Abstract
Purpose
The study was designed to evaluate the relationship between serum progesterone (P4) response after hCG administration and the number of oocytes retrieved and the embryo quality in fresh IVF cycles.
Methods
We conducted a retrospective cohort study of women aged 24–43 years who underwent first fresh IVF cycle from 2011 to 2013 at a single practice. We compared the post-hCG serum P4 level with values on the day of hCG trigger. Patients were analyzed in long and short protocols independently. In addition, patients were stratified by post-hCG P4 response. Number of oocytes retrieved and embryo quality were the primary outcomes of interest. Ordinary least square regression models and logistic regression analysis models were created to identify predictive factors associated with embryological outcomes while adjusting for potential confounders.
Results
Among the 2,978 IVF cycles, 2,484 patients were in long protocols, and 494 patients were in short protocols. After adjusting for patient age, rFSH duration, and basal FSH levels, the associations between P4 response after hCG administration and number of oocytes retrieved (P < 0.001) remained statistically significant in both long and short protocols. Additionally, mature oocyte rate, fertilization rate, good quality embryo rate, pregnancy rate and implantation rate were not significantly associated with the P4 increase when adjusting for the same factors. However, pregnancy rate and implantation rate from frozen-thawed cycles increased gradually across the seven groups.
Conclusions
Post-hCG P4 levels were positively associated with the number of oocytes retrieved, but did not affect oocyte or embryo quality. Our study suggests that the change in the post-hCG P4 level is another parameter that can be used by clinicians to assess the number of oocytes retrieved, and may further to estimate the pregnancy rate and live birth rate indirectly.
Keywords: Progesterone, HCG, Oocyte number, Embryo quality
Introduction
In recent times, the incidence of infertility has progressively increased due to potential factors such as life stressors and delayed child-bearing age [1]. The development of assisted reproductive technologies has dramatically improved the outcomes of in vitro fertilization/embryo transfer (IVF/ET) in recent years. However, the clinical pregnancy rate is still approximately 40 % per treatment cycle [2]. It has been recognized that parameters such as oocytes retrieved, endometrial thickness, number of embryos transferred and embryo quality are positively correlated with success of IVF-ET [3–6].
In contrast, factors associated with a reduced likelihood of IVF success include elevated basal FSH, advanced maternal age, low antral follicle number and premature luteinization [7, 8]. A large number of clinical studies have affirmed that the subtle progesterone rise on the day of hCG administration negatively affects the pregnancy rate in IVF [9–11]. It has been demonstrated that the pregnancy rate will drop dramatically when the progesterone level is over 1.5 ng/ml on the day of hCG administration [12–14]. However, only two studies have assessed the significance of P4 response after hCG administration on IVF cycle outcome of pregnancy rate [15, 16]. The two studies are of small sample size and do not focus on the influence of progesterone level to oocyte or embryo. In an effort to address these limitations and to explore whether progesterone level prior to oocyte retrieval was associated with the numbers of oocytes retrieved and embryo quality, our study was designed to evaluate the relationship between increase in P4 levels after hCG administration and laboratory outcomes including the number of oocytes retrieved, fertilization rate and the ratio of good quality embryos.
Material and Methods
Patient population
In this retrospective cohort study, all women aged 24–43 years who underwent fresh IVF with or without intracytoplasmic sperm injection treatment in the Assisted Reproductive Unit of Sir Run Run Shaw Hospital, Hangzhou, China between 2011 and 2013 were identified. Analysis was limited only to first IVF cycles. We excluded those cycles without accurate medical record numbers and missing data. Blood samples for progesterone measurement were obtained 12–16 h prior to hCG administration and repeated 10–12 h after hCG administration.
Controlled ovarian hyperstimulation protocals
The protocols were previously described [17]. Briefly, two ovarian stimulation protocols for IVF treatment (long and short protocols) and two types of recombinant follicle-stimulating hormone (rFSH; Gonal-F, Serono Laboratories, Aubonne, Switzerland or Puregon, N.V. Organon, Oss, The Netherlands) were used.
