The impact of down-regulation on obstetrics and perinatal outcomes in singleton pregnancies after IVF: a retrospective cohort study

Background Down-regulation has been widely used in IVF treatment; however, it lacks reports on the impact of down-regulation on obstetrics and perinatal outcomes. The purpose of this study is to evaluate the effect of down-regulation on obstetrics and perinatal outcomes. Methods This is a retrospective cohort study on 3578 patients achieving singleton pregnancy after their rst IVF attempt. The patients were grouped by the serum estradiol after down-regulation (E2D) into three groups: <30, 30-55, >55pg/ml. The cumulative live-birth rate, obstetrics and pediatric results were main outcome measures. General linear models and Chi-square test were performed for statistical analysis. Results The patients with E2D <30, 30-55, >55pg/ml had similar cumulative live-birth rate. The patients with E2D <30pg/ml had a lower risk for hypertension disorders than those with E2D 30-55pg/ml. No difference was found in the prevalence of placenta previa, placenta abruption, premature rupture of membrane, hemorrhage, gestational diabetes mellitus, or intrauterine growth restriction. The newborns of patients with E2D <30pg/ml had a lower risk for PICU attempt than those with E2D >55pg/ml. There was no difference in congenital anomaly or mortality rate. Conclusion We found no effect of down-regulation on cumulative live-birth rate. The patients with E2D<30pg/ml may have advantages in lower risks for maternal hypertension and newborns PICU attempt. mid-luteal GnRH-a long protocol; patients clinical after their rst stimulated cycle and/or subsequent frozen embryo transfer). Exclusion criteria: (1) patients involving donation freezing of gemmates; (2) patients undergoing PGD/PGS; (3) patients with hypertension, diabetes mellitus or immune diseases. All patients were followed up to the termination the total 3578 for analyses. study


Background
Pituitary down-regulation with a gonadotropin-releasing hormone-agonist (GnRH-a) is a common practice in the eld of In Vitro Fertilization (IVF). Down-regulation can avoid premature luteinizing hormone surge, favor follicle development, synchronize the growth of follicles and endometrium, and thus improve IVF success (1). In addition, down-regulation also has an advantage in treatment scheduling. Previous studies have shown the superiority of down-regulation, in terms of a lower cycle cancelation rate and a higher pregnancy rate (2). Our previous study showed that the degree of down-regulation was associated with the outcomes of ovarian response, pregnancy, and live birth. A serum estradiol level after downregulation (E2D) < 55 pg/ml is an optimal status for subsequent pregnancy (3).
It is estimated that births after IVF is over 1% of all births in the UK (4). There is a growing concern about the safety of IVF, in terms of obstetrics and perinatal outcomes (5)(6)(7). It has been reported that IVF increased risks for maternal disorders, e.g. placenta accreta, hypertensive disorders, and psychological disorders (5,8,9). Besides, it has been shown that IVF was associated with preterm birth, low birth weight, gender bias (10)(11)(12), and congenital anomalies (13).
To our knowledge, there is a lack of study analyzing IVF safety in different down-regulation conditions.
The objective of this study is to evaluate whether there is an association between down-regulation and obstetrics/perinatal outcomes in singleton pregnancies after IVF.

Study population
This is a retrospective cohort study on the patients undergoing their rst IVF treatment at our center, between January 2009 and December 2013. Inclusion criteria: (1) patients undergoing standard midluteal phase GnRH-a long protocol; (2) patients who achieved clinical pregnancy after their rst stimulated cycle (fresh and/or subsequent frozen embryo transfer). Exclusion criteria: (1) patients involving donation or freezing of gemmates; (2) patients undergoing PGD/PGS; (3) patients with hypertension, diabetes mellitus or immune diseases. All patients were followed up to the termination of pregnancy. Finally, the data of a total of 3578 patients were extracted for analyses. This study was conducted with the formal approval of the Institution Review Board (IRB) of Tongji Hospital. All patients in this study have given written consent to the inclusion of material pertaining to them. They have been fully anonymized before analysis.

