Effect of blood pregnancy test time on IVF clinical outcomes in urine β-HCG-positive patients: A retrospective cohort study

Abstract

Background

To evaluate the optimal time of blood pregnancy test for urine beta-human chorionic gonadotropin (β-HCG)-positive patients following embryo transfer.

Methods

A total of 1,106 women who underwent embryo transfer between January 2019 and December 2019 were divided into three groups based on the time of positive pregnancy test at the hospital: the ≤ 9 days group (n = 355), the 10–12 days group (n = 598), and the ≥ 13 days group (n = 153). Clinical pregnancy ratio, ectopic pregnancy rate, multiple pregnancy rate, early miscarriage rate, late pregnancy loss rate, live birth ratio, preterm birth rate, very preterm birth rate, gestational week of delivery, and congenital malformation rate of the three groups were compared.

Results

The time preference for pregnancy test was 10 days among patients with D3 embryo transfer and 11 days among those with blastocyst transfer. Patients in the ≥ 13 days group were older and had a higher proportion of previous childbirth(s). Patients in the ≤ 9 days group had a higher live birth ratio and a lower risk of early miscarriage than the other two groups; similar results were seen in a sensitivity analysis that excluded women aged over 35 years and those with previous childbirth(s). Nevertheless, no differences were observed in the clinical pregnancy ratio, ectopic pregnancy rate, late pregnancy loss rate, very preterm birth rate, gestational week of delivery, or congenital malformation rate for all ranges of pregnancy test time. The pregnancy test time to predict the early miscarriage and live birth based on receiver operating characteristic (ROC) curve was day 9 after embryo transfer. After excluding women aged over 35 years or those with previous childbirth(s), the optimal time to conduct pregnancy test based on ROC curve was day 9 or 10 after embryo transfer in predicting early miscarriage, the pregnancy test time to predict live birth should be conducted on day 9 after embryo transfer.

Conclusion

Patients with positive results for urine β-HCG after embryo transfer should receive blood pregnancy test on day 9–10 after embryo transfer, which probably facilitated the optimization of live birth.

Background

Since the birth of the world's first test-tube baby in 1978, constant innovations have been made in the in vitro fertilization–embryo transfer (IVF–ET) technology, both in terms of protocols and drugs, with the goal of alleviating patient suffering and increasing patient compliance. For instance, the long-acting gonadotropin-releasing hormone (GnRH)-antagonist protocol reduces the number of downregulation injections in patients [1], while the vaginal progesterone gel greatly lessens adverse reactions of progesterone injection [2]. Although technological innovations can reduce the partial physical stresses, literature shows that patients face varying psychological pressures at different stages of assisted pregnancy [3, 4]. As indicated by the comparison of patient stresses before and after embryo transfer and during pregnancy test, the psychological stress is particularly high during the pregnancy test. According to previous reports, a pregnancy test taken up to 14 days after an IVF protocol can make patients anxious and stressed [5, 6]. Such stress stems from facing the final outcomes after ovarian stimulation and oocyte retrieval or the upcoming fulfillment of years-long expectation following an excessively long infertility period [7].

Because patients cannot wait to learn about their IVF outcomes, the majority of them perform a urine beta-human chorionic gonadotropin (β-HCG) pregnancy test at home beforehand. Some patients even perform the pregnancy test just 5–6 days after embryo transfer. Patients with persistently negative test results often return to the hospital on the 14th day of transfer and undergo a blood β-HCG test to rule out false negatives. Patients with positive pregnancy test results are eager to know about the blood level changes and embryonic development. Furthermore, they are often unsure whether they should come to the hospital beforehand for blood tests and how far in advance the blood sampling should be performed. To our knowledge, no other studies have yet been conducted on this topic. Hence, in the present retrospective study, we intended to identify the time preference for pregnancy test among urine β-HCG-positive patients and evaluate whether their IVF and obstetric outcomes are affected by the time of blood pregnancy test at the hospital.

