Serum β-hCG, AMH, TSH, and PRL Levels in Predicting the Outcome of Early Pregnancy 14 Days after Embryo Transfer: A Prospective Cohort Study

Abstract

Background:

Pregnancy outcomes in assisted reproductive technology (ART) are influenced by the ability of the embryos to create a pregnancy and maternal factors such as hormone levels to maintain and develop the pregnancy. This study investigated the role of β-human chorionic gonadotropin (β-hCG), anti-mullerian hormone (AMH), thyroid-stimulating hormone (TSH), and prolactin (PRL) in predicting pregnancy outcomes and determined cutoff values in women undergoing ARTs.

Materials and Methods:

This prospective study included 153 women who underwent infertility treatment in the Department of Reproductive Medicine of the Yas Hospital, Tehran University of Medical Sciences (Tehran, Iran). Serum β-hCG level on day 14 after embryo transfer and serum AMH, TSH, and PRL were measured. Patients with β-hCG levels >25 mIU/ml were included in the study. Finally, any statistically significant relationships between these serum values and clinical pregnancy and live birth rate were investigated. To predict pregnancy outcomes with β-hCG levels, a receiver operating characteristic curve was used to estimate the β-hCG cutoff values.

Results:

Serum β-hCG levels were significantly higher in live-birth patients than in other patients. The β-hCG cutoff value for live births was 830 mIU/ml, with an AUC of 0.5920, sensitivity of 61.04%, and specificity of 56.58%. Serum AMH, TSH, and PRL levels did not show any significant results.

Conclusions:

Serum β-hCG levels on day 14 after embryo transfer can be used to predict the probability of live birth in patients undergoing infertility treatment.

INTRODUCTION

Human chorionic gonadotropin (hCG) is a glycoprotein produced during pregnancy that forms placental trophoblasts.[1] This illustrates a key embryonic signal necessary to maintain pregnancy. Other tissues, including hyperplastic and malignant cells such as choriocarcinoma and other neoplasms, also secrete it. HCG comprises two distinct glycosylated subunits: alpha and beta (β). The alpha subunit has 92 amino acids and is identical to the pituitary luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone (TSH). The β-subunit is different and contains 145 amino acids. This subunit is responsible for the biological activities of hCG.[2] As pregnancy progresses, the volume of trophoblastic tissue and serum levels of β-human chorionic gonadotropin (β-hCG) increase. Secreted β-hCG is a qualitative marker of trophoblastic function. It is generally accepted that low-serum β-HCG levels indicate an abnormal pregnancy, such as ectopic pregnancy or early delivery. However, there is no single threshold for evaluating pregnancy outcomes according to serum β-HCG levels.[3] In addition to β-HCG, hormones such as anti-mullerian hormone (AMH), TSH, and prolactin (PRL) play an important role in fertility, but their role in pregnancy outcomes is widely debated.[4,5,6]

Pregnancies after assisted conception and embryo transfer are at a greater risk of adverse outcomes than women who conceive naturally.[7] Premature birth, low birth weight, and birth complications are significantly increased after in vitro fertilization (IVF) compared with spontaneous pregnancies.[8] Several studies an association between serum β-HCG levels after embryo transfer and clinical pregnancy (CP) outcomes.[9]

In patients undergoing assisted reproductive treatment, the ability to predict the outcome earlier can reduce anxiety and help determine appropriate follow-up guidelines. This study aimed to determine the relationship between serum β-hCG levels on day 14 after embryo transfer, AMH, TSH, PRL levels, and pregnancy outcomes during infertility treatment cycles.

MATERIALS AND METHODS

This prospective study included 184 women who received infertility treatment in the Department of Reproductive Medicine of the Yas Hospital, Tehran University of Medical Sciences (Tehran, Iran) between June 2020 and December 2022. Thirty-one patients were excluded from the study due to incomplete information, and finally, research was conducted on the remaining 153 patients.

All women included in this study had a BMI below 30, were aged between 20 and 42 years, had at least two fetuses with grade A or B, and had no previous uterine abnormalities, submucous myoma, or polyps.