Clinical protocols
Down-regulation, ovarian stimulation, IVF, embryo culturing, and embryo transfer were performed as previously published (3). Brie y, a daily injection of 0.1 mg GnRH-a (Decapeptyl, Ferring, Switzerland or Diphereline, Ipsen, Australia) was initiated in the midluteal-phase of the preceding cycle. The ovarian stimulation with gonadotropins was initiated with rFSH (Gonal-F, Serono, Switzerland or Puregon, Organon, Netherlands). The starting dose of gonadotropin was 150-225 IU/d based on the age, antral follicle count (AFC), basal FSH, and body mass index (BMI). The dosage of GnRH-a was then reduced to 0.05 mg/day till the day of hCG triggering. The gonadotropin dose was adjusted according to the ovarian response, which is assessed by serum E2, progesterone (P), LH, and serial ultrasound scans. When at least 2-3 follicles developed to a diameter of ≥ 18 mm, 10,000 IU hCG was given to trigger the maturation of follicles. Oocytes were retrieved transvaginally 36-38 hours after the hCG injection. The fertilization method included IVF and ICSI. In a fresh cycle, typically no more than two best-quality embryos were transferred on day 3 after oocyte retrieval, and excessive available embryos were cryopreserved for subsequent FET cycles. An administration of 60 mg P was used as luteal phase support from the day of oocyte retrieval. The protocol for blastocyst culture, embryo vitri cation and warming, and FET have been described in the previously published literature (14).

Outcome measures
The primary measures were maternal and perinatal outcomes. The results of ovarian stimulation and pregnancy were also analyzed. A clinical pregnancy was diagnosed when the serum hCG level reached > 20 IU/l two weeks after transfer and the gestational sac was detected on ultrasound 5-7 weeks after transfer. A live birth was de ned as the completion of expulsion or the extraction of a live baby after 28th gestational week (15).

Grouping of patients with E2D
Patients were classi ed into the over (E2D < 30 pg/ml), optimal (E2D 30-55 pg/ml), and insu cient (E2D > 55 pg/ml) down-regulated group, according to the criteria published in our previous study (3). Statistical analysis SAS 9.2 (SAS, Inc., Cary, NC, USA) was used for statistical analysis. Numeric parameters were presented as mean ± SD. Categorical variables were presented as percentage (number). General linear models and Chi-square test were performed appropriately. Multiple comparisons were performed with Turkey or Bonferroni method. A P value < 0.05 was considered statistically signi cant.

Results
The data from a total of 3578 patients were analyzed. The demographic and clinical characteristics are shown in Table 1. There were differences in the age, BMI, baseline serum FSH level and AFC among the patients with E2D < 30, 30-55 and > 55 pg/ml, but these differences are very slight and of no clinical value. The duration and type of infertility were similar among the three groups.  Table 2 shows the ovarian stimulation parameters and pregnancy outcomes. The patients with E2D > 55 pg/ml had lower dosage of stimulation compared with those with E2D < 30 pg/ml (25.9 ± 7.9 vs. 29.0 ± 6.8 ampules, P < 0.01). The serum peak E2 level in the patients with E2D < 30 pg/ml was lower compared with those with E2D 30-55 pg/ml and > 55 pg/ml (4982.9 ± 2792.9 vs. 5471.7 ± 2890.8 and 5623.6 ± 2614.2 pg/ml, P < 0.01). The differences in the duration of rFSH, number of oocytes retrieved and number of good embryos have limited clinical value. The serum P level was similar among the three groups. The patients with E2D < 30, 30-55 and > 55 pg/ml obtained similar live-birth rate and miscarriage rate. Regarding the obstetrics outcomes, the patients with E2D < 30 pg/ml had a lower risk for hypertension disorders compared with those with E2D 30-55 pg/ml (0.46% vs. 1.31%, P = 0.04). The risks for placenta previa, placenta abruption, premature rupture of membrane, hemorrhage, gestational diabetes mellitus and intrauterine growth restriction were similar among the three groups. The newborns outcomes are shown in Table 3 and Fig. 1. The mean gestational week, percentage of preterm birth and very preterm birth were similar among the patients with E2D < 30, 30-55 and > 55 pg/ml. No differences were found in the mean birth weight, percentage of low birth weight and very low birth weight. The newborns from the group of E2D < 30 pg/ml had a lower risk for attending PICU (Pediatric Intensive Care Unit) compared with those of E2D > 55 pg/ml (2.1% vs. 4.6%, P = 0.04). There were no differences in the prevalence of congenital malformation and mortality among the three groups.