Methods

Results

In 2019, a total of 1,746 patients took blood HCG pregnancy tests at our hospital following fresh/frozen embryo transfer. Among them, 1,106 were eligible (Fig. 1) for our final analysis, with 355 categorized under ≤ 9 days group, 598 under 10–12 days group, and 153 under ≥ 13 days group. Figure 2 depicts the distribution of hospital pregnancy test times for the embryo transferred patients. For patients with D3 embryo transfer, the pregnancy test time tended to be the 10th day after transfer, while for those with blastocyst transfer, the most common pregnancy test time was the 11th day after transfer. The HCG levels on pregnancy test days in the three groups were 133.9 (76.3–212.9), 267 (116.4–532.4), and 559 (142.7–1,318.7) mIU/mL, respectively (P < 0.001). The 10–12 days and ≥ 13 days groups exhibited older ages and higher proportions of fresh embryo transfer than the ≤ 9 days group (P = 0.01 and P < 0.001, respectively). The number of antral follicles was the lowest in the 10– 12 days group (P = 0.004). In the FET procedure, the highest ratio of HRC with downregulation protocol was found in the ≤ 9 days group (P = 0.01), while the ≥ 13 days group exhibited the highest proportion of previous childbirth(s) and the greatest endometrial thickness on the day of transfer (P = 0.001 and P = 0.002, respectively). There were insignificant statistical differences between the three groups in terms of infertile duration, body mass index, maternal smoking, primary infertility rate, fresh cycle protocols, number of oocytes retrieved in fresh cycles, number of embryos transferred, and the good-quality embryo/blastocyst transfer rates. Table 1 lists all the background characteristics. 

The live birth ratio was the highest for the ≤ 9 days group and the lowest for the ≥ 13 days group (P < .001). The 10–12 days group exhibited the lowest rates of multiple pregnancy and preterm birth (P < .001 and P < .001, respectively). The early miscarriage rate was the highest in the ≥ 13 days group (P < .001). As shown in Table 2, the inter-group differences were not statistically significant in terms of clinical pregnancy ratio, ectopic pregnancy rate, late pregnancy loss rate, very preterm birth rate, gestational week of delivery, or congenital malformation rate. The aforementioned statistical significances remained the same in a sensitivity analysis that excluded women aged over 35 years and those with previous childbirth(s) (Table 3). 

In linear regression, pregnancy test time was incorporated into the model as a continuous variable, then, logistic regression was conducted to evaluate the relation of pregnancy test time with early miscarriage and live birth. The results suggested that an earlier pregnancy test time correlated with the lower early miscarriage rate (OR 1.26, 95% CI 1.14–1.38) and higher live birth rate (OR 0.82, 95% CI 0.76–0.88) (Table 4). In sensitivity analysis performed after excluding women aged over 35 years and those with previous childbirth(s), the earlier pregnancy test time continued to exhibit the gradually decreased early miscarriage rate (adjusted OR: 1.21; 95% CI: 1.09–1.35) and the gradually increased live birth rate (adjusted OR: 0.85; 95% CI: 0.78–0.93) (Table 5) 

Table 5 Association of pregnancy test time with early miscarriage and liver birth in the population after excluding women aged over 35 years and those with previous childbirth(s)

Moreover, we evaluated the pregnancy test time to predict early miscarriage and live birth rates using ROC curve for the general population. According to our results, the area under the ROC curve-1 (AUC) values were 0.614(95% CI: 0.57–0.66) and 0.598 (95% CI: 0.56–0.64), respectively, and the cutoff value was day 9. After women aged over 35 years and those with previous childbirth(s) were excluded, the ROC curve-2 (AUC) value for predicting early miscarriage rate, with cutoff value day 9 or 10, was 0.595 (95% CI: 0.54–0.65) and for predicting live birth rate, with cutoff value day 9, was 0.576 (95% CI: 0.53–0.62) ( Fig. 3 and Fig. 4).

