Possibilities to Estimate the Sex of the Fetus in Very Early Pregnancy

doi:10.21203/rs.3.rs-3134687/v1

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Possibilities to Estimate the Sex of the Fetus in Very Early Pregnancy

https://doi.org/10.21203/rs.3.rs-3134687/v1

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Background:Ultrasound examination of pregnant women to assess the anatomy and normal developmental parameters of the fetus has been the standard in obstetrics since the 1980s. There are medical situations in which the doctor overseeing the course of the pregnancy should know the sex of the fetus as early as possible. This is particularly true in the case of suspected cases of serious genetic and sex-linked diseases. Advances in medical diagnostics make it possible to diagnose the sex of the fetus as early in pregnancy as possible. However, some of the diagnostic methods used during this period are associated with risks of complications or high costs, which disqualify them as screening methods. This study is part of the current of research on the importance of physical fetal parameters in estimating fetal sex.

Methods: descriptive statistics, regression equations with t-tests and logistic regression model statistics were used. Fetal parameters were analyzed between 5 and 12 weeks of gestation. The sample consisted of 255 pregnant women.

Results: On the basis of CRL, FHR, FV and GSV parameters in 255 pregnant women between 5 and 12 weeks of gestation, attempts were made to build models for estimating the sex of the baby. The combination of gestational week (LMP) and fetal parietal length (CRL) gave the most promising, statistically significant results.

Conclusions: Early diagnosis of fetal sex is important especially in the context of X-chromosome-related genetic diseases. Such a diagnosis can be made with a sufficiently high probability on the basis of an ultrasound examination and the use of appropriate statistical models.

fetal sex

early pregnancy

ultrasound examination

LMP

CRL

There are only a few publications in the world literature on the methods and results of ultrasound fetal sex determination in the first trimester of pregnancy. Since the landmark publication by Efrat et al. in 2006 [1], which demonstrated the feasibility of fetal sex determination in the first trimester of pregnancy and indicated the method of examination, researchers working on this problem have been conducting research on the application of this method. The method used by Efrat et al. is to visualise the fetus with a transabdominal ultrasound transducer in the precise midshaft dimension and then measure the angle of the fetal genital cusp in relation to a line drawn through the skin of the lumbosacral region. An angle of less than 10 degrees corresponded to female sex, while male sex was recognised at an angle of more than 30 degrees. At an intermediate angle of 10–30 degrees, gender remained undetermined. The prerequisite for correct angle measurement was a neutral fetal position, i.e. not in flexion or upright. The aim of the study carried out on 656 12- to 14-week singleton pregnancies was to assess the feasibility and accuracy of the proposed method. Gender was successfully assigned in 613 fetuses (93%). The accuracy of male sex assignment in the entire study group reached 99–100% and was significantly higher than for female sex, where it reached 91.5%.

Studies using the same method were carried out by the following teams of researchers: Chelli D. et al (2009) [2] evaluated the feasibility and accuracy of fetal sex identification by transabdominal ultrasound in the first trimester of pregnancy. In 312 fetuses at 11–14 weeks' gestation, the angle of the genital cusp was measured in relation to a line drawn through the skin of the lumbosacral region. The feasibility of sex assignment was estimated at 89.7%. Accuracy increased with increasing CRL only in male fetuses. This is due to the observed fact that the measured genital cusp angle in male fetuses increased with increasing CRL, whereas it did not change significantly in female fetuses.

Lubusky M. et al (2012) [3] in 1222 patients at 12–14 weeks of singleton pregnancy found a close, directly proportional relationship between CRL increasing with gestational age and the feasibility and accuracy of sex determination. They found that with a CRL of less than 50 mm, corresponding to a pregnancy of less than 11 + 4 weeks, due to an assessment feasibility of 39.1%, with an accuracy of 30.5% (40.9% in males and 24.3% in females), the sex of the foetus could not be reliably predicted. The same parameters as the pregnancy progressed were as follows: CRL 50-54.9 mm (gestational age 11 + 4 to 12 + 0 weeks) feasibility 63.5%, accuracy 75% (89.1% in male and 66.7% in female), CRL 55-59.9 mm (gestational age 12 + 0 to 12 + 2 weeks) feasibility 90.5%, accuracy 96.6% (99.1% in male which already allows reliable determination and 93.5% in female). Only at CRLs above 60 mm (gestational age above 12 + 2 weeks) was the feasibility 97.4% and accuracy 100% in both sexes.

