Objective. The etiology of embryonic demise is multifactorial, with chromosomal abnormalities being the most common (40%). The purpose of this study is to evaluate the correlation between a serum biomarker, inhibine A, and an ultrasonographic parameter, the distance between yolk sac (YS) and embryo (DYSE), in assessing the prognosis of pregnancy evolutivity in the first trimester. Method. The study is a case-control prospective analysis that includes two groups of patients: 81 patients with first-trimester pregnancy in evolution, and 89 patients with embryonic demise, all of the patients having amenorrhea between 6 and 11 weeks. The endovaginal ultrasonographic exploration was performed to evaluate the distance between the lower pole of the embryo and the yolk sac. From each subject enrolled in the study, 20 ml of blood was collected for inhibin A dosing. Results. Regarding the DYSE in the case group, lower values were observed compared to the control group, the difference being statistically significant. In the statistical analysis of serum inhibin A values, statistically significant differences were observed between the two groups (p<0.05). Conclusions. DYSE has a high positive predictive value in identifying pregnancies with potentially reserved evolutivity, this study demonstrating that a DYSE<3 mm may lead to an unfavorable evolution of pregnancy. The low serum level of inhibine A is associated with an increased rate of non-viable embryos.
first trimester, ultrasonography, prognosis, inhibin A
Obiective. Etiologia decesului embrionar este multifactorială, anomaliile cromozomiale fiind cele mai frecvente (40%). Scopul acestui studiu este de a evalua asocierea dintre un biomarker seric, inhibina A, şi un parametru ultrasonografic, distanţa dintre vezicula vitelină (VV) şi embrion (DVVE), în evaluarea prognosticului evolutiv al sarcinii de trimestrul întâi. Materiale şi metodă. Studiul este o analiză prospectivă de tip caz-control care include două grupe de paciente: 81 de paciente cu sarcină de trimestrul întâi în evoluţie şi 89 de paciente cu sarcină oprită în evoluţie, amenoreea fiind cuprinsă între 6 şi 11 săptămâni. A fost efectuată o examinare ultrasonografică endovaginală pentru a evalua distanţa dintre polul inferior al embrionului şi VV. De la fiecare pacientă înscrisă în studiu, s-au colectat 20 ml de sânge pentru dozarea serică a inhibinei A. Rezultate. În ceea ce priveşte DVVE în grupul de caz, s-au observat valori mai scăzute comparativ cu grupul de control, diferenţa fiind semnificativă din punct de vedere statistic. În analiza statistică a valorilor inhibinei serice A, s-au observat diferenţe semnificative statistic între cele două grupuri (p<0,05). Concluzii. DVVE are o valoare predictivă pozitivă mare în identificarea sarcinilor cu evolutivitate potenţial rezervată, în acest studiu demonstrându-se că o DVVE<3 mm poate conduce la o evoluţie nefavorabilă a sarcinii. Nivelul seric scăzut al inhibinei A este asociat cu o rată crescută a embrionilor neviabili.
One in four pregnant women will miscarry at some time during her reproductive life, knowing that the incidence of early embryonic demise is higher compared with other early pregnancy complications(1). In more than 40% of cases, the etiology of embryonic demise are chromosomal abnormalities(2). Successful blastocyst implantation requires precise synchronization between the embryo and the uterine environment(3). Ultrasonographic examination is the method of choice in the diagnosis of embryonic demise(4). Ultrasonographic technique is dependent upon the skill of the operator and thus the results are not always consistently reproducible(5). In addition to this technique, another sensitive and specific biomarker is required to determine the pregnancy viability for early pregnancy termination(6). This pathology is associated with an increased emotional impact that leads to a standardized management(7).
It is well-established that high concentrations of inhibin A, activin A and follistatin are present in the maternal circulation throughout pregnancy. Placenta is a source for inhibin/activin subunits, dimeric proteins, follistatin and activin receptors throughout pregnancy. Inhibins (a-b dimers) and activins (b-b dimers) are members of the transforming growth factor beta (TGF-beta) family. Although the proteins and receptors are expressed in the placenta, the exact role of these proteins in pregnancy is yet unclear. However, maternal circulating concentrations of inhibins and activins are increased in pregnancy complications. The most common pregnancy complication is miscarriage, affecting 20% of pregnancies(8).
Ultrasound evaluation is the method of choice for assessing evolutivity prognosis of the early pregnancy. One in four women at some point in her reproductive lifetime loses a pregnancy(9). There are no prospective data to outline the guidelines for ultrasound diagnosis as accurate as possible for embryonic demise. The results are limited by the small number of patients and previous studies conducted long ago and by the variable reference standards for the diagnosis of early embryonic death. However, the ultrasonographic endovaginal technique has become a valuable tool in the worthy assessment of pregnancy and has helped to establish new elements about its evolution. The ultrasonography diagnosis of the first-trimester pregnancy with unfavorable outcome was based on a variety of elements, represented by the absence of yolk sac (YS), the lack of identification of the embryo or its cardiac activity, and abnormalities of the gestational sac (GS)(10).
