The gender of the fetus should be included in the prenatal ultrasound – echocardiographic examination reports in tertiary centres in 2021 – recommendations from the Polish Prenatal Cardiology Society

Is the sex of the fetus important for a pregnant woman?
There are few publications dealing with this subject. In 2010, Maaji et al. [1], based on a conducted survey, confirmed what the authors of this publication have noticed in everyday practice – that a vast majority of pregnant women (over 94% of respondents in the survey) want to know what gender their offspring will be. The percentage of pregnant women wishing to know the sex of fetuses varies depending on the country in which the survey is conducted [1-3]. However, most of the currently published studies indicate that women want to know the sex of their baby at the stage of pregnancy. A Swedish study of 2018 [3] shows additionally that the majority of couples deciding to have a child had previously talked about its gender, and some of them (approx. 5%) were also interested in the possible choice of gender.

Is fetal gender determination relevant for the ultrasound/ECHO investigator and for the obstetrician managing the pregnancy?
The answer is not unequivocal. Neither Polish nor American gynaecological and obstetric guidelines contain an obligation to determine the sex of the fetus during prenatal examinations. However, in 2019, the Guidelines for Prenatal Fetal Testing of the Danish Society of Fetal Medicine and the Nordic Federation of Societies of Obstetrics and Gynaecology introduced a significant change in the aspect of prenatal sex assessment. Accordingly, determining the sex of the fetus is an integral part of prenatal testing carried out in the second trimester of pregnancy for the detection of congenital defects in the fetus [4]. The aforementioned recommendations do not indicate the need to assess the sex of the fetus in the first trimester examinations. These are the first guidelines in the world to change the approach to gender determination during prenatal testing.

Fetal gender
During prenatal tests, genetic and phenotypic gender can be distinguished. Phenotypic sex is determined by the examining ultrasound specialist based on the appearance of the external genital organs (Figures 1, 2). Genetic sex requires invasive tests such as trophoblast biopsy amniocentesis, cordocentesis, or free fetal DNA in the maternal blood (non-invasive prenatal testing – NIPT) and subsequent molecular tests. The determinant of gender in humans is the sperm that fertilizes the egg cell. Depending on whether it has a 23, X or 23, Y karyotype, the gender of the fetus (46, XX or 46, XY) will be female or male, respectively. There are approximately 70 known genes responsible for sex differentiation in the human body. Their mutations can cause disorders of the process of differentiation of sexual characteristics. A flagship example is gonad dysgenesis syndrome (Swyer syndrome) [5], which consists of a function loss type mutation of the SRY gene, located on the Y chromosome, encoding a protein factor determining the development of the testicles. The above mutation results in the occurrence of a female phenotype in the individual with 46, XY karyotype.

Phenotypic sex characteristics in prenatal examination
Up to the 8th week of fetal life, the male and female genital organs are the same, and even during autopsy they cannot be distinguished. Between the 8th and 10th week of pregnancy, the production of testosterone, which initiates the differentiation of the penis and scrotum, begins. Between the 14th and 16th week of pregnancy, the development of the male genital organs begins – the penis with the urethra begins to be visible. After the 20th week, the testicles begin to descend into the scrotum, and in 93% of fetuses this process ends after the 30th week of pregnancy.
The second X chromosome, on which there is no SRY gene, and thus no testosterone is secreted, is involved in the development of the female genital organs. Between the 14th and 16th week of pregnancy, the clitoris is the same size as the penis (and therefore it is not recommended that the sex of the fetus be assessed at that stage of development). The labia are another structure visible during ultrasound examinations, testifying to the female sex. In addition, two X chromosomes are necessary for the appropriate differentiation of the ovaries and sex cells.
During prenatal diagnostic tests, there is a risk of a mistake and false recognition of the male sex in a female fetus. The abdominal attachment of the umbilical cord can be mistakenly considered as a penis. However, such an error is relatively rare and most often results from attempts to determine the sex of the fetus being made too early, especially in obese women. According to a 1985 study [6], erroneous gender identification accounted for less than 3% of cases, and all of these mistakes occurred before the 24th week of pregnancy. According to the same study, sex imaging was possible in 60% of cases at 18 weeks of gestation, and in 100% after the 20th week.
In the following decades there was development in both ultrasound scanners and the skills of the staff performing the scans. Hence, the correct recognition of fetal sex has become possible at increasingly earlier stages of pregnancy. As follows from the study by Gharekhanloo [7] of 2018, the percentage of correct sex determinations in fetuses during ultrasound examinations at the 11th and 12th weeks of pregnancy reached 91%. That was a significantly higher percentage of correctly diagnosed fetal sex compared to similar studies conducted in earlier years.
The breech position of the fetus is also a very important fetal factor, which can prove difficult in sex determinations for many, even experienced, ultrasound specialists, regardless of the gestational age. Such a position at the later stages of pregnancy can cause the fetus to effectively cover the perineal area with the limbs, which makes it impossible to assess its sex. The position that the fetus takes is very important at every stage of pregnancy and often affects the achievement of diagnostic success during prenatal tests [8].
Depending on the duration of pregnancy, the following characteristics can be observed in a healthy, well-developing fetus:
in the 1st trimester of pregnancy – whether it is a boy or a girl, but always taking into account the risk of misinterpretation of the image,
in the 2nd trimester of pregnancy – the penis and the scrotum (but without testicles in the scrotal sac) should be visible in boys,
in the 3rd trimester of pregnancy – the penis, scrotum, and both testicles should be visible in boys (from 28th week of pregnancy).