Discussion

Follow-up strategies for IVF patients slightly vary among reproductive centers. Most reproductive centers adopt blood pregnancy tests 14 days after embryo transfer [15, 16], while some centers implement hospital pregnancy tests on the 11th or 12th day after the transfer [17, 18]. In our center, HCG demonstrated spikes two to three times at 72–96-h intervals following pregnancy tests. Approximately 30 days after embryo transfer, a viability ultrasound was performed to determine whether a fetal heartbeat existed. If the HCG levels grew poorly, the number of follow-ups prior to the initial B-mode ultrasound should be increased to avoid the risk of ectopic pregnancy and threatened abortion. However, as a matter of fact, women undergoing IVF usually feel anxious due to their own infertility and the unpredictability of pregnancy progress. Such stress leads to several behavioral changes [19]. For instance, some patients start performing pregnancy tests just 5–6 days after embryo transfer, and if the urine β-HCG test is positive, they immediately go to the hospital for a blood test verification.

Stress fluctuates during IVF treatment; the period from oocyte retrieval to embryo transfer and the waiting time for pregnancy tests were specifically linked to high degrees of anxiety and stress [20, 21]. For patients, the stress of waiting is the direct cause of taking a pregnancy test beforehand. In this study, we found that patients whose pregnancy test time was ≤ 9 days from the day of embryo transfer were younger, indicating that younger patients were more eager to undergo a pregnancy test earlier. In contrast, older patients showed varying expectation levels and complied better, probably because of higher previous childbirth ratios, with many of them taking pregnancy tests within the prescribed period. The ≥ 13 days group was not inferior to the other two groups in terms of factors conducive to embryo implantation, such as endometrial thickness, blastocyst transfer rate, and good-quality embryo transfer rate. Thus, we may consider that the reason for late pregnancy test time was linked to the difference in patient compliance. Additionally, despite the earlier implantation requirement following blastocyst transfer, the overall pregnancy time preference of blastocyst transfer patients was later than the D3 embryo transfer patients, suggesting that the pregnancy test time was decided based on patient compliance.

For the general population, patients taking pregnancy tests on days 9 or 10 after transfer exhibited the highest clinical pregnancy rate, low early miscarriage rate, and high live birth rate. Even after women aged over 35 years and those with previous childbirth(s) were excluded, the population taking pregnancy test on day 9–10 after transfer exhibited a lower early miscarriage rate and higher live birth rate. The too-early testing behavior did not necessarily predict a favorable pregnancy outcome. The reasons are stated as follows: 1. Some patients are detected positive for urine HCG in an extremely early period after transfer, suggesting that the embryo cleaves rapidly, with an advanced implantation compared with an embryo developing at a normal rate. The further development potential of rapidly-cleaving embryo remains controversial, and some scholars suggest that the rapidly-cleaving embryo is associated with a markedly increased chromosomal abnormality rate. It is interesting that the fast cleaving rate is related to similar chromosomal patterns to the slowly-cleaving counterparts. Embryos that exhibit 9 blastomeres after insemination for 62 h may experience chromosomal abnormality at an identical probability to that of their slowly-cleaving counterparts. Compared to embryos that contain 8 cells, those containing 9–11 cells have a lower development potential [22]. 2. As suggested in some articles, stress level in the IVF process affects reproductive outcome [23]. An early pregnancy test time demonstrates that the patient is anxious to some extent, and when she obtains the positive result, she may be worry about the blood HCG level. As confirmed by a study concerning the association of emotion with early miscarriage, emotional health deficiency increases the risk of miscarriage [24]. The reasons for poor pregnancy outcome at a late pregnancy test time may be due to the following reasons. Uterine receptivity occurs around 5–7 days after ovulation and lasts for 4 days, when embryo implantation commences. To ensure a successful implantation, the growth and maintenance of blood vessels should be coordinated at the maternal-embryo interface, so that the environment offered is nourished [25]. Patients with a pregnancy test time around 9–10 days are in the stage of embryonic post invasion and persistent uterine decidualization [26–28]. If these patients come to the hospital for a blood pregnancy test during this period, they can receive individualized luteal phase support (LPS) and other medications (e.g. low-molecular-weight heparin or glucocorticoid adjustments) based on the results of hormone levels, D2 dimers, immunoassays, and other tests, which may better facilitate endometrium remodeling, including an influx of uterine-specific NK cells and the exclusion of maternal B and T cells [27]. This is beneficial for continuous embryo implantation. However, if the test time around 9th-10th day is missed, the patient may miss the optimal luteal support adjustment time, leading to a poor pregnancy outcome.

