Ankyloglossia in Monochorionic Diamniotic and Dichorionic Diamniotic Twins: A Cross-Sectional Study

Abstract

Objective:

To determine the prevalence of ankyloglossia in diamniotic monochorionic and diamniotic dichorionic twins, as well as to verify the relationship between gender and type of pregnancy.

Study Design:

A cross-sectional observational study, carried out with 52 pairs of dichorionic/diamniotic twins and 49 monochorionic/diamniotic pairs. The data collection was carried out through the analysis of medical records and the results of the Neonatal Screening of the Tongue Frenulum Assessment Protocol in Babies, and corresponded to the period of 2 years (2020–2022). Statistical analysis of data was performed, considering the significance value of 5%. The study was approved by the Human Research Ethics Committee of the institution.

Results:

The statistical analysis of multiple logistic regression between the two groups of twins (Mono/Di and Di/Di) according to the socioeconomic, demographic, and clinical-epidemiological profile was statistically significant for some variables. The prevalence of ankyloglossia, according to the type of twin pregnancy, showed a statistically significant difference. There was no statistical difference in relation to sex and ankyloglossia, or between couples diagnosed with ankyloglossia according to the type of pregnancy.

Conclusion:

Monochorionic/diamniotic twins had a higher prevalence of ankyloglossia, regardless of gender.

Introduction

The simultaneous development in the uterus of two or more fetuses is called multiple pregnancy, and can be dizygotic (DZ), when there is the fertilization of two eggs by two spermatozoids, or monozygotic (MZ) when fertilization occurs from a single egg and spermatozoid.¹ Thus, MZ twins will show the same genetic similarity, while DZ twins will have about half of their genetic makeup in common.²

Multiple pregnancies, according to chorionicity and amnionicity, can be classified as dichorionic/diamniotic (Di/Di), in which each fetus has its own placenta and its own amnion, not being identical, and may present equal or opposite sexes; monochorionic/diamniotic (Mono/Di), in which the fetuses share a single placenta and each twin has its own amnion, being necessarily identical and of the same sex; and monochorionic/monoamniotic (Mono/Mono), in which they share a single placenta and a single amnion, being necessarily identical and of the same sex.³ Multiple pregnancies account for 3% of all births, of which approximately one third are monochorionic twins.⁴

In multiple pregnancies, maternal complications are 3–7 times more frequent, increasing the risk for a pathological pregnancy.^1,5,6 Worldwide, 2–3% of all live births present some clinically significant morphological abnormality,⁷ which can be chromosomal or structural abnormalities, and one of the risk factors is twinship.^8,9

Twin studies have traditionally been used to understand the influence of epigenetic characteristics on the etiology of anomalies.¹⁰ Among the possible anomalies, we can mention ankyloglossia, defined as a congenital condition that occurs when remnant embryological tissues, which did not undergo apoptosis during embryonic development, restrict tongue movements.¹¹ This anomaly has an autosomal dominant hereditary trait with incomplete penetrance^12–14; however, there is a scarcity of studies relating ankyloglossia and twin pregnancy, hence the need for this study.

Thus, the aim of this study was to determine the prevalence of ankyloglossia in diamniotic monochorionic and diamniotic dichorionic twins, as well as to verify the association between gender and type of pregnancy.

Methods

Ethical assessment

The study was analyzed by the Ethics Committee on Human Research of the Centro Universitário Integrado de Saúde Amaury de Medeiros (CISAM), affiliated to the University of Pernambuco, Recife, Brazil, receiving a favorable decision under no. 5.602.952 and CAAE: 54175121.1.0000.5191, and complies with the various international ethical guidelines. The informed consent was waived because this was a retrospective analysis of the electronic medical records of individuals who had left CISAM.

Type of study

This is a quantitative cross-sectional observational study, developed at the CISAM, located in the city of Recife, Pernambuco, Brazil.

Participants and criteria

For this study, 332 charts of twin newborns, born preterm or full term, admitted to the ward or neonatal unit and evaluated by the institution's speech-language pathology (SLP) team, were selected. The cutoff period for data collection was from January 2020 to January 2022.

