Impact of Assisted Reproductive Technology on Embryonic Health Screening Tests: A Case-Control Study


Shirin Soltani ORCID 1 , * , Akbar Karimi 1 , Zahra Khashavi 2 , Nasibeh Roozbeh 3

1 Department of Biology, Payame Noor University, Tehran, Iran

2 IVF Center of Omeleila Hospital, Bandar Abbas, Iran

3 Mother and Child Welfare Research Center, Hormozgan University of Medical Sciences, Bandar Abbas, Iran

How to Cite: Soltani S, Karimi A , Khashavi Z , Roozbeh N . Impact of Assisted Reproductive Technology on Embryonic Health Screening Tests: A Case-Control Study, Hormozgan Med J. Online ahead of Print ; In Press(In Press):e103645. doi: 10.5812/hmj.103645.


Hormozgan Medical Journal: In Press (In Press); e103645
Published Online: November 14, 2020
Article Type: Research Article
Received: April 25, 2020
Revised: July 11, 2020
Accepted: July 29, 2020
Uncorrected Proof scheduled for 25 (1)


Background: More than one million children throughout the world are born by using fertility techniques. In this process, human intervention and laboratory conditions may have an impact on the growth and development of the fetus.

Objectives: The present study aimed to consider the effect of assisted reproductive techniques on the results of embryonic health screening tests.

Methods: In this case-control study, among clients who went to the Infertility Center of the Ome Leila Hospital in Bandar Abbas Province, we selected 200 pregnant women who were impregnated via assisted reproductive techniques as the case group and 200 natural pregnant women as the control group by sample random sampling. Checklists were filled out and data analyzed by the SPSS version 21 and chi-square tests by using an interview with pregnant women and investigating the embryonic screening tests.

Results: Most of the participants in the study were housewives and had primary infertility. Their BMI was normal. There was no significant difference between the final results of the embryonic screening test in the two groups at the first trimester (P > 0.05). However, in the second trimester, there was a significant difference between them. The positive results were higher in the case group in comparison with the control group (P = 0.001). The mean of all types of screening tests in the first trimester in both groups was not different, significantly (P < 0.05), although in the second trimester, the mean of all the testis, including BHCG (P = 0.006) and AFP (P = 0.018) in both case and control groups, was significantly different. The mean of BHCG and AFP was higher in the case group, while the mean of Estradiol and Inhibin, NB, and NT were not really different (P < 0.05).

Conclusions: Our study showed that embryonic screening tests, particularly the BHCG and AFP tests, would be affected by applying the assisted reproductive techniques. For the couples who had a history of infertility and used reproductive methods, screening tests are essential in the first and second trimester.

1. Background

Recent scientific researches have revolutionized the possibility of fertility for infertile couples. More than one million children worldwide are born using assisted fertility techniques. In this process, human intervention and laboratory conditions may affect the growth and development of the fetus (1, 2), early diagnosis of a congenital anomaly at the beginning of pregnancy and termination can decline the birth of disabled infants (1). Since congenital anomalies such as trisomy 13, 18, and 21 result in economic, social, and cultural burdens for families and society, early diagnosis in the first trimester of pregnancy and termination can prevent these disadvantages. Down syndrome -in most cases- is a non-hereditary genetic condition (2, 3). Prenatal screening tests are conducted to check and diagnose these three disorders (4, 5). Embryonic health screening tests can be fulfilled from the beginning of the 11th week up to 13 week + 6 days. All people under 35 years old and over are advised to be screened. The stages include:

1) Providing history and conducting genetic council before examinations.

2) Performing ultrasound for measuring the nuchal translucency (NT) in the first trimester.

3) Measuring the pregnancy-associated plasma protein-A (PAPP-A) and human chorionic gonadotropin (hCG) in the maternal serum.

4) Assessment of the risk ratio by using software that is approved by the FMF.

5) Genetic council after examinations to interpret and justify the results and provide the necessary information and answer questions (4-6).

Screening for the second trimester is conducted between the 15th and 20th weeks of pregnancy and includes:

1) Measuring the nuchal translucency (NT) and the nasal bone.

2) Measuring three biochemical markers in the mother’s serum (alpha-fetoprotein, free B-HCG, and non-conjugated estradiol).