There were no differences in the late-term miscarriage rate or the gestational week of delivery among the three groups, suggesting that women with a relatively stable early pregnancy showed no differences in pregnancy outcomes during later gestation. The stability of early pregnancy under identical blastocyst transfer rate and good-quality transfer ratio, i.e., under equal development potentials of embryos, can be improved in the following ways: 1. Strengthening the post-transfer lifestyle and psychological support. Some treatments or therapies, including relaxation training, medical clowning, coping cards, and hypnosis during embryo transfer, can be used to relieve the anxiety and stress caused by the uncertainties of pregnancy outcomes [29–32]. We should not object to the patients performing an early pregnancy test at home but help them in properly accepting the self-tested urine HCG results. 2. Progesterone administration remains controversial regarding dosages, routes, and timing [33–35]. In our reproductive center, a combination therapy with varying routes is used. For patients with deficient serum progesterone levels under a single treatment, our therapy seems a plausible option for maintaining adequate progesterone exposure. Besides, it can eliminate the increase in early miscarriage rates resulting from improper medications, drug allergies, and incompatible luteal support. There was no difference in the number of women with natural cycle FET among the three groups, and the luteal support protocols were consistent for each procedure of fresh/frozen embryo transfer. According to some literature, the serum progesterone measurements on the day of pregnancy test are linked to the ongoing pregnancy rate and live birth ratio [36–38]. In our study, the progesterone levels on the day of pregnancy test did not differ among the three groups. Nevertheless, Manuel Alvarez et al. argued that the appropriate time point for initiating an individualized LPS should be the day prior to blastocyst transfer [39]. Further prospective research is required to determine whether progesterone detection one week following the transfer and the corresponding adjustment of luteal support protocol is necessary.

This study has some shortcomings, regarding the fact that there are no data on pregnancy tests prior to the test at the hospital (self tests), and this is a factor that interferes with the final conclusion, which is a limitation of retrospective analysis. The cause analysis of early miscarriage (especially the embryo karyotype abnormalities, which are the most frequent cause of miscarriage during the first trimester) is lacking in the present study because not every miscarriage patient can accept the embryo chromosomal analysis. Replication and extension of our study can be a valuable direction for future prospective research.

Conclusions

In conclusion, taking a pregnancy test soon after embryo transfer is a common phenomenon. Such early pregnancy tests, especially on day 9–10 after embryo transfer, do not adversely affect the pregnancy outcomes. However, it is not advisable that patients return to the hospital for a blood pregnancy test only 14 days after the transfer.

Abbreviations

BMI: body mass index; AFC: antral follicle count; GnRH: gonadotropin-releasing hormone; HRC: hormonal replacement cycle; FET: frozen embryo transfer; HCG: human chorionic gonadotropin; IVF: In vitro fertilization; CI: Confdential interval; OR: odds ratio.

Declarations

Ethics approval and consent to participate The study was approved by the Scientific Ethics Committee of Chengdu Women’s and Children’s Central Hospital (reference: B2021-7). The Ethics Committee waived the need for written informed consent due to the retrospective nature and patients’ data were used anonymously. All procedures performed in the study were in accordance with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. 

 Consent for publication Not applicable. 

 Availability of data and materials The dataset supporting the conclusions of this article is included within the article. 

Competing Interests The authors report no conflicts of interest.

Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Authors’ contributions XK supervised the entire study, including the procedures, conception, design, and completion. XK and FW performed the acquisition of data, analysis of data. DC wrote the manuscript. YHL and FW revised the manuscript. All the authors take full responsibility for the work. The author(s) read and approved the final manuscript.

 Acknowledgements We wish to thanks to all the authors who contributed to this article.

Xue Ke, Yonghong Lin, Fang Wang

Chengdu Women’s and Children’s Central Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, Sichuan, China

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