In the frozen embryo transfer (FET) cycle, on the second or third day of the menstrual cycle, estradiol 6 mg/day was started for the patients and sonographic evaluation of the endometrial thickness was performed, after which the drug dose was adjusted. When the endometrial thickness was ≥8 mm, 50 mg intramuscular injection of progesterone was administered twice a day. Three days later, two or three grade A or B eight-cell embryos were transferred under ultrasound guidance using the Cook (Cook Medical, USA) catheter. In the fresh embryo transfer and FET, serum β-hCG assays were done 14 days after embryo transfer. Patients with β-hCG levels >25 mUI/ml were included in the study. Serum β-hCG assays was performed by the enzyme-linked immunofluorescence method. CP was confirmed after observing the gestational sac using ultrasonography.

Statistical analysis

Statistical analysis was performed through the GraphPad Prism8 software. Student’s t-test or Mann–Whitney test was used for statistical comparisons. Statistical significance was set at P <0.05. The receiver operating characteristic (ROC) curve was used to estimate the β-hCG cutoff values. The area under the curve (AUC) was calculated, and cutoff values were determined when the Youden index (sensitivity + specificity − 1) was the largest.

RESULTS

Pregnancy results of patients with various β-hCG levels

A total of 153 women with β-hCG levels above 25 mUI/ml were enrolled in this study. Their average age and BMI are, respectively, 32.6 ± 4.97 and 25.17 ± 4.5. β-hCG levels were divided into 10 groups shown in Table 1. There were no significant differences in age, BMI, TSH, AMH, and PRL between these groups, but pregnancy results were better in patients with high serum β-hCG levels. When the β-hCG level was less than 400 mIU/ml, the mean live birth rate (LBR) was only 27.9%; however, when the β-hCG level was ≥400 mIU/ml, the mean LBR was 53.5%.

Table 1.

Characteristics and clinical outcomes of patients with different serum β-hCG level (mUI/ml)

BHCG		≤100		101–200		201–300		301–400		401–500		501–600		601–1,000		1,001–2,000		2,001–4,000		4,001–6,000
Clinical pregnancy [n (%)]		3/8 (37.5)		3/5 (60)		6/9 (66.6)		4/6 (66.6)		7/8 (85.5)		13/14 (92.8)		36/50 (72)		18/27 (66.6)		20/22 (90.9)		2/4 (50)
Live birth [n (%)]		2/8 (25)		1/5 (20)		3/9 (33.3)		2/6 (33.3)		4/8 (50)		8/14 (57.1)		28/50 (56)		12/27 (44.4)		14/22 (63.6)		2/4 (50)
AGE		32		31		34.14		36.33		29.62		31.38		32.3		33.15		34.4		32.33
BMI		25.25		26.4		26.8		22.9		24.04		25.01		24.78		24.51		24.49		30.2
TSH		1.96		2.85		3.04		3.22		2.58		2.54		2.1		2.33		2.21		2.92
AMH		2.7		4.43		5.29		2.4		2.18		4.61		3.82		5.01		3.31		2.49
PRL		11.26		15.9		15.4		12.25		11.48		29.56		13.9		14.1		23.6		–

Characteristics of pregnancy with live birth versus nonlive birth

Baseline characteristics, including β-hCG, age, AMH, PRL, TSH, and body mass index (BMI), were comparable for different pregnancy outcomes. As shown in Table 2, there was no statistically significant difference between CP and LBR of patients with age, AMH, PRL, TSH, and BMI; however, the serum level of β-hCG in patients with live births was significantly higher than in patients with nonlive births (P = 0.0493), as well as in patients with CP and nonlive births (P = 0.0323). However, no significant association was found between β-hCG levels and CP (P = 0.6708) [Figures 1 and 2].

Table 2.