Pituitary down-regulation was initiated with triptorelin 50 u/day (Ferring Pharmaceuticals Ltd., Kiel, Germany) from the mid-luteal phase in the long protocol, and from the second day of the menstrual cycle in the short protocol. Luteal support consisted of intramuscular progesterone in oil 40 mg/day for 1 day, starting on the night of oocyte retrieval, and then 80 mg/day for at least 2 weeks.
Endocrine hormone measurements
Blood samples were obtained for FSH, luteinizing hormone (LH) and oestradiol measurement on Day 3 of the IVF treatment cycle. Additionally, serum progesterone level was measured on the morning of hCG administraton (hCG-day P4) and repeated 10–12 h after hCG administration (post-hCG P4). Hormone assay was performed within 2 h of collection using a Beckman Coulter immunoassay analyzer (DXI800, California, USA), in accordance with the manufacturer’s instructions. The inter-assay coefficients of variation for FSH, LH, E2 and P4 were 10.0, 5.4, 12.0 and 10.0 %, respectively, and the intra-assay coefficients of variation were 5.0, 3.8, 8.0 and 6.3 %, respectively. All the assays were performed in the same laboratory within the IVF unit.
Categories of groups
Progesterone response was calculated as the ratio change of post-hCG P4 to hCG-day P4. Patients were analyzed in long and short protocols independently. In addition, in order to explore the results more specifically, patients were stratified into seven groups according to P4 response to hCG administration: Group 1: the ratio of post-hCG P4 to hCG-day P4 was 0–1; Group 2: the ratio was 1–2; Group 3: the ratio was 2–4; Group 4: the ratio was 4–6; Group 5: the ratio was 6–8; Group 6: the ratio was 8–10; Group 7: the ratio was >10.
Embryo transfer
Patients underwent Day 3 embryo transfer. Patients with failed fertilization and severe ovarian hyperstimulation syndrome did not undergo embryo transfer. Embryo cryopreservation was performed on Day 3 if there were residual embryos after transfer.
Measurements of outcomes
The number of oocytes retrieved, the ratio of mature oocytes, fertilization rate, the ratio of good quality embryos, pregnancy rate and implantation rate were measured and analyzed according to the ratio of post-hCG P4 to hCG-day P4 levels. Good quality embryos were graded as described by Volpes [18]: Day 3 embryos that reached the eight cell stage with <20 % fragmentation. Good quality embryo rate was defined as the number of good quality embryos divided by the number of all embryos. The pregnancy rate was identified with the presence of an intrauterine gestational sac confirmed by transvaginal ultrasonography. The implantation rate reflects the number of gestational sacs visualized on transvaginal ultrasound divided by the number of embryos transferred.
Statistical analysis
Ordinary least square regression models were employed to evaluate the associations variables of interest and number of oocytes retrieved, MII oocyte rate, fertilization rate, good quality embryo rate and implantation rate while adjusting for potential confounders. Logistic regression analysis was conducted to identify independent correlates between pregnancy rate and each possible confounding factor. Data analysis was conducted using Statistical Package for the Social Sciences Version 16.0 (IBM Corp., New York, NY, USA). A p- value <0.05 was considered to indicate significance.
Results
We analyzed a total of 2,978 fresh, first IVF cycles. With patients divided according to the ovarian stimulation protocols, a summary of important embryological aspects of the treatment outcome is presented in Table 1. Ordinary least square regression models and logistic regression analyses were used to analyze the influence of increase in post-hCG P4 levels on oocyte and embryo development. After adjusting for patient age, rFSH duration, and basal FSH levels, the associations between P4 response after hCG administration and number of oocytes retrieved (P < 0.001) remained statistically significant in both long and short protocols. Additionally, mature oocyte rate, fertilization rate, good quality embryo rate, implantation rate and pregnancy rate were not significantly associated with the increase of P4 levels when adjusting for the same factors (Table 1). Patient age and basal FSH levels were negatively associated with the number of oocytes retrieved (Table 2).
Table 1.