The identification of other prognostic ultrasonographic elements has also been required. In the early embryonic development, the embryo is immediately revealed adjacent to YS, the embryonic structure called ‘yolk stalk’ is not yet developed. The separation of the embryo to the YS is due to the development of both this structure and the embryo. Thus, the distance between the embryo and YS is increased due to the growth of ‘yolk stalk’. For embryos with crown-rump length (CRL) less than or equal to 5 mm, there must be no separation of the YS(11). When the embryo reaches 5 mm, there is a physiological separation between the embryo and YS due to the development of this structure. The absence of separation of the embryo and YS, when the CRL exceeds 5 mm and the embryo is with cardiac activity, is considered an unfavorable prognostic factor(12). M.R. Filly et al. argue that this ultrasonographic parameter is valuable in anticipating an unfavorable evolutivity of the pregnancy, whether used alone or in combination with other abnormal features(11).
The aim of this study was to investigate the potential value of various novel biochemical markers in combination with clinical and ultrasound findings for the prediction of pregnancy evolutivity in the first trimester.
Materials and method
The study is a case-control prospective analysis that includes 170 patients in the first trimester of pregnancy without associated pathology. The patients were followed for a period of two years, between 2016 and 2018. The patients were grouped in two lots: the control group of 81 first-trimester pregnancy patients, and the case group of 95 pregnant patients with a potentially reserved evolutivity pregnancy with amenorrhea of 6-11 weeks. The latter showed unfavorable prognostic ultrasonographic signs, respectively a distance between YS and embryo (DYSE) less than 3 mm at a gestational age at which the separation would have already occurred. For six of these, after repeated ultrasounds, it wasn’t confirmed the prediction of an unfavorable evolution. Thus, the case group included 89 patients with embryonic demise.
Endovaginal (a 6.5 MHz nominal frequency probe) ultrasound examination (Toshiba Aplio 300 device) was performed in dorsal decubitus with bent knees in order to identify predicted evolutivity prognostic factors: CRL, GS size, appearance and size of YS, and the distance between the lower embryo pole and the YS. There were on average 2-3 serial examinations, performed at regular intervals of 3-5 days, also by the same examiner, until the final diagnosis of embryonic demise. The measurements of DYSE were performed using the sagittal section and evaluating three different distances from the inferior pole of the embryo and YS, the smallest from those three distances being taken into account.
From each subject enrolled in the study, 20 ml of blood was collected by venipuncture into anticoagulant-free tubes for inhibin A dosing.
The participation of the patients in the research activities was made only after the informed consent. Also, the working methodology has been approved by the Ethics Commission of the “Iuliu Haţieganu” University of Medicine and Pharmacy, Cluj-Napoca, no. 85/24.03.2016.
For normal distribution, the Shapiro-Wilk test was used. In the case of normal distribution data, the t (Student) test was used, and in the case of non-uniform distribution values or ranks, the non-parametric Mann-Whitney (U) test was used. The significance threshold for the tests used was =0.05 (5%), =0.01 (1%), or =0.001. The Pearson correlation coefficient (r) was used to detect the correlation between two continuous quantitative variables (normal). In the case of non-uniform distribution variables, Spearman () correlation coefficient was used. The analysis of correlation coefficients was performed using Colton’s rule. Linear regression was the method used to obtain the mathematical equation of the dependence of a continuous variable on another variable. Statistical processing was performed with the Stats Direct v.2.7.2 program, Open Epi 3.03 and the Excel application (from the Microsoft Office 2010 package).
In the statistical analysis of DYSE, statistically significant differences were observed between the two groups (p<0.001).
In the statistical analysis of inhibin A (INHA) values, statistically significant differences were observed between the two groups (p<0.001) – Table 1.
Inhibins are glycoprotein hormones, which are under two molecular forms, inhibin A and inhibin B(13). Classically, inhibin is known to have a negative feedback effect on pituitary follicle-stimulating hormone secretion. The fetoplacental unit produces inhibin throughout pregnancy(14). Inhibin A is the predominant molecular form of inhibin in maternal circulation from four weeks of gestation. Although the precise biological function of inhibin A in pregnancy is unclear, it is obvious from recent studies that inhibin A could be a better marker of placental function than human chorionic gonadotropin because of its shorter half-life(15). The possible clinical applications for the measurement of inhibin A in early pregnancy could be in predicting miscarriage, Down’s syndrome, preeclampsia, and fetal growth restriction in the first and/or second trimester before the onset of the clinical symptoms(13). The source of inhibins, factors controlling inhibin production, the possible functions of inhibin, and the use of inhibin measurement in normal and high-risk pregnancy are reviewed(14).