Exclusion criteria were twins whose corresponding pair died at birth or during hospitalization; unclassified or inconclusive pregnancies as to chorionic type and amnionicity; Mono/Mono pregnancies (due to low incidence); incomplete or absence of neonatal screening in the medical record; absence of neonatal screening in any of the twins; and questionable screening results (scores between 5 and 6); as well as babies who had some craniofacial, syndromic, or genetic anomaly.

For the classification of twin pregnancies, the obstetric ultrasound report was considered, as well as the confirmation in the delivery description present in the medical chart. The pregnancies were classified as Mono/Di and Di/Di.

Assessment instrument

In this study, all newborns were evaluated using the Newborn Screening Protocol for Evaluation of the Tongue Frenulum in Infants.¹⁵ This screening was validated and proved accurate for diagnosing ankyloglossia in infants.

The screening is composed of seven items that evaluate the anatomical and functional aspects related to the lingual frenulum, namely: lip posture at rest; tongue positioning tendency during crying; shape of the tip of the tongue when lifted during a crying or lifting maneuver; and possibility of visualizing the frenulum of the tongue; thickness of the frenulum, as well as its attachment to the sublingual (ventral) side of the tongue and to the floor of the mouth. Each subitem has scores in which the sum total allows for the diagnosis of ankyloglossia. When the sum of the items evaluated is ≤4, it is considered normal; between 5 and 6, doubtful, with the need for reassessment when the baby is 30 days old; and 7 or more is considered altered, in which the lingual frenulum restricts tongue movements.

Data collection

Data were collected through access to the participants' electronic medical records, which store all hospital records held at the institution during the hospitalization period. The search was carried out using the medical record number provided by the archive sector. The variables collected were as follows: level of maternal education; self-declared color of the postpartum woman; parity; way of delivery; prenatal care; type of twin pregnancy; classification of estimated intrauterine growth at birth; classification of birth weight, according to World Health Organization (WHO) criteria; Apgar score at first and fifth minute; type of breastfeeding described on the day the lingual frenulum screening was performed (exclusive or nonexclusive); gender of the newborn; and diagnosis of ankyloglossia.

Two researchers collected the data. The participants were coded using Arabic numerals, for protection and confidentiality of information.

Statistical analysis

Data were entered into a Microsoft Excel^® spreadsheet, in its MS-Office 2016 version. The description was presented by the observed frequency and percentage. Simple and multiple logistic regression with the forward variable selection method associated the classification of twins and sex with possible risk or protective factors. A significance value of 5% was considered. To obtain the results, the statistical package IBM SPSS Statistics (Statistical Package for the Social Sciences), in its version 24.0, was used.

Results

Of the 332 medical records analyzed, 130 were excluded after applying the exclusion criteria (6 pairs without classification and/or inconclusive as to the type of chorionicity and amnionicity, 1 pair died, in 7 pairs, one of the twins died, in 14 pairs, one of the twins was unscreened, in 32 pairs, both were unscreened, in 2 pairs, one of the twins had doubtful results, and 3 pairs had mono/mono classification).

In this study, 202 medical records of 101 pairs of newborn twins (52 pairs of Di/Di twins and 49 pairs of Mono/Di twins) were analyzed.

Statistical analysis of multiple logistic regression between the two groups of twins (Mono/Di and Di/Di) according to socioeconomic, demographic, and clinical-epidemiological profile showed a statistically significant relationship for the variables: gestational age (p = 0.013) and birth score first minute Apgar (p = 0.039) described in Table 1. Thus, an NB with first minute Apgar ≥7 is 53% less likely to have been classified as Mono/Di compared to one with first minute Apgar <7. The term newborn has these chances reduced by 60% compared to the preterm newborn.

Table 1.