3) Also, in some laboratories, the fourth biochemical marker, so-called Inhibin A, is measured. This method is nominated Quadruple test that determines whether the baby is at risk for specific chromosomal disorders and neural tube defects (NTD) or not) (6).

Studies have shown that children who are born via fertility treatment, particularly IVF, have more congenital anomalies than the babies from normal-conception (3-7). Hunt et al. (8) showed that PAPP-A levels in those who have been treated by assisted fertility methods, such as IVF, were higher than those who became pregnant spontaneously. It could be a result of the ICSI technique through microinjection that makes bleeding in zona pellucida so that the placenta and biochemical tests in the first trimester can be affected (8). Human intervention and laboratory conditions in assisted reproductive techniques may affect the fetus growth and development. The more aggressive is the method, the more likely will be the disorder in the fetus (2).

The underlying reasons behind conducting this research include the significance of early diagnosis of congenital malformations (particularly in couples who have been waiting for a child many years and paying much more expenditures), the lack of study in our country about embryo screening tests in the first and second trimesters among infertile women who have applied fertility methods, comparison with natural-pregnant women, and contradictory results.

2. Objectives

Therefore, the purpose of the study was to compare the results of embryonic health screening tests in pregnant women impregnated with assisted reproductive techniques and natural pregnant women in Bandar Abbas City.

3. Methods

This case-control study was conducted between 1/4/2017 and 30/11/2017. The study population included pregnant women referred to the Ome Leila Fertility and Infertility center in Bandar Abbas City. At first, 400 pregnant women referred to the center. Based on the statistical population size and Cochran’s formula at 95% confidence level:

Equation 1.n=z2pqd21+1N(z2pqd2-1)

Two hundred pregnant women with assisted reproductive techniques as case group and 200 natural pregnant women as a control group were selected by a simple random sampling method. The inclusion criteria comprised: Iranian women with the Persian language, single pregnancy, having no specific disease related to pregnancy, and at the 21st - 42nd week, performed both screening tests (the first-trimester screening and Quadruple test in the second). Enough information about the goals of the study was provided, and then written consent was taken.

To gather data, a researcher-made checklist was used whose validity had been previously considered. It contains personal and demographic information (such as mother and father’s occupation and age, maternal body mass index, father’s smoking, the length of marriage, infertility history, duration of infertility, cause and type of infertility, serum levels of embryo screening tests, family history of marriage, and a mother’s medical history). This information was obtained by interviewing with mothers, considering tests and sonographies, and health records. At one laboratory (Dr. Sobhani’s Laboratory in Bandar Abbas) the related tests were evaluated. In the first trimester, two markers (PAPP-A and Free BhCG), beside NT marker in ultrasonography (NT), were simultaneously measured. Then, in the second trimester, four biochemical markers were measured (alpha-fetoprotein, free B-HCG, non-conjugated estradiol, and inhibin A). After the analysis of the results, a number was found, which was equivalent to the probability of embryonic chromosomal abnormalities (Down syndrome, Trisomy 18, and 13). Based on this number, we were able to decide.

To calculate the risk of disease, MOM (multiple of the median) software was used as an assessing indicator of the ratio of measured marker to the expected marker in a specific pregnancy week. Then, the data were encoded and analyzed by SPSS 21-version. Statistical tests such as ANOVA, t-test, and chi-square were used. The significance level in all stages was (P = 0.05).

4. Results

In the present study, the mean age of women was 31.3 ± 6.26 years, and the men were 35.6 ± 6 years (Table 1).

Table 1. Demographic Data in Both Case and Control Groupsa
Age of the woman, y31.3 ± 6.2628 ± 6.0
Age of the man, y35.6 ± 632 ± 6.1
The length of marriage, y5.17 ± 8.865.0 ± 4.9
The length of infertility, y7.3 ±5.153.06 ± 1.17

aValues are expressed as mean ± SD.

Most women were housekeepers who had primary infertility and no history of smoking or underlying disease. Most of them had a normal BMI.

The results showed no significant difference (P > 0.05) in the final findings between two groups in the first trimester. However, in the second one, it was higher in the case group (P = 0.001) (Table 2).