Manzanares S. et al (2016) [4] attempted to assess the feasibility and accuracy of determining fetal sex on transabdominal ultrasound at 11–13 + 6 weeks of gestation, and the factors. that affect the accuracy of the assessment. The method of examination was to assess the angle of the genital cusp in the sagittal plane. We were able to determine the sex of 608 out of 672 fetuses (90.5%) including correctly in 532 cases (87.5%). The accuracy of fetal sex prediction was supported by an increase in fetal parietal length (CRL) above 55.7 mm, gestational age above 12 + 2 weeks, and a BMI of the pregnant woman below 23.8.

Hsiao CH. et al. (2008) [5] modified the method of Erfat et al. by additionally assessing cross-sections of the genital tubal region. The results they presented were similar to the studies presented above. During transabdominal ultrasound examinations in 496 patients with singleton pregnancies between 11 and 13 + 6 weeks of gestation, they estimated the feasibility of sex determination at 88.9% (441 of 496) and the overall accuracy at 91.8%. The accuracy of identification increased with gestational age, from 71.9% at 11 weeks, through 92% at 12 weeks, to 98.3% at 13 weeks, respectively.

Arfi A. et al. (2016) [6] proposed their own different method of fetal sex identification subject to the measurement of the anogenital distance, i.e. the distance between the caudal end of the fetus and the base of the genital cusp. This distance, from the 11th week of pregnancy onwards, varies according to the sex of the fetus. The study included 310 singleton pregnancies at 11–14 weeks. A cut-off point of 4.8 mm was established at which the sex of 87% of male fetuses and 89% of female fetuses was correctly determined. A cut-off of 4.8 mm or more allows the identification of a male fetus, while less than 4.8 mm characterises female foetuses. The authors assessed their method as accurate, but encourage the study of a larger case series to validate it.

The studies conducted and described in the literature differ in the methods used to assess the fetus, such as viewing the genital region of the fetus and using cross-sections in addition. At the same time, as ultrasound technology has developed and improved, high-frequency transducers, transvaginal transducers, as well as virtual reality and 3D apparatus Bogers et al [7] have been used in studies.

The identification in 1997 by Lo et al [8] of cell-free fetal DNA (cffDNA) in the plasma and serum of pregnant women, as a source of fetal genetic material, makes it possible to identify Y-chromosome sequences and thus to determine the sex of the fetus early, reliably and minimally invasively, avoiding conventional invasive prenatal diagnostic methods. The most common technique currently used to detect and identify cffDNA sequences is the polymerase chain reaction (PCR). Among the many known types of PCR, quantitative real-time PCR is the most popular [9], as it combines high sensitivity with a closed detection system, thus minimising the risk of sample contamination [10].

Most studies that assessed fetal sex from cffDNA in maternal blood were based on the detection, the first used to determine fetal sex, of the SRY gene [11] or the DYS14 marker located on the TSPY gene. Although only a single copy of the SRY gene exists in the genetic material, Lo et al. in 1998 demonstrated its usefulness in confirming the presence of cffDNA in maternal blood [12]. In contrast, the DYS14 marker sequence exists in several copies and is easier to detect in plasma, at concentrations up to 10 times lower than SRY, in the male fetus [13]. In a procedure for detecting fetal DNA, Zhong et al, found greater sensitivity of PCR amplifying the DYS14 sequence than PCR amplifying the SRY sequence [14]. Because the X-chromosome-associated amelogenin gene (AMELX) differs by 6 base pairs in intron 3 from the Y-chromosome-associated gene (AMELY), PCR diagnosis of these genes can also, with near 100% accuracy, be used to determine fetal sex [15]. The fetus is estimated to be female if no Y-chromosome DNA is detected.