Relationship Between the Socioeconomic, Demographic, and Clinical-Epidemiological Profile According to the Type of Twin Pregnancy

Dependent variable—classification of twins—Mono/Di		Di/Di		Mono/Di		p ^a	OR	95% CI to OR
Dependent variable—classification of twins—Mono/Di		n	%	n	%	p ^a	OR	Inferior limit	Superior limit
Apgar first minute score	<7	8	7.69	19	19.79	—	1	—	—
Apgar first minute score	≥7	96	92.31	77	78.57	0.013	0.47	0.26	0.85
Classification according to gestational age	Preterm newborn	53	50.96	70	72.92	—	1	—	—
Classification according to gestational age	Term newborn	51	49.04	28	28.57	0.039	0.40	0.16	0.95

Multiple logistic regression with forward selection method.

(1) Reference category; significant if p < 0.050.

Variables included in the model: maternal education level, self-declared color, parity, prenatal care, mode of delivery, classification according to gestational age, gender of the twins, classification according to the intrauterine growth curve, weight classification, Apgar first minute score, Apgar score of fifth minute, and breastfeeding.

The reference category of the dependent variable is the Di/Di classification.

CI, confidence interval; Di/Di, dichorionic/diamniotic; Mono/Di, monochorionic/diamniotic; OR, odds ratio.

When comparing the data regarding ankyloglossia and the type of twin pregnancy, there was a statistically significant relationship (p = 0.002) presented in Table 2. Thus, a twin with present ankyloglossia is 4.4 times more likely to be classified as a Mono/Di twin compared to a twin without ankyloglossia.

Table 2.

Relationship of Ankyloglossia According to the Type of Twin Pregnancy

Dependent variable—classification of twins—Mono/Di		Di/Di		Mono/Di		p ^a	OR	95% CI to OR
Dependent variable—classification of twins—Mono/Di		n	%	n	%	p ^a	OR	Inferior limit	Superior limit
Ankyloglossia	Absent	98	94.23	77	78.57	—	1	—	—
Ankyloglossia	Present	6	5.77	21	21.43	0.002	4.45	1.71	11.58

Simple logistic regression.

(1) Reference category; significant if p < 0.050.

The reference category of the dependent variable is the Di/Di classification.

CI, confidence interval; Di/Di, dichorionic/diamniotic; Mono/Di, monochorionic/diamniotic; OR, odds ratio.

There was no statistically significant relationship when comparing the data regarding sex and ankyloglossia, according to the type of twin pregnancy (Table 3).

Table 3.

Relationship Between Sex and Ankyloglossia According to the Type of Twin Pregnancy

Dependent variable—classification of twins—Mono/Di			Female		Male		p ^a	OR	95% CI to OR
Dependent variable—classification of twins—Mono/Di			n	%	n	%	p ^a	OR	Inferior limit	Superior limit
Di/Di	Ankyloglossia	Absent	53	94.64	45	93.75	—	1	—	—
Di/Di	Ankyloglossia	Present	3	5.36	3	6.25	0.846	1.18	0.23	6.13
Mono/Di	Ankyloglossia	Absent	38	84.44	39	73.58	—	1	—	—
Mono/Di	Ankyloglossia	Present	7	15.56	14	26.42	0.196	1.95	0.71	5.36

Simple logistic regression.

(1) Reference category; significant if p < 0.050.

The reference category of the dependent variable is the female gender.

CI, confidence interval; Di/Di, dichorionic/diamniotic; Mono/Di, monochorionic/diamniotic; OR, odds ratio.

There was no statistically significant relationship when comparing the type of twin pregnancy and the presence of ankyloglossia in both babies of the pair or just one baby of the pair (Table 4).

Table 4.

Relationship Between the Type of Twin Pregnancy and the Presence of Ankyloglossia in Both Babies of the Pair or Just One Baby of the Pair

Dependent variable—classification of twins—Mono/Di		Di/Di		MONO/DI		p ^a	OR	95% CI to OR
Dependent variable—classification of twins—Mono/Di		n	%	n	%	p ^a	OR	Inferior limit	Superior limit
Ankyloglossia	Both twins	1	20.00	9	75.00	0.064	12.00	0.87	166.40
Ankyloglossia	Only one twin	4	80.00	3	25.00	—	1	—	—

Simple logistic regression.

(1) Reference category; significant if p < 0.050.

The reference category of the dependent variable is the Di/Di classification.