Table 2. Results of Embryo Screening Tests in the First and Second Trimester in Both Case and Control Groupsa
Test ResultCaseControlTotalP Value
First trimester0.372
Positive10 (2.5)11 (2.8)21 (5.3)
Negative152 (38)221 (55.3)373 (93.3)
Intermedia4 (1)2(0.5)6 (1.5)
Second trimester0.001
Positive21 (5.3)5 (1.3)26 (6.5)
Negative136 (34)221 (55.3)357 (89.3)
Others (intermediate and non-performing)9 (2.3)8 (2)17 (4.3)

aValues are expressed as No. (%).

Although, in the first trimester, the mean of all types of tests was not significantly different (P > 0.05), in the second trimester, the mean of screening tests, including BHCG (P = 0.006) and AFP (P = 0.018), was higher in the case group than the control group. However, there was no significant difference in the mean of Estradiol, Inhibin, NB, and NT tests in the both (P < 0.05) (Table 3).

Table 3. Types of Embryo Screening Tests in the First and Second Trimester in Both Case and Control Groupsa
VariableCaseControlP Value
1st trimester
PAPPA2609.47 ± 5434.573265.78 ± 4433.670.201
BHCG786.80 ± 4193.05812.23 ± 3449.840.947
NT1.16 ± 0.341.22 ± 0.630.252
Yes164 (41)234 (58.5)
No2 (0.5)0 (0)
2nd trimester
ESTRADIOL4.44 ± 16.653.01 ± 2.440.203
BHCG32090.62 ± 43828.9621903.65 ± 15117.890.006
Inhibin269.46 ± 279.4921903.65 ± 15117.650.153
AFP51.99 ± 65.66239.35 ± 124.280.018

aValues are expressed as mean ± SD.

5. Discussion

Based on the cause of infertility, it is possible to become pregnant for infertile women by using assisted reproduction technology. Various studies have shown that children born through infertility treatments, particularly IVF, have more congenital anomalies than normal births (7-12). Prevention of chromosomal abnormalities is only possible via doing the screening tests, detecting malformations, and terming this pregnancy with family consent. The purpose of this study was to determine the effect of assisted reproduction technology on screening tests in two groups: one included women impregnated by assisted reproductive methods, and the other one included natural pregnant women. The present study did not illustrate any significant difference in the use of these techniques on the screening tests in the first trimester. Bellver et al. (13) found that assisted reproductive methods did not affect the first-trimester biochemical tests. Ghisoni et al. (2003 - 2000) showed that the first-trimester biochemical tests such as free BhCG, PAPP-A levels, and NT were not significantly different among natural pregnant women or those impregnated by using assisted reproductive techniques (14). In the second trimester, our study showed that there was a significant difference between the case and control groups. The probability of congenital anomalies was higher in the case group. The results showed that the mean between groups in the first trimester did not have any significant difference. However, in the second trimester, the mean of tests, including BHCG and AFP, was not significantly different. It was higher in the case group than the control. The results of Raty et al. (15), which were similar to this study, showed that by using IVF and ICSI, IVF in particular, BHCG was higher than in the control group. The level of AFP had approximately the same level. The number and time of embryo transmission may be effective on the marker level (15). One of the hypotheses is that in assisted reproductive techniques, the dosage of gonadotrophin can change the level of markers (16). The high levels of BHCG and AFP markers could indicate the possibility of Down syndrome in fetuses resulted from assisted reproductive technology. This study showed that the use of assisted reproductive techniques did not have any impacts on the mean of Estradiol and INHIBNE, NB, and NT.

5.1. Conclusions

The results of our study showed that embryonic health screening tests, particularly BHCG and AFP markers, will be affected by the use of assisted reproductive techniques. For couples who have a history of infertility and using these techniques, screening tests are essential in the first and second trimesters.




  • 1.

    Pour Javad M, Dadgir S, Shakeri M, Mahmoudi M. S. tudy of the results of triple test and obtaining median serum markers for early diagnosis of fetal abnormalities in the first trimester of pregnancy. Iran J Obst Gynecol Infertil. 2010.

  • 2.

    Mozafari Kermani R, Arabi N, Shahzade Fazeli S, Azeri A. Physical health status of children after fertility treatment. J Army Univ Med Sci Islam Republ Iran. 2010;8(1):56-65.