Comparison of characteristics of patients with different pregnancy outcomes

		CP+/CP−P		Significantly different		LB+/LB−P		Significantly different		LB+/CP+/LB−P		Significantly different
β-hCG		0.6708		No		0.0493		Yes		0.0323		Yes
AMH		0.8787		No		0.8140		No		0.8857		No
PRL		0.5906		No		0.6875		No		0.4907		No
TSH		0.2980		No		0.0730		No		0.1562		No
BMI		0.1338		No		0.5665		No		0.9503		No
AGE		0.8831		No		0.5272		No		0.5009		No

Student’s t-test or Mann–Whitney test was used for statistical comparisons. Statistical significance was set at P<0.05. CP=Clinical pregnancy, LB=live birth rate

Figure 1.

Statistical comparison of β-HCG levels (a) in cases with and without clinical pregnancy, (b) in clinical pregnancy cases with and without live birth, and (c) in cases with and without live birth

Figure 2.

Statistical comparison of (a) age, (b) AMH levels, (c) BMI, (d) prolactin levels, and (e) TSH levels in cases with and without live birth

Prediction of pregnancy outcomes with β-hCG level

The ROC curve was used to predict pregnancy outcomes based on the β-hCG levels. The threshold for live births, regardless of the presence or absence of CP, was 830 mIU/ml with an AUC of 0.5920, a sensitivity of 61.04%, and a specificity of 56.58%. In the case of CP, the β-hCG cutoff value for live births was 830 mIU/ml with an AUC of 0.6174, sensitivity of 65.91%, and specificity of 56.58% [Figure 3, Table 3].

Figure 3.

Receiver operating characteristics (ROC) curve of β-hCG (a) in cases of clinical pregnancy with or without of live birth and (b) in cases with or without of live birth

Table 3.

Receiver operating characteristics (ROC) of β-hCG levels to predict pregnancy outcomes

		Estimate criterion		AUC		J †		Sensitivity		Specificity		P ‡
LB+/CP+/LB−		<830.0		0.6174		123.49		65.91		56.58		0.0326
LB−/LB+		<830.0		0.5920		118.62		61.04		56.58		0.0494

^†Youden index. ^‡Significance level P (area=0.05). AUC: Area under curve

DISCUSSION

In this study, the effects of AMH, PRL, TSH, and β-hCG levels on the pregnancy outcomes of patients, whose serum β-hCG levels were >25 mIU/ml 14 days after embryo transfer, were investigated. In our study, no significant difference was observed between LBR and AMH, PRL, TSH levels, age, and BMI; however, pregnancy outcomes were poor in patients with low β-hCG serum levels at baseline. A significant correlation was observed between the β-hCG level and LBR, but this correlation was not observed for the CP rate. It appears that β-hCG levels can indicate progression or nonprogression of pregnancy.

These results are consistent with those of previous studies. Porat et al.[10] compared pregnancy outcomes between IVF cycles with initial βhCG ≤150 mIU/ml on day 13 and pregnancies with an initial β-hCG level >150 mIU/ml. Poor pregnancy outcomes were significantly higher in pregnancies with initial β-hCG levels ≤150 mIU/ml. Sidora Hashemi et al.[11] compared serum β-hCG levels 16 days after embryo transfer between ongoing and ectopic pregnancies. The level of β-hCG in the ongoing pregnancy group was significantly higher than in the ectopic pregnancy group. They recommended that women with β-hCG levels below 278 IU/l should be closely monitored. Y. Zhang and coworker also found a strong association between serum β-hCG levels and reproductive outcomes.[12] Despite the consensus on this association, reports of cutoff values for predicting pregnancy outcomes are highly variable. In this study, the threshold for live births was 830 mIU/ml, with an AUC of 0.5920, a sensitivity of 61.04%, and a specificity of 56.58%. Povoa and coauthors examined the value of hCG level at day 13 post embryo transfer. The results of their study showed that singleton pregnancies with an initial hCG concentration <85 IU/l had an 89% risk of first-trimester loss. Those with hCG levels >386 IU/l had a 91% chance of live birth.[13] Another study was performed on fresh and frozen cycles to investigate β-hCG levels on postovulatory days (POD) 12 and 14. The cutoff values of POD 12 and 14 for live birth were 43.5 mIU/ml and 101.6 mIU/ml. In a study by Al Mamari et al.,[14] pregnancy outcome was evaluated based on day 16 for single embryo transfer cycles, and the cutoff value for live birth was reported as 213. Zhang et al.[12] remarked β-hCG cutoff values on day 13 for predicting biochemical pregnancy/CP, presence/absence of adverse pregnancy outcomes, and singleton/twin live birth as 89.6, 241.1, 585.9, and 981.1 mIU/l. They observed significantly higher LBRs in the group with the highest β-hCG concentration. This disagreement in the cutoff values of β-hCG can be due to reasons such as the testing day, number and quality of embryos, embryo stage on the day of transfer, fresh versus FET, procedure of fertilization and freezing/thawing, transfer protocol, embryo biopsy for preimplantation genetic testing, and cause of infertility.[15] For example, some studies show that FET leads to increased hCG levels and frozen-thawed embryo transfer leads to improved pregnancy outcomes. Also, In FET cycles, blastocyst transfer leads to higher hCG levels than frozen cleavage-stage transfer.[16,17]