The lowest sensitivity, in detecting the SRY gene between 11 and 12 weeks of gestation, of 65.9% z was described by Picchiassi et al [16]. The authors attributed the low detection rate to the fact that the SRY gene has only one copy and the cffDNA is low in the first trimester of pregnancy. However, a similar study covering the fifth to ninth week of pregnancy, presented by Ren et al. in 2007, had a sensitivity of 85.4% [17]. Finally, the arguments presented by Picchiassi et al. were refuted by the studies of Costa et al [18, 19], and Kiolialexi et al [20] in 2012, which found a sensitivity of 100% between 8 and 13 and 6 and 11 weeks of gestation, respectively.

Another, less commonly used, method of identifying the sex of the fetus is the detection of foetal cells in the mother's blood and the analysis of the DNA they contain [21, 22]. However, this method finds a limitation due to the prolonged presence of fetal cells circulating in the mother's blood long after birth and even after miscarriage especially when the doctor has not been informed about previous pregnancies [23, 24].

In the search for fetal sex, trophoblast villi biopsy (CVS) is highly controversial. Trophoblast villi biopsy (CVS) is performed on medical indication and at the request of the pregnant woman in order to know the karyotype and sex of the fetus as early as possible. Fetal tissues for the diagnosis of the sex of the foetus or the diagnosis of a genetic disease can be obtained by invasive tests such as amniocentesis or chorionic villus sampling (CVS). Chorionic villus sampling under ultrasound guidance was the first approved technique for fetal genetic diagnosis. A pregnant woman may opt for these tests for a number of reasons, such as determining the sex of the foetus in hereditary X-chromosome-related diseases or in congenital adrenal hyperplasia.

CVS provides reliable fetal karyotype results from as early as the seventh week of pregnancy, thus enabling early termination of pregnancy when medically necessary. The main reason for false results, which are rare with this method, is contamination of the trophoblast tissue examined with maternal cells during chorionic villus sampling [25]. Hence, ultrasound fetal sex determination can be a valuable adjunct to the CVS procedure allowing for a significant reduction in the risk of a false result. Prenatal counselling must take into account the risks associated with invasive procedures. CVS can be difficult to perform. Moreover, this test should not be performed before 11 weeks of pregnancy due to the risk of pregnancy loss and the risk of limb reduction [26, 27]. For this reason, most women have concerns and are reluctant to undergo the villi sampling procedure.

In 2007, Mujezinovic et al. conducted a systematic review of all publications on complications after transabdominal CVS performed between 10 and 14 weeks' gestation [28]. The fetal loss rate at 14 days post-procedure was 0.7 per cent and reached an additional 1.3 per cent by 24 weeks' gestation. The overall fetal loss rate was therefore 2%.

In 2010, Tabor et al. stated in a review of the literature that the miscarriage rate associated with the CVS procedure is 0.5-1% [29]. In developing countries and with low levels of medical care, this rate can be as high as 5% [30]. Because of the complications presented, invasive biopsy testing should only be used to obtain a definitive prenatal diagnosis.

AIM OF THE STUDY

Given the controversy surrounding the CVS method and the cost of cell-free fetal DNA (cffDNA) testing, the aim of the study was to identify the possibility of identifying the sex of the fetus in early pregnancy based on the CRL dimension. The study was based on the supposition that there may be a statistically significant relationship between fetal parietal length (CRL) in the first trimester of pregnancy and fetal sex.

This conjecture is based on the increase in testosterone levels secreted by Leydig cells developed in the nucleus accumbens, observed from the 7th week of pregnancy in male fetuses. The anabolic effect of testosterone should result in noticeably faster growth of male fetuses compared to female fetuses.