  • 3.

    Ishraqi M. Medical ethics. 1. Tabriz, Iran: Islamic Azad University Press; 1988.

  • 4.

    Solook M. Medical and divine responsibility. . Tehran, Iran: Culture Publishing; 1990. p. 30-2.

  • 5.

    Sa'idi A. Investigating the results of triple trials and obtaining the median index of serum markers for the early diagnosis of fetal anomalies in the first trimester of pregnancy. University of Tehran; 2014.

  • 6.

    Moderesi M, Karami F, Noori M. Non-invasive methods for screening Down syndrome through maternal blood. J Modern Genetics. 2015;10(2):135-50.

  • 7.

    Naghizadeh S, Ebrahimpour Mirza Rezaie M, Fath Negad Kazemi A, Azari S, Alizadeh S. Assessing and comparing result of amniocentesis and triple marker tests to detection the aneuploidies. J Urmia Nurs Midwifery Fac. 2015;13(7):596-604.

  • 8.

    Hunt LP, McInerney-Leo AM, Sinnott S, Sutton B, Cincotta R, Duncombe G, et al. Low first-trimester PAPP-A in IVF (fresh and frozen-thawed) pregnancies, likely due to a biological cause. J Assist Reprod Genet. 2017;34(10):1367-75. doi: 10.1007/s10815-017-0996-1. [PubMed: 28718082]. [PubMed Central: PMC5633581].

  • 9.

    Hasanzadeh R, Naghizadeh S, Azari S, Ebrahimpour Mirza Rezaei M. Diagnosis of Aneuploidies by amniocentesis in high risk cases of first trimester screening test. Iran J Obstet Gynecol Infertil. 2014;17(119):18-26.

  • 10.

    Ong CY, Liao AW, Spencer K, Munim S, Nicolaides KH. First trimester maternal serum free beta human chorionic gonadotrophin and pregnancy associated plasma protein A as predictors of pregnancy complications. BJOG. 2000;107(10):1265-70. doi: 10.1111/j.1471-0528.2000.tb11618.x. [PubMed: 11028579].

  • 11.

    Niemimaa M, Heinonen S, Seppälä M, Hippeläinen M, Martikainen H, Ryynänen M. First-trimester screening for Down’s syndrome in in vitro fertilization pregnancies. Fertil Steril. 2001;76(6):1282-3. doi: 10.1016/s0015-0282(01)02871-0.

  • 12.

    Elejalde BR, de Elejalde MM, Acuna JM, Thelen D, Trujillo C, Karrmann M. Prospective study of amniocentesis performed between weeks 9 and 16 of gestation: its feasibility, risks, complications and use in early genetic prenatal diagnosis. Am J Med Genet. 1990;35(2):188-96. doi: 10.1002/ajmg.1320350210. [PubMed: 2309757].

  • 13.

    Bellver J, Lara C, Soares SR, Ramirez A, Pellicer A, Remohi J, et al. First trimester biochemical screening for Down's syndrome in singleton pregnancies conceived by assisted reproduction. Hum Reprod. 2005;20(9):2623-7. doi: 10.1093/humrep/dei107. [PubMed: 15905286].

  • 14.

    Ghisoni L, Ferrazzi E, Castagna C, Levi Setti PE, Masini AC, Pigni A. Prenatal diagnosis after art success: The role of early combined screening tests in counselling pregnant patients. Placenta. 2003;24:S99-S103. doi: 10.1016/s0143-4004(03)00178-4.

  • 15.

    Raty R, Virtanen A, Koskinen P, Anttila L, Forsstrom J, Laitinen P, et al. Serum free beta-HCG and alpha-fetoprotein levels in IVF, ICSI and frozen embryo transfer pregnancies in maternal mid-trimester serum screening for Down's syndrome. Hum Reprod. 2002;17(2):481-4. doi: 10.1093/humrep/17.2.481. [PubMed: 11821299].

  • 16.

    Maymon R, Shulman A. Comparison of triple serum screening and pregnancy outcome in oocyte donation versus IVF pregnancies. Hum Reprod. 2001;16(4):691-5. doi: 10.1093/humrep/16.4.691. [PubMed: 11278220].

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