DOI: https://doi.org/10.21203/rs.3.rs-1808174/v1
The effect of premature P elevation on assisted reproductive technology outcomes has historically been a matter of some debate. In previous studies, there were conflicting results regarding the impact of elevated progesterone on pregnancy outcomes in different ovarian stimulation protocols, different ovarian response and embryos transferred. In addition, most studies have focused on significantly elevated progesterone levels, such as above 1.5ng/ml, 1.75ng/ml, or even 2ng/ml. However, few studies have focused on slightly elevated progesterone level, and many scholars have previously concluded that it has no adversely effect on pregnancy outcomes.
Clinical data of patients were collected who underwent early follicular phase prolonged protocol cycle IVF/ICSI-ET treatment. The women were divided into two groups according to the progesterone level on HCG trigger day : Group 1:<1.0ng/ml; Group 2: 1.0ng/ml-1.5ng/ml. Differences in baseline characteristics, ovulation promotion situation and embryo culture were compared between the two cohorts. After adjusting for confounding factors using multivariate logistic regression analysis, the clinical pregnancy outcomes were compared between the two groups.
A total of 743 subjects were included in this study, of which 587 were included in Group 1 and 156 were included in Group 2. In terms of pregnancy outcome, Group 2 had a significantly lower clinical pregnancy rate and live birth rate per cycle than Group 1 (64.1% vs 75.7%, P < 0.05; 63.5% vs 72.7%, P < 0.05). After correcting for female BMI, total dose of Gn, days of stimulation, et al using multivariate logistic regression analysis, slightly elevated progesterone levels (P > 1.0ng/ml) remained a risk factor for pregnancy failure in fresh single D5 blastocyst transfer under an early follicular phase prolonged protocol cycle.
The slightly elevation progesterone level on the HCG trigger day may have a negative effect on the clinical pregnancy in early follicular phase prolonged protocol cycles, even in ETs with a good prognosis.
In women who undergo ovarian stimulation, the incidence of progesterone elevation on HCG trigger day fluctuated at 38% although the application of gonadotropin-releasing hormone (GnRH) analogues could significantly reduce the incidence of premature luteinization[1].
Because of the high variability of assays used for progesterone measurement, making it difficult to compare the results of different studies and there is no uniform standard for the definition of elevated progesterone levels on HCG trigger day.
To our knowledge, almost all studies found that the significantly elevated progesterone level (P>1.5ng/ml) on the HCG trigger day have detrimental effect on clinical outcomes[2,3]. But what is the relationship between a slight increase in progesterone level of 1.0–1.5ng/ml on trigger day and the clinical outcomes? Few studies explored this question. If there is no adverse effect, the freeze-all strategy would prolong the time of TTLB and increase the burden on the patients. The objective of this study was to evaluate whether slightly elevated progesterone level(1.0-1.5ng/ml)on the trigger day has an adverse effect on clinical pregnancy under an early follicular phase prolonged protocol cycles.
Study population and design
This study was reviewed and approved by the Institutional Review Board and the Ethics Committee of Henan Provincial People's Hospital, China(approval number: SYSZ-LL-2021091501). The study was based on the Declaration of Helsinki, as revised in 1983. All participants in this study signed a written informed consent form.
This was a retrospective study which collected data from 743 cycles of fresh IVF/ICSI-ET under an early follicular phase prolonged protocol from January of 2017 to October of 2020 at a single reproductive center.
The inclusion criteria including: (1)20-40 years old; (2)single D5 blastocyst transfer; (3)the endometrial thickness on the day of trigger was greater than 6 mm; (4)progesterone level was≤1.5ng/ml on the trigger day. The exclusion criteria were as follows: (1)endometrial polyp, uterine anomaly, endometriosis, adenomyosis, hydrosalpinx; (2)incomplete data; (3)the times of egg retrieval≥3 times; (4)anyone of the couple has chromosomal abnormalities (including chromosome polymorphisms); (5)patients with testicular sperm retrieval.