In this study, our aim was to investigate a possible role for inhibins as markers of early pregnancy viability in patients with embryonic demise. The early diagnosis of a complicated or poor pregnancy outcome could aid in counseling and management of this group of patients.
Recent studies suggest that inhibine A measured in early pregnancy may be a powerful predictor of pregnancy outcome in natural conceptions. Decreased inhibine A levels lead to abortion. Therefore, this prospective study was designed to detect the relation between serum inhibin A and the viability of the pregnancy during the first trimester. Our data suggest the possibility that the early stages of pregnancy may be particularly sensitive to inhibine A deficiency. The study conﬁrmed that inhibin A levels in embryonic demise were signiﬁcantly lower than in healthy controls, as already reported by Lockwood et al.(16). So, the evaluation of inhibin A levels may therefore be helpful in the management of early pregnancy problems. When evaluated in a larger population, according to the different gestational ages, or longitudinally in the same patients(17), inhibin A was found to be signiﬁcantly lower in patients who had an embryonic demise than in healthy patients.
There is also a study which demonstrated that serum concentrations of inhibin A were 70% lower in women with clinical symptoms of sporadic miscarriages compared to controls matched for gestational age, consistent with other previous studies(18). These observations suggest that circulating levels of inhibin A in miscarriages may be decreased because of reduced placental mass and/or decreased secretion by the trophoblasts into the circulation and/or reduced luteal function rather than any changes in gene expression or protein production in the placenta. In our study, the patients had no symptoms, but they had the unfavorable ultrasound prognostic factors, leading to the idea that their pregnancies were at risk.
There are also some patients that express differing inhibin A values in a normal pregnancy. In a study, nearly half of the patients in the normal pregnancy group were found to have a lower range of inhibin A values (<180 pg/ml). This was also observed in an earlier study(19).
It may be possible that the patients with normal intrauterine pregnancy and a low inhibin A concentration may have a poor outcome, as reported earlier(20,21).
In our study, 6.7% of viable pregnancies had serum inhibin level <17 ng/mL, while 20.7% of non-viable pregnancies had serum inhibin A level >80 ng/mL; the serum inhibin A at a cut-off level of 16.48 ng/mL was 99.3% sensitive for the diagnosis of non-viable pregnancy. Also, in this study, 1.1% of viable pregnancies had a serum inhibin A level <84.18 ng/mL, while 4.8% of non-viable pregnancies had a serum inhibin A level >84.18 ng/mL.
Future trials and large population studies are needed to support our findings and to establish the cut-off values of serum inhibin A to differentiate between viable and non-viable pregnancies.
There are studies that had identified a DYSE of less than 3 mm in size, evidenced in pregnancies with CRL>5 mm and an embryo without cardiac activity, and considered that this was defining for the diagnosis of embryonic demise(22). In the absence of the specific ultrasonographic signs of unfavorable prognosis of the first-trimester pregnancy, such as hypotonic GS, increased size of YS and embryonic bradycardia, identifying a DYSE<3 mm at an early stage of pregnancy could increase the accuracy of the diagnosis(9).
Prospective data on which precise prognostic factors can be established to determine a series of decisive predictive factors in the ultrasonographic diagnosis of the first-trimester pregnancy with a potentially reserved evolutivity are insufficient. Transvaginal ultrasound has become an important tool for assessing the first-trimester pregnancy, and it helped to identify new parameters of pregnancy evolutivity.
The data from the present study shows that DYSE is an ultrasonographic feature that indicates earlier embryonic demise compared to specific ultrasonographic parameters, which certainly confirms the absence of pregnancy evolution in a more advanced gestational age. For higher diagnostic accuracy, it is important that this parameter be correlated with a serum marker. The clinical value of the study results consists in the possibility of using these markers to establish a management in the very early stages of pregnancy, for patients with ultrasonographic parameters considered to be of unfavorable prognosis. By this association, the originality of this study is outlined: the correlation of this ultrasound parameter with an important hormone, which is highly used in literature in the prediction of some pathological entities that appear late in pregnancy, but which is known to have an important role in the first trimester.
DYSE has a high positive predictive value in identifying potentially reserved evolutivity of pregnancies, this study demonstrating that a DYSE<3 mm leads to an unfavorable evolution of pregnancy. Also, correlating this ultrasound parameter with a serological one, respectively with the inhibin A serum level, brings valuable information about pregnancy viability in the first trimester.
The low serum level of inhibin A is associated with an increased rate of non-viable embryos.
Conflict of interests: The authors declare no conflict of interests.
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