Regression analysis of post-hCG P4 to hCG P4 ratio and embryological outcome
| Overall (Regression coefficient) | P-value | Long protocol (Regression coefficient) | P-value | Short protocol (Regression coefficient) | P-value | |
|---|---|---|---|---|---|---|
| No. of oocytes retrieveda | 0.0343 | <0.001 | 0.03 | <0.001 | 0.0468 | <0.001 |
| Mature oocyte rate (%)a | −0.00108 | 0.63 | −0.000463 | 0.845 | −0.00633 | 0.502 |
| Fertilization rate (%)a | −0.00104 | 0.581 | 0.000154 | 0.931 | −0.0056 | 0.646 |
| Good quality embryo rate (%)a | −0.000926 | 0.598 | −0.000952 | 0.608 | −0.00433 | 0.416 |
| Implantation rate (%)a | −0.00602 | 0.230 | −0.00724 | 0.177 | −0.0215 | 0.275 |
| Pregnancy rate (%)b | −0.0170 | 0.554 | −0.0328 | 0.289 | −0.0419 | 0.729 |
aOrdinary least square regression
bLogistic regression analysis
Table 2.
Regression analysis of other factors and oocytes retrieved number
| Overall (Regression coefficient) | P-value | Long protocol (Regression coefficient) | P-value | Short protocol (Regression coefficient) | P-value | |
|---|---|---|---|---|---|---|
| Post-hCG P4 to hCG P4 ratio | 0.0343 | <0.001 | 0.03 | <0.001 | 0.0468 | <0.001 |
| Age of patient | −0.0153 | <0.001 | −0.0130 | <0.001 | −0.0222 | <0.001 |
| Day 3 FSH levels | −0.0243 | <0.001 | −0.0221 | <0.001 | −0.0331 | <0.001 |
In order to analyze the distribution of patients, patients were stratified into seven groups according to the ratio change in post-hCG P4 to hCG P4 levels. We found the increased ratio of post-hCG P4 to hCG P4 levels were between 2 and 8 in 79.3 % patients. The higher the post-hCG P4 to hCG P4 ratio, the more oocytes retrieved (Table 3). Moreover, the fertilization rate increased gradually across the seven groups. And in frozen-thawed cycles, 1,979 patients had frozen embryo transfer. The average number of embryos transferred was similar among seven groups. However, pregnancy rate and implantation rate from frozen-thawed cycles increased across the seven groups (Table 3).
Table 3.
IVF outcomes in seven groups stratified by serum Progesterone response to hCG dministration
| Group 1 | Group 2 | Group 3 | Group 4 | Group 5 | Group 6 | Group 7 | |
|---|---|---|---|---|---|---|---|
| No. of fresh cycles | 4 | 99 | 772 | 967 | 622 | 265 | 249 |
| No. of oocytes retrieved | 3.0 ± 2.2 | 3.6 ± 2.6 | 7.2 ± 4.4 | 11.2 ± 5.8 | 14.1 ± 6.7 | 16.3 ± 7.2 | 17.7 ± 7.7 |
| Mature oocyte rate (%) | 89 | 87.1 | 84.7 | 83 | 86 | 83 | 85.8 |
| Fertilization rate (%) | 33.3 | 41 | 51.8 | 55.4 | 66.4 | 76.3 | 83.7 |
| Good quality embryo rate (%) | 60 | 47.6 | 47.7 | 48.5 | 46.3 | 48.3 | 46.7 |
| No. of frozen-thawed cycles | 2 | 54 | 439 | 642 | 425 | 211 | 206 |
| Mean number of embryos transferred | 1.5 ± 0.7 | 1.6 ± 0.5 | 1.7 ± 0.5 | 1.8 ± 0.5 | 1.8 ± 0.4 | 1.8 ± 0.4 | 1.9 ± 0.4 |
| Pregnancy rate (%) | 50 | 41 | 51 | 57.5 | 58.6 | 68.7 | 68 |
| Implantation rate (%) | 33.3 | 27.3 | 36.3 | 42.1 | 42.4 | 51.7 | 53.3 |
values are the mean ± SD unless otherwise noted
Discussion
In this large cohort study, we found a significant association between serum P4 response the day after hCG administration and oocytes retrieved. The higher the post-hCG P4 levels, the more oocytes retrieved in both long and short protocols. There was no association of post-hCG P4 levels with other embryological aspects such as mature oocyte rate, fertilization rate and good quality embryo rate.