Patient-related clinical data that strictly met the inclusion and exclusion criteria were retrieved through the software database of the Clinical Assisted Reproductive Technology Management System in the department. 743 women involved were divided into two groups according to the progesterone level: Group 1:<1.0ng/ml (n=587); Group 2: 1.0<P<1.5ng/ml (n=156).
Progesterone measurement
Serum progesterone levels were measured on the day of HCG injection. Samples were tested with a microparticle enzyme immunoassay Axsym System (Abbott Cientifica S.A., Madrid, Spain), and the lower limit of detection for the assay was 0.05ng/ml. Besides the internal quality control checks performed daily by the institution laboratory, the assays were calibrated whenever a new reactive batch was used or whenever an outcome outside the normal range was observed.
Ovarian stimulation and embryo culture
Long-acting GnRH-a 3.75 mg (Triptorelin Acetate for injection, IPSEN PHARMA BIOTECH, H20140298) was administered intramuscularly on days 2 to 4 of the menstrual cycle, and blood hormone levels and ultrasound were measured 28 to 30 days later to determine whether the criteria for down-regulation had been met. After down-regulation was achieved, ovarian stimulation was started with a starting dose of 75iu to 300iu, depending on the patient's age, AFC, basal sex hormones, AMH, BMI and previous ovarian responsiveness. When≥18mm in diameter follicles accounted for 50-60% of the dominant follicles, HCG trigger was given at a dose of 4000-12000 IU.
Depending on the quality of male semen, IVF or ICSI techniques were selected for fertilization, followed by transfer to a culture medium for embryo culture. The Embryonic oocyte cleavage was observed 70-72h after fertilization and blastocyst scoring was used by the Gardner scoring system.
Luteal phase support
Routine luteal support was performed from the day of egg retrieval, with oral dydrogesterone tablets(Duphaston, Abbott Biologicals B.V., H20170221) 30mg and progesterone sustained-release vaginal gel(Fleet laboratories Limited, H20140552) 90mg placed vaginally. On the 5th day after egg retrieval, high scoring blastocysts were selected for embryo transfer according to the embryo status and patient's specific condition.
Peripheral blood was taken from the patient 14 d after embryo transfer to measure β-hCG level. The transvaginal ultrasounds were given to evaluate embryo implantation after the 28th-35th day of fresh SBT, and The pregnancy sac was detected as clinical pregnancy. If clinical pregnancy was confirmed, continue luteal support until about 12 weeks of gestation, if embryo loss or loss of fetal heartbeat occurs, mark as abortion.
Statistical analysis
Continuous variables of normal distribution were expressed as the mean±standard deviation (mean±SD) and the t test was selected to conduct the statistics. Variables of non-normal distribution were presented as median±quartile range (median±QR) and Mann-whitneyU test was selected to conduct the statistics. Classified variables were expressed as frequency and percentage(%) and the Chi-Square test was used to compare the percentage. Multiple logistic regression analysis was used to correct all confounding factors. SPSS23.0 was implied to carryout the statistical analysis, and P<0.05 was considered as statistically significant.
In this study, we analyzed 743 fresh SBT cycles under an early follicular phase prolonged protocol and compared the patients’ clinical outcomes between the two groups. The demographic data, such as age, infertility duration, BMI, AMH, et al were summarized in Table 1. Baseline information was similar across the two groups, except with regards to BMI; BMI decreased as P levels increased on the day of trigger.
When performing COS under the prolonged protocol, the Group 2 had higher total dose of Gn(2608.29±969.91 vs 2301.12±914.51, P=0.001), at the same time,the days of stimulation were higher (12.39±3.14 vs 11.68±2.29, P=0.001). E2 level on the day of trigger were higher in Group 2 (2216.01±932.93 vs 1732.49±763.99, P=0.001)(Table 2).