Ultrasound examinations were performed using a GE Voluson 730 ProV (GE Healthcare Ultrasound). Fetal sonographies were performed by a Fetal Medicine Foundation-certified operator. The study was performed in 225 singleton pregnancies aged 5.3–12.0 weeks and CRL < 6mm. A 4–9 MHz transvaginal probe was used for transvaginal scans performed before 11 weeks of gestation, while a 5.0 MHz convex probe was used for transabdominal scans performed after 11 weeks of gestation. The total scanning time did not exceed 10 min.

Gestational age (GA) was determined by the last menstrual period or date of assisted reproductive technology and was corrected by assessing the CRL of the fetus if the difference was greater than 7 days, there were irregular periods, a short time since the last pregnancy, the pregnant woman was still breastfeeding or had taken oral contraceptives for less than 3 months. In all pregnancies studied, the following were measured:

- fetal parietal length (CRL),

- fetal heart rate (FHR),

- fetal volume (FV),

- gestational follicle volume (GSV).

All measurements were taken in triplicate and the mean values from these measurements were used in the analysis. As the cephalic and caudal parts of the embryo cannot be distinguished in pregnancies below 7 weeks, the greatest length of the embryo was measured instead of the CRL. From week 7 onwards, the CRL was measured in the sagittal section of the embryo, taking care not to include the yolk sac in the measurement. GSD was calculated as the average of 3 perpendicular diameters, with markers placed on the inner edges of the trophoblast. GSV was calculated using the ellipsoidal formula:

Volume (V) = 1/6 π × length × width × height of gestational follicle.

The fetal heart rate (FHR) was calculated by the ultrasound software as the number of heart contractions per minute, after marking with electronic markers the distance between two heart contractions, on a frozen B-mode image [31]. The criteria for excluding a fetus from the FHR assessment were bradycardia or tachycardia, cardiac arrhythmias, and thyroid disease, cardiac arrhythmias and use of beta-blockers in the pregnant woman.

The inclusion criteria for the pregnant woman in the study were: a singleton pregnancy with a viable embryo with no observable significant abnormalities, at a gestational age (GA) of 5.0 to 12.0 weeks. In all pregnancies meeting the inclusion criteria, GA was calculated based on the Robinson and Fleming formula using CRL [32].

STATISTICAL MODEL

1. Women's physical characteristics. Selected physical characteristics of the women were described: age, weight, height, BMI. To ensure representativeness, efforts were made to ensure that the women studied were a relatively homogeneous group in terms of physical characteristics. The study group of women was divided into women who had given birth to a boy and women who had given birth to a girl. The statistical description of the physical characteristics of the women used classical measures and positional measures of the description of homogeneity: mean, standard deviation, minimum, lower quartile, median, upper quartile, maximum.

2. Linear regression models. Four linear regression models were used to represent the relationship between the parameters CRL, FHR, FV and GSV and gestational week LMP separately in the group of male fetuses and female fetuses:

CRL_male = a₀ + a₁LMP_male; CRL_famale = b₀ + b₁LMP_female

FHR_male = a₀ + a₁LMP_male; FHR_famale = b₀ + b₁LMP_female

FV_male = a₀ + a₁LMP_male; FV_famale = b₀ + b₁LMP_female

GSV_male = a₀ + a₁LMP_male; GSV_famale = b₀ + b₁LMP_female

Regression coefficients a₁ and b₁ were evaluated, which determined by how much, on average, the value of the dependent variable (CRL, FHR, FV, GSV) changes when the independent variable increases by 1 (here by 1 week of LMP). The above models were applied to fetuses with LMPs in the ranges:

[5–12),
[5–7],
(7–12).

When comparing the models for male fetuses and female fetuses, the significance of the difference in a1 and b1 increments was additionally tested. The p-value t-Stat values are reported here. Values of p-0.05 are treated as indicating the presence of significant differences in the increments of the dependent variable for male fetuses and female fetuses.