This is the first large retrospective study investigating the role of serum P4 response the day after hCG administration on embryological outcome. The embryo quality and quantity were important in predicting the outcome of IVF. The greater number of embryos and the better the embryo quality, the more likely it is that a higher cumulative pregnancy rate is achieved. However, it has been recognized that the risk of ovarian hyperstimulation syndrome will increase as the number of follicles and oocytes increase, and thus may lead to patients losing the chance of fresh embryo transfer. With the development of new stimulation protocols and laboratory micromanipulation techniques, it is important to evaluate the significance of changes in post-hCG P4 levels in light of current practice. Only two studies have examined this question recently [15, 16]. Burns et al. performed a retrospective study of 114 cycles undergoing IVF [15]. Progesterone concentrations on the day after hCG were inversely associated with clinical pregnancy by multiple logistic regression analysis. Subsequently, Check et al. conducted a retrospective review of IVF cylces stratified into three progesterone groups: low, normal and high [16]. They found that either too little or too much of a rise in serum progesterone on the day after hCG injection can adversely effect implantation. However, these studies had not analyzed the influence of post-hCG P4 levels on oocytes or embryos, which our study has investigated.
One of main strengths of our study is the large sample size including 2,978 fresh, first IVF cycles within a single institution. Regression analysis were used to detect the role of rise change in post-hCG P4 levels on oocyte retrieved, MII oocyte rate, fertilization rate and good quality embryo rate. We found that the fertilization rate increased gradually across the seven groups, it may mean that as the progesterone goes up, oocyte quality improves and therefore resulting in a higher fertilization rate. Moreover, the distribution of patients was also calculated according to the rise change in post-hCG P4 levels. We found the increased ratio of post-hCG P4 to hCG P4 levels were between 2 and 8 in 79.3 % patients, and the number of oocytes retrieved were between 6 and 15 in that range of ratio change. Ji et al. has found that the optimal number of oocytes for achieving a live birth is between 6 and 15 [19]. It indirectly pointed out that the rise change in post-hCG P4 levels could predict the outcome of IVF in some extent. However, our study was a retrospective research which often requires the analysis of data originally collected for reasons other than research purposes and we did not examine E2 levels change after hCG administration and recognize these are the limitations.
In natural menstrual cycles, P4 is typically synthesized as LH increases and reaches its peak at the time of implantation. In contrast, gonadotrophin-stimulated cycles produce multiple follicles and oocytes at different stages of maturation, and may thereby exhibit different responses to hCG administration. As the follicles develop and mature, the concentration of FSH and LH receptors on granulosa and theca cells increase [20], the response of follicles to FSH and LH increase. Moreover, hCG shares the same hCG/LH receptor with LH and its effect is 80 times that of LH, so the use of long-acting hCG may induce increased P4 production. The more follicles develop, the more P4 product. Before the administration of hCG, the P4 levels may rise slightly as premature luteinization occurs, but change in rise of P4 does not appear to be obvious as the change after hCG administration. So the degree of change in rise of post-hCG P4 levels has the greater predictive value of the number of oocytes retrieved. Numerous studies have confirmed that the subtle rise of P4 levels on the day of hCG administration does not affect the embryo quality [21], and our research had found that the evident rise of P4 levels the day after hCG administration did not influnce the fertilization rate and good quality embryo rate as well. The outcome of frozen embryo transfer cycles we evaluated was an extra measure of embryo quality in a different implantation setting. The pregnancy rate and implantation rate of frozen-thawed cycles were not influenced by the rise of post-hCG P4 levels. This showed us that the preovulation elevated progesterone does not affect oocyte or embryo quality.
In summary, our study suggests that post-hCG P4 levels can be used as an additional component, along with age and ovarian function, to assess the number of oocytes retrieved, and may further to estimate the pregnancy rate and live birth rate indirectly.
Acknowledgments
The authors sincerely thank other investigators and physicians who made contributions to this study. This work was supported by grants from the National Natural Science Foundation of China (No. 81270657), the Science and Technology Department Program of Zhejiang (2013C33145), the health department Program of Zhejiang (No. 2012RCA036) and the Natural Science Program of Zhejiang (Y14H040012). There is no conflict of interest in this article.
Footnotes
Capsule Preovulation elavated progesterone was positively associated with the number of oocytes retrieved, but does not affect oocyte or embryo quality.
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