The laboratory results showed that Group 2 had higher number of retrieved oocytes, double pronuclear (2PNs) and MII oocytes (13.78±4.36 vs 12.41±4.26, 9.28±3.15 vs 8.41±3.13, 12.21±3.87 vs 11.05±3.86 respectively; all P < 0.001) (Table 3). The comparison of the grades of blastocyst cavity expansion,inner cell mass, and trophoblastic layer showed no significant differences. Blastocyst cavity expansion was almost the 4th degree of the two group.
Slightly elevated progesterone level leaded to significant lower clinical pregnancy rate and live birth rate than that of group with normal progesterone level (75.5% vs 64.1%, 72.7% vs 63.5% respectively; all P <0.05) (Table 4, Figure 1).
In order to eliminate the Interference of variables with statistical differences between the two groups on the Clinical pregnancy rate and live birth percentage, we performed a logistic regression analysis. After correction for BMI, total dose of FSH, days of stimulation, E2 level on the day of HCG, number of obtained oocytes, et al, slightly elevated progesterone level remained a risk factor for lower clinical pregnancy and live birth rates(Table 5).
Despite some controversial reports, most studies, to date, strongly support the idea that a premature rise in progesterone levels on HCG trigger day during ovarian stimulation is associated with reduced implantation and pregnancy rates following fresh embryo transfers. However, almost all studies focused on the effect of significantly elevated progesterone level on the trigger day on the clinical outcomes after IVF/ICSI cycles[3-6]. This study assessed the clinical outcomes between slight elevation progesterone level 1.0-1.5ng/ml with normal progesterone level<1.0ng/ml for the first time.
The mechanism causing elevated progesterone on the trigger day during COH is unclear, which is currently believed to be related to the following factors[2,7-10]: (1)excess mature follicles; (2)high Gn dose: Bosch et al. found that for every 75 U increase in FSH, the risk of elevated progesterone on HCG days increased 1.44-fold; (3)High LH; (4)prolonged ovarian stimulation; (5) different ovulation promotion protocols: GnRH agonist protocols were most associated with extreme elevations of progesterone. In the present study, total dose of Gn, days of stimulation, E2 levels on the HCG day and number of oocytes retrieved in Group 1 were all higher than these in Group 2 (1.0<P<1.5ng/ml), consistent with the results of most of the above studies. Also, the women in Group 1(P<1.0ng/ml) had a higher BMI and there was a statistical difference between the two groups(23.49±3.54 vs 22.45±2.99, P=0.002), suggesting that women with low BMI are more likely to have elevated progesterone. It is possible that the higher the BMI, the higher the aromatase expression and, consequently, the higher the estrogen production, which could impact the production of progesterone[11].
Although the “freeze-all” strategy can avoid the effects of high progesterone levels on pregnancy outcomes, there is no uniform expert consensus on the safety and long-term prognosis of this strategy. Therefore, careful selection of the dose of ovulation stimulating drugs and reduction of the duration of stimulation time are particularly important to prevent premature elevated progesterone on HCG day in order to improve the transplantation rate of fresh cycles.
There are different threshold values defining elevated progesterone,and retrospective studies have demonstrated that progesterone levels greater than 1.5–2.0ng/mL are associated with reduced pregnancy rates and fresh embryo transfer should be cancelled[2,9,12]. Although most clinicians think progesterone level<1.5ng/ml have no negative effect on outcomes, the present study found that the clinical pregnancy outcome was poor when the serum progesterone level higher than 1.0ng/ml under early follicular phase prolonged protocol cycles, the same with another study[13].
According to previous studies, elevated progesterone may affect ART outcomes in several ways. Huang et al. found that elevated progesterone levels on HCG days had a significant negative effect on the rate of high-quality embryos(P < 0.05)[14]. However, there are some studies that take the opposite view. Based on various markers of oocyte and embryo quality, the data strongly support that there is no negative influence of premature progesterone elevation on oocyte and embryo quality[14-16]. Therefore, the conclusion is yet to be confirmed by a large prospective study. In this study, there was no significant difference in embryo quality between the two populations, and both were dominated by high-quality blastocysts.