3. Estimation of fetal sex probability. The estimation of the probability of the sex of the child was based on a logistic model. This probability was defined as LMP combinations of gestational week LMP and one of the parameters CRL, FHR, FV and GSV, assumed to count the probability of male sex:

$$P\left(X=male\right)=\frac{{e}^{{a}_{0}+{a}_{1}LMP+{a}_{2}CRL;FHR;FV;GSV}}{1+{e}^{{a}_{0}+{a}_{1}LMP+{a}_{2}CRL;FHR;FV;GSV}}$$

This model was supplemented with appropriate quality statistics. Values of p-0.05 for the relevant statistics denote the significance of the model as a whole or also of its parameters. A graphical interpretation of the model obtained is provided for easier interpretation.

1. Physical characteristics of women. The study group of women is characterised in Table 1 using descriptive statistics. They are women aged between 16 and 41 years, with a weight between 40 and 90 kg, a height between 150 and 182 cm and a BMI between 15.43 and 34.63. At the time of the study, the sex of the fetus was unknown. After termination of the pregnancy, the patients were divided into two groups according to the characteristics at the time of the ultrasound examination. It turns out that the groups are similar to each other, i.e. the respective statistics do not differ significantly.

Table 1

Physical characteristics of women
Statistics	Age (years)		Weight (kg)		Hight (cm)		BMI
Statistics	Male sex	Female sex	Male sex	Female sex	Male sex	Female sex	Male sex	Female sex
Mean	26.19	26.31	61.95	64.81	165.33	167.20	22.63	23.20
St.dev.	5.24	5.19	10.06	10.23	5.93	5.85	3.32	3.59
Min	18.0	16.0	40.0	42.0	150.0	152.0	17.58	15.43
Lower quartile	22.0	22.0	55.0	58.0	161.0	164.0	20.20	20.80
Median	26.0	26.0	61.0	63.0	165.0	168.0	22.04	22.45
Upper quartile	29.0	31.0	68.0	72.0	170.0	170.0	24.74	24.92
Max	41.0	37.0	87.0	90.0	181.0	182.0	32.81	34.63

According to literature reports, fetal sex studies in women with a high BMI may be unreliable. Here, the number of women with a BMI of 25 or more accounted for about ¼ of the total sample population, and a maximum BMI of 34.63 was not an obstacle to the study.

2. Relationship of CRL, FHR, FV, GSV parameters with gestational week of LMP. Based on linear regression models, the effect of LMP gestational week on the values of CRL, FHR, FV and GSV parameters was determined, these relationships are illustrated in Fig. 1. All the correlations turn out to be positive, which means that the parameters CRL, FHR, FV and GSV increase with increasing LMP gestational week. These relationships are shown as linear, as for such a range of LMP variability from week 5 to week 12, the model proved to be the best fit to the empirical data. However, fetuses grew at different rates, so many fetuses deviate in plus or minus to the line of the theoretical relationship. Sometimes the differences are significant.

The rate of fetal growth was divided by sex and LMP range (Table 2) across the range [5–12] and [5–7] and (7–12). This study identifies differences in the growth rates of male and female fetuses before and after week 7 of pregnancy. In the literature, week 7 is indicated as the cut-off point from which the growth rate of male fetuses becomes faster than that of female fetuses, which can provide a basis for gender identification.

In terms of CRL size, a faster growth rate for female fetuses was observed up to week 7, with an average of 0.4489 cm per week, while for male fetuses it was 0.2581 cm per week. However, these results are not significantly different (p = 0.3472). By contrast, after week 7, the situation becomes reversed, with male fetuses growing at an average of 0.8534 cm per week and female fetuses at 0.5433 cm per week, which already proves statistically significant (p = 0.0002).

The strongest FHR growth was observed up to week 7, and after week 7, FHR growth was already slower. Despite quite large differences between male and female fetuses, no statistical difference could be found. Over the entire studied interval [5–12] of the LMP, the average FHR increment for male fetuses was 11.1056 per week and for female fetuses 10.3633 per week, which is not significantly different (p = 0.7497).