Discussion
This study compared the ovarian performance, pregnancy results, and outcomes of obstetrics and newborns from the patients with an E2D of < 30, 30-55, > 55 pg/ml. We found that E2D was associated with ovarian stimulation, but did not in uence the live-birth rate. The patients with E2D < 30 pg/ml were less likely to suffer hypertension disorders and their babies had a lower risk for PICU attempt.
Down-regulation is a common practice in IVF treatment. Previous studies showed that down-regulation with GnRH-a increased the clinical pregnancy rate after IVF; however, this result is obtained from fresh IVF cycles (2,16). In a recent study, we de ned E2D < 30 pg/ml as an over down-regulation, 30-55 pg/ml as a suitable down-regulation, and > 55 pg/ml as an insu cient down-regulation, and we found that an over or suitable down-regulation elevated the cumulative pregnancy and live-birth rates (3). Currently, it lacks reports on the effect of down-regulation on obstetrics and pediatrics outcomes.
The result of this study shows that the degree of down-regulation is negatively associated with ovarian stimulation e ciency. With the decrease of E2D, more aggressive and longer stimulation is needed, but the peak E2 level, the number of oocytes and good embryos are lower. When pituitary is aggressively suppressed, its function recovers slower, and more stimulation is needed to initiate follicle-genesis.
Our previous study has demonstrated that a su cient down-regulation (E2D ≤ 55 pg/ml) resulted in a higher cumulative clinical pregnancy rate. The present study showed that the live-birth and miscarriage rates were similar between the patients with E2D ≤ 55 and > 55 pg/ml. This result can help clinicians in common counselling. Although an insu cient down-regulation (E2D > 55 pg/ml) indicates a lower chance of conception, extra concerns about miscarriage is not needed when a clinical pregnancy has been achieved.
Since the rst IVF baby (17), the number of children born after IVF has risen rapidly in these 40 years. Globally, there is an increasing attention to the safety of IVF, in terms of obstetrics and perinatal outcomes. However, the results of previous studies are inconsistent. Some studies showed that IVF was a generally safe procedure (18)(19)(20). In contrast, some studies found increased risks for newborns or mothers after IVF (9,21). In an IVF treatment, superovulation or culture conditions may contribute to these adverse outcomes (22). Regarding maternal outcomes, we found that E2D was not associated with the occurrence of placenta previa, placenta abruption, premature rupture of membrane, hemorrhage, gestational diabetes mellitus, or intrauterine growth restriction; however, an E2D of < 30 pg/ml indicated a lower risk for hypertension disorders of pregnancy. The quality of oocytes/embryos may be associated with the pathogenesis of hypertension disorders. Indeed, abnormal cytotrophoblasts cause inadequate remodeling and atherosis of spiral artery, result in ischemia and hypoxia of placenta, which is central to the pathogenesis of this disease (23). Regarding perinatal outcomes, we found that the gestational week, birth weight, sex ratio, congenital anomaly rate, and mortality rate were similar in the patients with different E2D. The babies of patients with an E2D of < 30 pg/ml were less likely to need PICU treatment. This result indicates that an over down-regulation may bene t follicular recruitment and development, improve embryo/fetus quality, and thus reduce the risk for PICU attempt.
The limitation of this study is its retrospective nature. In addition, only the singleton pregnancy was analyzed. Further study can be performed to con rm these ndings.

Conclusions
We found no effect of down-regulation on cumulative live-birth rate. The patients with E2D < 30 pg/ml may have advantages in lower risks for maternal hypertension and newborns PICU attempt. Availability of data and materials: the data are included within the manuscript.

Competing interests:
there is no con ict of interest.