CI, confidence interval; Di/Di, dichorionic/diamniotic; Mono/Di, monochorionic/diamniotic; OR, odds ratio.

Discussion

Studies involving twins are relevant to map the importance of the role of genetics in the presence of anomalies, as well as to determine their prevalence in individuals who share the same genes.¹⁶ In the searched databases, no study on ankyloglossia in twins was found, which proves the originality of this research.

A study¹⁷ carried out in China analyzed data from 161,076 twins and found a lower percentage of preterm infants (45.9%), which differs from the data found in this study (72.92%). A population-based historical cohort study¹⁸ conducted in Finland analyzed 1,034 pregnancies and reported that Mono/Di twins had lower Apgar scores at 1 minute compared to Di/Di, although the mean scores were above 7, confirming our findings.

The similarity of the anatomy of the tongue was already described in a study that compared the anatomical features of the tongue in MZ and DZ twins, to determine whether the tongues, like any other anatomical structure, could be used to reliably predict the kinship.¹⁹ As a methodology, photographs of the participants' language were paired. The authors reported that MZ twins present high similarity in tongue morphology when compared with DZ twins, although aspects related to the lingual frenulum that could characterize ankyloglossia were not considered.

A survey carried out in Nigeria evaluated 33,659 newborns, of which 1,453 were twins, and reported that the occurrence of ankyloglossia was the most common anomaly related to defects of the gastrointestinal system in the study twins.²⁰ However, the classification according to the type of twin pregnancy has not been described.

The prevalence of ankyloglossia in singleton newborns has already been well described in national^21–23 and international studies,^24–28 with variations between 7.1% and 22.54%, corroborating the percentage of 13.40% found in twins Mono/Di in this research.

The literature reports that monochorionic twins are at greater risk of developing congenital and/or structural anomalies, including the nervous and urinary systems, followed by the musculoskeletal and circulatory systems. As an example, congenital heart defects often associated with twin-to-twin transfusion syndrome, which is a common complication in monochorionic twins due to sharing a single placenta.^29–31

Research indicates the possible association of the gene T-box transcription factor 22 (TBX22)^32,33 and SRY (sex determining region Y)-box 2 (Sox2)³⁴ with ankyloglossia, being considered an anomaly of chromosomal origin; therefore, it should be expressed in both twins due to monozygosity. The results found in this study corroborate* the discussions presented in other studies, which report discordance of alterations between monochorionic twins.^35,36 The cause of these phenotypic differences between MZ twins is unknown, being attributed to undefined environmental factors.³⁷

Studies carried out in different countries sought to characterize ankyloglossia, so that some found a higher prevalence of this anomaly in male newborns,^22,23,27 others showed a similar prevalence.³⁸ In the twins in this research, there were no significant relationship, although Mono/Di twins of the male sex had a higher occurrence, suggesting that men seem to be more susceptible to ankyloglossia.

When comparing the pairs of Di/Di twins, a lower occurrence of ankyloglossia was observed in both babies of the same pair. Conversely, in the pairs of Mono/Di twins, the occurrence of ankyloglossia was higher in both babies of the same pair. However, there was no statistical significance, probably due to the small number of pairs analyzed in each type of twin pregnancy.

As limitations of this study, we can mention the lack of karyotyping of the participants, to confirm the zygosity for the differentiation of MZ and DZ twins, in addition to the lack of information in medical records about the methods of conception (whether by assisted reproductive technology, ovarian stimulation, or natural causes), which may be associated with the occurrence of multiple pregnancies and congenital anomalies. Thus, we suggest carrying out karyotyping and researching conception methods for future studies on this topic.

Conclusion

Mono/Di twins have a higher prevalence of ankyloglossia, regardless of gender.

Footnotes

Authors' Contributions

A.L.F.F.: study design, data acquisition, analysis and interpretation of data, and drafting the work; G.N.R.S.: data acquisition, analysis and interpretation of data, and drafting the work; M.P.M.: analysis and interpretation of data and drafting the work; R.L.C.M.: critical review for relevant intellectual content, data analysis and interpretation, and drafting the work; M.G.P.S.: critical review for relevant intellectual content, final approval of the version to be published.

Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

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