The parameter that differs between male and female fetuses turns out to be FV. Up to week 7 of the LMP, the results are almost identical (p = 0.9817), as FV development for male fetuses averaged 0.2400 per week and for female fetuses averaged 0.2447. However, from week 7 to week 12 of pregnancy, FV development of male fetuses averaged 0.9943 per week and FV development of female fetuses averaged 0.6432 per week, which was statistically significantly different (p = 0.0197).

Similar conclusions as for FV can be drawn from the observation of the behavior of GSV according to the week of gestation of the LMP. Up to week 7 of the LMP, the results are not different (p = 0.1042), although the GSV development of male fetuses averaged 7.4414 per week and that of female fetuses averaged 1.9575. The difference, although apparently large, cannot be considered significant due to the significant variation in GSV values within sex. On the other hand, from the 7th to 12th week of gestation, due to the more stable results within the sexes, the GSV development of male fetuses turns out to be close to being considered statistically significantly faster than that of female fetuses (p = 0.0602), it averaged 12.0728 per week and that of female fetuses averaged 7.1019 per week.

Table 2

Tests of significance for differences in growth rates of male and female fetuses
Parametr	LMP	Gender	N	Reg. coeff.	p-value
CRL	[5–12)	male	126	0.7616	0.0001
	[5–12)	female	99	0.5480	0.0001
	[5–7]	male	27	0.2581	0.3472
	[5–7]	female	30	0.4489	0.3472
	(7–12)	male	99	0.8534	0.0002
	(7–12)	female	69	0.5433	0.0002
FHR	[5–12)	male	83	11.1056	0.7497
	[5–12)	female	62	10.3633	0.7497
	[5–7]	male	21	12.4835	0.3277
	[5–7]	female	27	24.8161	0.3277
	(7–12)	male	62	7.5176	0.4818
	(7–12)	female	35	5.1322	0.4818
FV	[5–12)	male	95	0.7525	0.0165
	[5–12)	female	79	0.5168	0.0165
	[5–7]	male	24	0.2400	0.9817
	[5–7]	female	28	0.2447	0.9817
	(7–12)	male	71	0.9943	0.0197
	(7–12)	female	51	0.6432	0.0197
GSV	[5–12)	male	97	9.3855	0.0458
	[5–12)	female	78	6.0262	0.0458
	[5–7]	male	24	7.4414	0.1042
	[5–7]	female	27	1.9575	0.1042
	(7–12)	male	73	12.0728	0.0602
	(7–12)	female	51	7.1019	0.0602

3. Estimating the probability of fetal sex. Greater male fetal growth in the early weeks of pregnancy may be the basis for estimating fetal sex. Based on a logistic regression model, a discussion was undertaken on the possibilities of estimating fetal sex before 12 weeks of gestation based on combinations of (CRL; LMP), (FHR; LMP), (FV; LMP) and (GSV; LMP). The statistics of the models performed are shown in Table 3 and graphically illustrated in Fig. 2.

The models presented concern the estimation of the probability of a male fetus, in particular:

- values above 0.5 indicate higher probability of male sex and lower probability of female sex,

- values below 0.5 indicate a lower probability of male sex and a higher probability of female sex,

- a value of 0.5 indicates an inconclusive situation, 50% probability of the male sex and 50% probability of the female sex,

- a cut-off value of 1 means 100% probability of male sex and 0% probability of female sex,

- a cut-off value of 0 means 0% probability of male sex and 100% probability of female sex.

The statistically best model for estimating the probability of fetal sex concerns the CRL parameter. This model as a whole is statistically significant (p = 0.0029), the effect of the CRL parameter itself is also significant (p = 0.0073). The CRL parameter in this model has the highest odds ratio value of 2.0090. Such high odds ratio parameters are not observed in the other models. This value means that, at a constant LMP, a CRL value of 1 higher results in a 2 times higher probability that the fetus is male.

The model for the GSV parameter is also relatively good. Here, although the model as a whole is not statistically significant (p = 0.1041) and the effect of the GSV parameter is also not significant (p = 0.1061), the significance levels obtained are close to the limit of being considered significant. An odds ratio parameter of 1.0221 means that, at constant LMP, a GSV value greater than 10 results in a 1.24-fold higher probability that the fetus is male.