What can interfere with the proper development of fetal sex?
Endocrine disorders in the fetus can cause disturbances of the process of proper differentiation of external sexual characteristics. Elevated levels of androgens in female fetuses can cause enlargement of the clitoris, adhesion of the labia, or lead to the development of a persistent genitourinary sinus. On the other hand, a reduced level of androgens in male fetuses may lead to underdevelopment of the penis (microphallus), hypospadias (as a result of incomplete connection of the urethra with the penis), cryptorchidism, bipartite scrotum, or the presence of a vaginal pouch. The development of sexual characteristics in the fetus, and thus their visibility in ultrasound examination, may be affected by the maternal use of hormonal drugs in the first trimester of pregnancy: progesterone (to maintain an endangered pregnancy) and androgens (for treatment of endometriosis). Some maternal conditions, such as acne, hirsutism, and infertility, are associated with higher androgen concentrations [9-12].

What ultrasound abnormalities can we observe in the course of prenatal examination of the fetus?
Ambiguous fetal gender image (hermaphroditism) – the sex of the fetus visible, but unequivocal interpretation is difficult.
Scrotal hernia and scrotal oedema.
Hypospadias.
Microphallus.
Undescended testicles (> 30 weeks).
Cloacal anomalies.
If any of the aforementioned irregularities are observed, the following questions should be answered:
Is it an isolated defect?
Can it indicate a monogenic defect?
Are there any other features that indicate a chromosomal defect?
Should a congenital malformation syndrome be suspected?

Hermaphroditism
There are special situations in which we see the genitals of the fetus but, despite this, the determination of sex is difficult. A hermaphrodite is an individual in whom ultrasound male and female sexual characteristics can be observed – the outlines of both the testicles and the ovaries are noticeable. Female pseudohermaphroditism is a situation when there is a masculinization of a female genetic individual (46, XX), because of the influence of androgen stimulation in utero. Such an exposure may occur in the course of congenital hypertrophy of the adrenal cortex, when a pregnant woman is taking exogenous androgens or when she has a virilizing tumour.
An analogous example is male pseudohermaphroditism in an individual with a male karyotype (46, XY), in which the features of insufficient masculinization with phenotypically visible features of testicular tissue are noticeable. This may be a consequence of insufficient testosterone synthesis or defects in testosterone receptor proteins [13, 14].

Scrotal oedema and scrotal hernia
One of the most common abnormalities that we observe in prenatal ultrasound is the occurrence of swelling around the testicles (Figure 3). A black, crescent-shaped lesion is then visible in the scrotum, which sometimes, as systemic abnormalities increase, occupies a large volume. Such a finding requires an increase in the diagnostic vigilance of the ultrasound specialist to be able to answer the question: is the swelling a result of cardiovascular failure, or is it a consequence of intrauterine infection in the fetus? Other features assessed during the examination, such as: the appearance and thickness of the placenta, the shape and size of the heart, the contractility of the ventricular muscle, the occurrence of atrioventricular regurgitation, the occurrence of anatomical defects of the heart, intestinal echogenicity, the size of the thymus gland, as well as the medical and epidemiological history of the pregnant woman, can be helpful in answering this question [15].

Hypospadias
A valuable diagnostic tip during difficult diagnostic cases of fetal sex determination is 3D imaging of the perineal area. During such visualization, it is possible to assess the distance between the anus and the genitals, which is shorter in boys. This marker is particularly useful in fetuses with microphallus observed in a 2D study [16]. Short and insufficiently developed, curved, or poorly visible in ultrasound examination penis and glans (an image of the so-called “hooded” prepuce) may suggest hypospadias (Figure 4-6). In order to diagnose hypospadias, it is also worth using 3D imaging, which improves the sensitivity and specificity of the examination. The advantage of the combination of 2D + 3D imaging in the diagnosis of hypospadias was confirmed in 2019 by Li et al. in “Scientific Reports” [17].

Other congenital urinary tract abnormalities
In addition to malformations of the genital organs, anomalies of the urinary system can be observed during ultrasound examination of the perineal area in fetuses. One such case was described by Łosińska in 2018 in Prenatal Cardiology. Urinary bladder exstrophy (Figure 7) was visible in the form of a large tumour that caused bulging of the skin integuments above the labia of the fetus [18]. Other congenital defects that are described during prenatal examination include the co-occurrence of congenital megalourethra with congenital imperforate anus [19].

Summary

Prenatal diagnostics is always more difficult than postnatal diagnostics. Any abnormality found during prenatal examinations should increase the diagnostic vigilance of the examiner. The perceived anomaly can be isolated or may herald subsequent ones that may manifest themselves during that examination or a few weeks later. Some anomalies will be a part of well-known genetic syndromes (relatively rarely) or may (relatively often) constitute an uncharacteristic set of anomalies.
In the case of finding non-cardiac defects in the fetus, including the discussed topic “fetal sex visible but difficult to interpret unambiguously”, the echocardiographic diagnosis: Normal Heart Anatomy and Normal Heart Study is a favourable prognostic factor.
The birth of a newborn in the case when the fetal gender is doubtful and difficult to name should take place in a referral centre where comprehensive medical assessment by qualified medical personnel (specialists in the field of clinical genetics, paediatric endocrinology, paediatric urology) is possible in a short time. Laboratory tests will include, among others, genetic reassessment, hormone concentrations, metal ion concentrations, and 24-hour urine collection. In such difficult cases, the final determination of the neonate’s sex in the medical records should be postponed until the diagnostic process is completed. Correct early diagnosis is a chance for optimal growth, as well as the highest possible quality of life at the subsequent stages of development.

Conclusions

Fetal gender should be assessed by prenatal ultrasound and echocardiographic examination at the reference centre.

Conflict of interest

The authors declare no conflict of interest.

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