Currently, AMH is considered the first and most sensitive marker of reproductive aging. In assisted reproductive technology, it is predictive of the ovarian response to ovulation stimulation and the onset of menopause. However, evidence regarding its ability to predict CP, live birth, and miscarriage is inconclusive.[4] This study, like many studies in this field, pointed out the inability of AMH to predict pregnancy outcomes.[18,19,20] However, other studies have reported similar results. Miyagi and coauthors reported that AMH predicts live births among older, but not younger, women.[20] Guo et al.[21] showed that higher baseline AMH levels in women with polycystic ovary syndrome (PCOS) resulted in a lower LBR, clinical pregnancy rate, and normal fertilization rate but did not influence the cumulative live birth rate. In addition, Arslanca et al.[22] reported that in assisted reproductive treatment, PCOS women with higher AMH levels had a higher risk of preterm delivery than PCOS patients with lower AMH levels.

Although there is much evidence supporting the effect of PRL on reproductive pathology, and hyperprolactinemia is a known cause of infertility through inhibition of gonadotropin production, most studies have rejected the association between PRL and pregnancy outcomes. However, few studies on IVF treatment have reported that higher PRL levels are associated with better pregnancy outcomes. Factors such as stress, infertility treatment protocol, and the timing of PRL level measurement can be the cause of this difference in the results.[5]

The TSH threshold and its role in pregnancy outcomes in cases of IVF/ICSI are widely arguable.[6] The reference range varies by ethnicity; reference levels are slightly higher in Caucasian women than in African and Asian women. In addition, some studies have reported the effects of age and smoking on TSH levels. It seems that in order to reach an agreed conclusion about the effect of TSH on pregnancy outcomes, other contributing factors must be excluded.[23]

The number and quality of oocytes decrease with increasing age.[24] In this study, the patients are the same in terms of the number and quality of the embryos, so no relationship was found between age and pregnancy outcomes.

Increased BMI has detrimental effects on female fertility.[25] To avoid the effect of overweight on pregnancy outcomes, all women included in this study had a BMI below 30.

In this study, attempts have been made to minimize other factors affecting pregnancy outcomes such as age, BMI, number, and quality of embryos, but there are still other intervening factors such as cause of infertility, fresh or frozen embryos, and the level of stress in patients. Further research with a sufficient sample size is needed to minimize the potential risks associated with adverse pregnancy outcomes and fewer live births.

CONCLUSION

In conclusion, among the investigated variables (β-HCG-AMH-TSH-PRL), only the β-hCG level was associated with pregnancy outcomes. As the serum β-hCG level increased on day 14 after embryo transfer, the LBR also increased, whereas no such relationship was observed with the CP rate. The β-hCG cutoff value for live births was 830 mIU/ml, with an AUC of 0.5920, a sensitivity of 61.04%, and a specificity of 56.58%.

Ethics approval and consent to participate

The Ethics Committee of Tehran University of Medical Sciences Tehran, Iran, approved our study protocol (approval number: IR.MEDECINE.REC.1400.853), and written consent was obtained from all participants.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

This study was not financially supported.