Models including FHR and FV parameters as a whole are statistically insignificant (p = 0.1060 and p = 0.2847), the effect of FHR and FV parameters on sex alone also proves statistically insignificant (p = 0.4573 and 0.2486).

Table 3

Logistic regression model statistics
N = 225	Const. B0	LMP	CRL	N = 145	Const. B0	LMP	FHR
N = 225	Chi2( 2) = 11,718 p = 0,0029			N = 145	Chi2( 2) = 4,4890 p = 0,1060
Estimation	1,3326	-0,2744	0,6977	Estimation	-2,2908	0,1889	0,0077
Std. Error	1,2449	0,1941	0,2576	Std. Error	1,2605	0,1835	0,0103
t(222)	1,0705	-1,4140	2,7088	t(222)	-1,8173	1,0292	0,7453
p	0,2856	0,1587	0,0073	p	0,0713	0,3052	0,4573
Wald Chi2	1,1459	1,9995	7,3377	Wald Chi2	3,3026	1,0592	0,5555
p	0,2844	0,1574	0,0068	p	0,0692	0,3034	0,4561
Odds ratio	3,7911	0,7600	2,0090	Odds ratio	0,1012	1,2079	1,0077
N = 174	Const. B0	LMP	FV	N = 175	Const. B0	LMP	GSV
N = 174	Chi2( 2) = 2,5126 p = 0,2847			N = 175	Chi2( 2) = 4,5258 p = 0,1041
Estimation	0,0425	-0,0013	0,2254	Estimation	0,0943	-0,0144	0,0219
Std. Error	1,1338	0,1542	0,1947	Std. Error	1,1370	0,1531	0,0135
t(222)	0,0375	-0,0086	1,1577	t(222)	0,0829	-0,0942	1,6245
p	0,9702	0,9931	0,2486	p	0,9340	0,9251	0,1061
Wald Chi2	0,0014	0,0001	1,3402	Wald Chi2	0,0069	0,0089	2,6389
p	0,9701	0,9931	0,2470	p	0,9339	0,9250	0,1043
Odds ratio	1,0434	0,9987	1,2528	Odds ratio	1,0989	0,9857	1,0221

For models containing the FHR and FV parameters, the graphs shown (Fig. 2) cannot be reliably interpreted, as is evident from the description above. In contrast, models containing the CRL parameters, and to a lesser extent GSV, can be interpreted, as is done below.

From the values of the logistic regression model of the combination of LMP and CRL parameters, it follows that the higher the value of the CRL parameter with a lower value of LMP, the higher the probability that the fetus will be male and the lower the probability that the fetus will be female. Conversely, the lower the value of the CRL parameter with a higher value of the LMP parameter, the less likely the fetus will be male and the more likely the fetus will be female. A line of equal probability connects the points from (LMP = 7;CRL = 0.85) to (LMP = 12; CRL = 2.85). Above this line, there is a higher probability of a male fetus and below this line a female fetus.

In contrast, the model containing the GSV parameter is approximately parallel to the LMP axis, meaning that in gender prediction GSV is independent of LMP, and the higher the GSV the higher the probability of a male fetus regardless of LMP. The line of equal probability runs from (LMP = 5; GSV = 0.5) to (LMP = 12; GSV = 3.8). Above this line a male fetus is more likely and below this line a female fetus is more likely.

Differently from female fetuses, male genital differentiation is preceded by maturation of the gonads. The male phenotype depends on the presence of testosterone and antimaternal hormone secreted in the testes of the fetus, whereas the female phenotype depends on their absence. The onset of testicular differentiation occurs in the 6th-7th week of gestation, when the sex cords formed by the Sertoli cells are formed. In the 8th week of pregnancy, testosterone-producing Leydig cells, differentiated from mesenchyme, are observed in the testis. Testosterone production is stimulated by high levels of HCG. The absence of testicular development at 6–8 weeks of gestation is evidence of a primary, temporarily inactive ovary [33].