On the other hand, elevated progesterone may affect endometrial tolerance. By examining the transcriptome profile of peri-implantation window endometrium, Liu et al. found that in endometrium with high progesterone levels on HCG day, the expression profile of genes related to natural killer cell-mediated cytotoxic pathway was significantly different compared to normal levels of endometrium[17].
Some clinicians have proposed a question: are good patient and embryo characteristics protective against the negative effect of elevated progesterone level? Studies showed that high progesterone(>1.5ng/mL) did not change the pregnancy rate in cases of hyperresponders[18,19]. In the present study, analyzed the data including maternal age (30.78±3.76, 30.94±3.87), AMH (4.21±2.92, 4.01±2.85), AFC (15.42±5.57, 14.47±5.13), Basal FSH (6.44±1.85, 6.55±1.82), number of oocytes retrieved (12.41±4.26, 13.78±4.36), suggesting that the vast majority of women in both cohorts could be classified as high-response patients with a good prognosis. However, a slight increase progesterone(>1.0ng/mL) on the day of HCG still had a negative impact on pregnancy outcome. This result may suggest that the negative impact of premature P elevation on pregnancy rates is an endometrial effect, as opposed to an oocyte effect.
Furthermore, some studies hypothesized only in cases of D3 embryo transfer, there would be interference from progesterone(P>1.5ng/mL) on the HCG day, the implantation rate would drop by 50%; the same would not occur in the transfer of blastocysts[8]. In contrast, in this study, D5 single blastocyst transfer was used in all these women, yet the high progesterone group (P>1.0ng/mL) still had lower clinical pregnancy rates and live birth rates. The results of this study suggest that on the fifth luteal day, the endometrium still has not recovered from the effects of high progesterone and its impact on pregnancy outcome still persists.
An important strength of the present study was that it is the first study to have compared the ART outcomes between women with progesterone<1.0ng/ml and progesterone>1.0 to<1.5ng/ml under an early follicular phase prolonged protocol. Also both groups used the same COS protocol and all underwent D5 single blastocyst transfer, avoiding the influence of relevant confounding factors. In addition to comparing implantation rates and early pregnancy rates, an extended comparison of the differences in live birth rates between the two groups was also performed. Another significant factor to consider in assessing any study on P level is the accuracy and precision of the assay[20]. The internal and external quality control of the assays utilized in our laboratory ensured that these standards were maintained. Weaknesses of this study include its retrospective design and small sample.
The present study suggests the slight elevation progesterone level on the HCG trigger day may have a negative effect on the clinical outcomes of SBT cycles under an early follicular phase prolonged protocol even with a good prognosis. The application of “freeze-all”strategy could improve the pregnancy outcomes in case of slightly elevation progesterone level on the HCG trigger.
The key to ovarian stimulation in IVF is an individualized therapy, which means the duration of COS should not be prolonged or exceeded beyond the optimal standard of final oocyte maturation and the doctor should select the appropriate Gn dose at the same time.
IVF-ET: in vitro fertilization and embryo transfer
ICSI-ET: intracytoplasmic sperm injection and embryo transfer
TTLB: Time to Live Birth
SBT: single blastocyst transfer
COS: controlled ovarian stimulation
ART: assisted reproductive technology
Ethics approval and consent to participate: This study was reviewed and approved by the Institutional Review Board and the Ethics Committee of Henan Provincial People's Hospital, China(approval number: SYSZ-LL-2021091501). All participants in this study signed a written informed consent form.
Consent for publication: Not applicable
Availability of data and materials:The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.
Competing interests: The authors declare that they have no competing interests.
Funding: This work was supported by the Henan Provincial Medical Science and Technology Tackling Program Provincial-Ministerial Co-construction Project (SBGJ202001002).
Authors' contributions: Longlong Wei was a major contributor in writing the manuscript and analyzed all patients data. Yan Zhao and Chunyu Xu collected all patients information and analyzed all patients data. Cuilian Zhang performed conception and design of work, drafting and revising manuscript. All authors read and approved the final manuscript.
Acknowledgements: Not applicable
Table 1-5 are available in the Supplemental Files section.