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Introduction

Obstetric anal sphincter injuries (OASIs) are a severe complication of vaginal delivery that can lead to considerable morbidity and severely diminished quality of life in a substantial

0.1% and 34.9% among studies and countries, depending on the population number of women [1]. Despite surgical repair, a considerable number of women continue to experience some degree of anal incontinence that may lead to distressing and disabling complaints, and accompanying social and hygienic problems, isolation, lowered self-esteem, negative effects on sexual function, and diminished quality of life [1-4].

The prevalence of OASI varies between examined, parity, type of episiotomy used, and incidence of operative vaginal delivery [5-8]. Although most risk factors are associated with the mechanical forces encountered during vaginal birth, OASI is usually unpredictable and is currently unpreventable, because it is associated primarily with non-modifiable risk factors [9, 10].

A tendency toward OASI occurrence, unrelated to mechanical forces, had been reported within families. The Medical Birth Registry of Norway has reported that OASI risk increases if a woman’s mother or sister experienced OASI during a vaginal birth [11], thus suggesting that a genetic tendency might predispose individuals to this complication. However, the genetic predisposition to OASI, if any, had not been investigated.

Pelvic organ prolapse (POP), a connective tissue disorder, has been associated with a subset of single-nucleotide polymorphisms (SNPs) in connective tissue genes [12-19]. OASI, similarly to POP, results from the same mechanical forces during vaginal birth on the same pelvic muscles and connective tissue, thereby potentially affecting the pelvic floor and leading to similar symptoms of anal incontinence. The hypothesis in this study was based on the assumption that the genetic variations in connective tissue identified in women with POP might also contribute to OASI development. Accordingly, we selected ten SNPs known to have the strongest associations with POP, prioritizing those located within genes involved in extracellular matrix remodeling and connective tissue integrity. By examining the frequency of these specific variants among women who experienced OASI, we aimed to bridge the genetic gap between pelvic floor relaxation and acute birth trauma. Identifying these associations may improve OASI risk stratification prior to a trial of labor, potentially facilitating targeted prevention strategies in high-prevalence regions.

Material and Methods

This prospective case-control study was conducted at a university teaching hospital from October 2020 to December 2023. The institutional review board approved the trial protocol on August 31, 2020 (#0147-19-EMC). Informed consent was obtained from all participants in the study. The study was registered at ClinicalTrials.gov (identifier NCT04047433).

The study cohort included primiparous women with a vaginal delivery. The study group comprised women diagnosed with 3^rd or 4^th degree perineal tear (OASI) after a vaginal birth, whereas the control group comprised women who had a vaginal birth without OASI. The control group was matched to the study group by age and date of delivery in a 1:2 ratio. OASIs were diagnosed and graded according to a standardized classification of perineal lacerations [20] by a physician in the delivery ward. The diagnosis and grade of the tears were confirmed in all cases by a senior physician specializing in pelvic floor surgery or by an obstetrician specializing in suturing tears of this nature. The exclusion criteria included patients who were multiparous, had a known metabolic or connective-tissue disorder (e.g., Ehlers–Danlos syndrome), or had known neurologic disorders. Patients who underwent episiotomy or assisted vaginal delivery (e.g., vacuum or forceps delivery) were also excluded.

After delivery, peripheral blood samples (5 mL) were obtained from eligible cases and controls who agreed to participate and provided signed informed consent. All blood samples were transferred to the institutional genetic laboratory facility for DNA extraction and storage. DNA was extracted with a Qiagen kit according to the manufacturer’s protocol and stored at a temperature of -20ºC. The DNA samples were examined for ten SNPs in genes encoding proteins associated with extracellular matrix structure. A pair of primers amplified approximately 250 base pairs including the SNP site of interest. Amplification of the products was performed with Sanger sequencing on an Applied Biosystems 3500 machine.

Demographic and obstetric variables and outcomes were collected from each patient records at admission, delivery, and discharge.

The primary outcome was the incidence of the selected SNPs among women diagnosed with OASI compared with those in the control group.

Sample Size Calculation

The sample size was calculated based on published data indicating a 60% SNP prevalence in women with POP compared to 30% in healthy controls [17]. To detect this specific 30% difference—representing a large genetic effect—with 80% power and a two-sided alpha of 0.05, a sample size of 93 women (1:2 ratio; 31 study, 62 control) was required. Consequently, this study was designed to identify substantial differences in genotype frequency rather than subtle genetic variations.

Statistical Analysis

The associations between the demographic variables and the two groups were tested via independent t-tests for continuous variables and the χ2 test or Fisher’s exact test in the case of small cell size for categorical variables. The associations of SNPs with OASI were tested at the allele and genotype levels, with the χ2 test or Fisher’s exact test in the case of small cell size. Genotype and allele frequencies in cases and controls were analyzed for associations with the χ2 test on 2×2 and 2×3 contingency tables. The overall SNPs and the heterozygote and homozygote averages between groups were tested with independent t-tests. Tree analysis revealed no combinations of SNP types appearing more distinctly in the study group versus the control group. The associations among demographic variables, the ten types of SNPs, and the degree of OASI (3^rd or 4^th degree) were tested with the χ2 test or Fisher’s exact test in the case of small cell size for categorical variables. Statistical analysis was performed in SPSS v. 27 software (IBM). The threshold for significance was set at p <.05 (two tailed).