Early diagnosis of fetal sex is important especially in the context of X-chromosome-related genetic diseases.

Several attempts to assess fetal sex on the basis of ultrasound performed during the first trimester of pregnancy have been presented in the world literature of the last 15 years [e.g. 1–6]. These studies all focused on the final weeks of the first trimester and covered a range of 11-13.6 weeks of gestation. The authors assessed the distance between the caudal end of the fetus and the base of the genital cusp [6], the angle of the genital cusp measured in the sagittal plane [1–5], or the CRL and genital area of the fetus together [3].

The authors of the presented studies agree that the accuracy of fetal sex assessment increases significantly with gestational age. They set the cut-off point for reliable results at 11 weeks' gestation [6], 12.2 weeks' gestation and CRL 56.7 mm [4] and 11.4–12 weeks' gestation and CRL 50.0-54.9 mm [1–3].

In daily medical practice, the embryo length or crown-rump length (CRL) of the fetus is considered the most important parameter in estimating the age of early pregnancy. The CRL is solely dependent on the age of the fetus and no socioeconomic variable influences its magnitude [34]. Transvaginal ultrasound allows an earlier measurement of parietal length than transabdominal sonography. Ultrasound assessment of fetal age by measuring CRL in the first trimester is now recommended for all pregnant women, including those with a certain date of last menstruation [35, 36]. The difference in the determination of gestational age between the CRL method and the last menstrual method can exceed 7 days [37, 38]. CRL has the highest reproducibility and lowest margin of error compared to other biometric parameters [35].

This study was inspired by a 2012 publication by Ben-Haroush et al. [39]. The authors presented a study that aimed to determine whether parietal-peripheral length (CRL) differs between male and female fetuses early in the first trimester of pregnancy. Measurements were taken at 43–45 days GA. In linear regression analysis, male sex was found to be a significant (p = 0.011) predictor of higher CRL according to the formula:

CRL(mm) = -23,851 + GA(days) x 0,621 + 0,334 x Sex

(Female = 1, Male = 2), R2 = 0,512, p < 0,001.

This leads to the conclusion that male fetuses are larger than female fetuses early in the first trimester, which is consistent with the study conducted in this paper.

The action of fetal testosterone is important for the larger CRL of male fetuses in the first weeks of pregnancy. Of course, the action of other factors cannot be ruled out as well, which requires further research. Surprising and difficult to explain may be the fact found in observations that up to the 7th week of pregnancy female fetuses develop faster than male fetuses. In contrast, the fact that up to 7 weeks of gestation the development of male fetuses is weakly dependent on gestational age, and that after 7 weeks the weekly CRL gain of male fetuses is greater than that of female fetuses confirms the assumptions and findings of Ben-Haroush et al [40]. In line with the predictions and results of other authors, no significant relationships between gestational follicle volume, fetal volume and fetal heart rate and fetal sex were found in the present study. In contrast, the present study shows that it is possible to predict fetal sex with high probability based on CRL examination earlier than previously thought.

Ethics approval and consent to participate

All methods were carried out in accordance with relevant guidelines and regulations.

All experimental protocols were approved by a named institutional and/or licensing committee. The study protocol was approved by the Bioethics Committee at the District Medical Chamber in Koszalin (KB 1/10/2021 of October 27, 2021).

Informed consent was obtained from all subjects and/or their legal guardian(s).

Consent for publication

Not applicable

Availability of data and materials

Data available upon request. Contact person Anna Szczepanska-Przekota, [email protected]

Competing interests

The authors declare that they have no competing interests.

Funding

Research conducted with the authors' own funds.

Authors' contributions

JB (Author A) and JB (Author D) designed the research study. JB (Author A) performed the research. MB (Author B) provided help and advice. AS-P (Author C) analyzed the data. JB, AS-P, JB (Author A, C and D) wrote the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript.

Acknowledgements

Not applicable

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No competing interests reported.

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Possibilities to Estimate the Sex of the Fetus in Very Early Pregnancy

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