natural Science Foundation of China [NSFC82022044, NSFC82272391].