Results

Overall, 93 eligible women provided informed consent to participate. Three were excluded from the final analysis because of logical issues. The final analysis included 30 and 60 women in the study and control groups, respectively.

Basic demographic, obstetric, and intrapartum variables are presented in Table 1. All variables were comparable between groups, except for neonatal birthweight, which was significantly higher in the study group than the control group (p=0.007).

The occurrence of SNPs in the study cohort is presented in Table 2. No significant differences in the frequencies of each SNP or the overall SNP average were observed between groups. Of the 90 women included, 61 (67.8%) were homozygous for one or more SNPs: 19 of the 30 women in the study group (63.3%) and 42 of 60 (70.0%) women in the control group. No significant differences were observed in the distribution of the number of homozygous SNPs between groups (p=0.56) or the average in each group (mean difference: -0.003, p=0.83). The women were heterozygous for zero to six SNPs, and no significant difference was observed in the average number of heterozygous SNPs between groups (mean difference: -0.023, p=0.48).

Tree analysis revealed that no combinations of SNP types appeared more prominently in the study group than the control group. Furthermore, the number of branches without a patient did not differ significantly between groups.

Of the 30 women with OASI, eight (26.7%) had 4^th degree OASI. The distribution of the ten SNP types did not significantly differ according to tear severity. The distribution of homozygotes and heterozygotes was also not significantly different between groups.

Data are presented as n (%) or OR (95 CI) Chi square testFisher’s exact test first row, normal/heterozygote; second row, normal/homozygoteZFAT, Zinc-Finger gene in Autoimmune Thyroid disease; MMP; Matrix Metallo-Proteinase

Discussion

Herein, the incidence of known POP-associated SNPs in women who experienced OASI after a vaginal birth was comparable to that in a control without OASI. This finding also applied to both the heterozygous state and the homozygous state. Additionally, the distribution of the ten SNP types did not differ according to OASI severity. Neonatal birthweight was higher among women who experienced OASIs than those who did not. This outcome was largely expected.

OASI is an unpredictable and unpreventable complication. To date, no evidence is available to counsel women regarding the probability of such an event before a trial of vaginal birth. Although understanding of a genetic basis might improve counseling, the association between OASI and a specific genetic origin remains uncertain.

POP, similarly to OASI, occurs in the same anatomic region, and both disorders result from similar mechanical forces leading to symptoms associated with anal incontinence [21]. These aspects, combined with the known associations between several SNPs and POP, suggest that a similar genetic basis might underlie both disorders. Diminished elastin content and expression have been observed in vaginal and supportive pelvic tissues in women with POP [22–27]. Polymorphisms in matrix metalloproteinases (MMPs) genes, such as MMP-9 [28], have been associated with POP. Fibulin-5, an inhibitor of MMP-9 and a major contributor to elastic fiber assembly, has been reported to protect against POP in a murine model [29] but has been associated with POP among Russian women [30]. Elevated presence of polymorphisms in type 3 collagen has been reported among Dutch and Chinese women with POP [14,15], but not in a mixed Brazilian population [16]. Another polymorphism in type 3 collagen has been associated with elevated risk of POP among Korean women [17]. POP has also been associated with laminin, a glycoprotein in the extracellular matrix [18].

In the current study, we examined SNPs previously shown to have the strongest association with POP, but the findings did not support associations with OASI. Our findings might be simply explained by the absence of a genetic basis associated with OASI, despite prior reports of a familial relationship and suggestions of a possible genetic link [11]. Alternatively, although these SNPs were previously established to be associated with POP, they might have weaker effects on OASI. Because our study was specifically designed to detect only large genetic differences, it is possible that we did not identify more modest genetic effects that contribute to OASI risk in a multi-factorial manner. The interplay between genetic predisposition and mechanical birth forces suggests that while these specific SNPs do not show a large effect size here, they may still play a subtle role within a larger polygenic framework.

Additionally, we emphasize that the current study did not evaluate several additional SNP variants encoding extracellular matrix proteins, which might potentially be linked to OASIs. Finally, as previously described [14–16], the absence of a relationship in one geographic region does not preclude a positive association in another region of the world.

Limitations

Our study has several limitations. We examined the ten most relevant SNPs. Because other POP-associated SNPs were not examined, possible associations cannot be ruled out. Additionally, we included only women with spontaneous vaginal deliveries. Women who had an episiotomy or operative vaginal birth were excluded, to lessen the influence of both factors on OASI occurrence; thus, the results might not be applicable to this group of women. Finally, because the study was conducted at a single facility, the results might not be generalizable. The lack of operative deliveries or episiotomies in our cohort, while reducing confounding variables, limits our ability to generalize these findings to all clinical settings. Furthermore, variations in obstetric practices and the specific ethnic makeup of our population may influence the prevalence of these SNPs, potentially affecting the reproducibility of this research in different geographic regions or healthcare systems.

Conclusion

OASI is influenced by a combination of mechanical factors during vaginal birth and obstetric practices (e.g., episiotomy and operative vaginal delivery). These environmental and procedural factors are likely to play a more direct role in OASI than POP, a condition that tends to develop in older women. Nonetheless, the absence of a difference in the incidence of SNPs examined between women with versus without OASI, does not necessarily and unambiguously rule out actual associations. The findings of this novel study may pave the way to future genetic research on OASI, such as searching for other related SNPs, or the use of other genetic technology, such as next-generation sequencing, to identify novel rare genetic variants that disrupt the normal function of extracellular components.