INTRODUCTION
Luteal phase support (LPS) has been shown to be an integral part of ovarian stimulation protocols in vitro fertilisation (IVF) cycles and there is a consensus on the need for progesterone supplementation in the luteal phase of all stimulated IVF cycles.[1 2 progesterone to optimize the peri-implantation milieu cannot be overemphasized.
Various theories have been postulated for LPD in IVF cycles. LPD could be due to removal of granulosa cells during oocyte retrieval, prolonged pituitary suppression due to agonist administration in the GnRH agonist cycles or due to supraphysiological levels of steroids, which directly inhibit the LH release via negative feedback actions at the hypothalamic-pituitary axis level (GnSAF).[3
Progesterone is important as it helps in the secretory transformation of the endometrium by modulating the expression of giant mitochondria, subnuclear glycogen deposits, pinopodes, and nucleolar channel systems (NCSs), which facilitates the process of implantation.[4 Progesterone also enables implantation by promoting the immune system to produce non-inflammatory T-helper-2 cytokines. In the presence of sufficient progesterone , CD56 cells synthesize progesterone induced blocking factor (PIBF) which is a mediator having substantial anti-abortive activities.[5 et al. [6 progesterone increases nitric oxide production bringing about an improvement of blood flow and oxygen to the endometrium. Progesterone also reduces the contractility of the myometrium at the time of implantation, which is believed to promote implantation.
The importance of progesterone during the process of implantation and early pregnancy cannot be over emphasized and it seems plausible that LPD might be a cause of failing implantation and early pregnancy loss.[7 8
However, it is unclear if there is a cutoff level of progesterone in the luteal phase that may influence the outcome of IVF-ET cycles and whether increasing plasma progesterone at this stage can be considered as a rational approach to improve rates of implantation.
We conducted a prospective cohort study earlier at our center on 147 women undergoing fresh and frozen embryo transfers (ETs) and found that serum progesterone levels less than 15 ng/mL in the mid luteal phase had a negative impact on pregnancy outcome in both fresh and frozen ET cycles.[9
Aims and Objectives:
The aim of this study was to determine if incrementing progesterone supplementation based on the levels of serum progesterone (P) measured on the day of ET has an impact on the pregnancy outcome in fresh and frozen ET cycles.
MATERIALS AND METHODS
Source of data
A total of 220 women undergoing ET in both fresh and frozen cycles between February 2017 and August 2017 were enrolled in our study after obtaining written and informed consent. Out of these, 205 met the inclusion criterion and were included in our study group. Ethical clearance was obtained.
Design
This was a prospective cohort study.
Inclusion criterion
Patients less than 40 years undergoing ET (both fresh and frozen cycles) during the study period were included in our study.
Exclusion criterion
The exclusion criteria of the study were recurrent implantation failures, poor embryo quality, patients wherein a change in LPS was required due to inability to tolerate vaginal/injectable progesterone .
Methodology
GnRH antagonist protocol was followed in all the patients enrolled in our study. Dose of gonadotropins (rFSH [follitropin alpha or beta and highly purified HMG]) was decided based on the patient’s age, body mass index (BMI), ovarian reserve, and previous response. GnRH antagonist (Cetrolix Intas) was started according to the flexible protocol. Injection human chorionic gonadotropin 10,000 IU was given as the trigger in all the patients included in our study.
LUTEAL SUPPORT
LPS in the fresh ET cycles was vaginal micronized progesterone 400 mg twice a day and Injectable progesterone in oil 50 mg intramuscular twice a week.
In the frozen ET cycles LPS was vaginal micronized progesterone 400 mg twice a day and Injectable natural progesterone 50 mg intramuscular alternate day for patients with BMI less than 35. Injectable natural progesterone 50 mg intramuscular was given daily in cases having BMI more than 35.
Serum progesterone levels were measured on the day of ET after collecting 2cc of blood in plain vacutainer and analyzed after centrifugation using automated chemiluminescence microparticle immunoassay.
Patients undergoing fresh and frozen ETs were both divided into two groups based on serum progesterone levels on the day of ET. Group A1 included women who underwent a fresh ET and had serum progesterone less than 15 ng/mL on the day of ET, whereas group B1 included women undergoing a fresh ET with serum progesterone levels more than 15 ng/mL on the day of ET Group A2 and B2 included women who underwent frozen ET and had serum progesterone levels on the day of ET less than and more than 15 ng/mL, respectively [Figure 1 ].
Figure 1: Division of patients in two groups based on their serum progesterone levels on the day of transfer
If serum progesterone levels were found to be less than 15 ng/mL on the day of ET, the dose of progesterone supplementation was incremented (in fresh cycles we increased the dosage of injectable progesterone 50 mg from twice a week to 50 mg alternate days and in frozen cycles the dose of injectable progesterone was incremented to 50 mg daily from 50 mg alternate day) [Figure 2 ].
Figure 2: Protocol followed for division into subgroups based on serum progesterone and progesterone incrementation
A correlation between the groups with serum progesterone less than and more than 15 ng/mL on the day of ET with the pregnancy outcome was then made. Urine pregnancy test and βHCG were done 14 days after ET. A pregnancy test was considered positive if the βHCG levels exceeded 25 IU. Clinical pregnancy was defined by the presence of a gestation sac with fetal pole. Biochemical pregnancy was defined as a fall in βHCG levels without the appearance of a gestation sac.
Statistical analysis
Continuous variables are presented as mean ± standard deviation (SD), and categorical variables are presented as absolute numbers and percentage. For all statistical tests, chi-square tests were used for qualitative analysis and t test was used for comparison of means. All data analysis was carried out by the SPSS program for Windows, version 21.0. A value of P < 0.05 was taken to indicate a significant difference.
In fresh ET cycles we observed that there was no difference in the age, BMI, AMH, starting dose, the total days of stimulation, estrogen on the day of trigger, number of oocytes retrieved and the number of embryos transferred between the groups having progesterone less than 15 ng/mL (Group A1) and more than 15 ng/mL (Group B1) on the day of ET.
However, the progesterone level on the day of trigger was significantly lower in the group with progesterone less than 15 ng/mL [Table 1 ].
Table 1: Demographic profile of women undergoing fresh embryo transfer
In fresh ET cycles out of the 62 women recruited in the study eight patients had serum progesterone <15 ng/mL on the day of ET, which was incremented after the results of progesterone levels were obtained, whereas rest of the patients had progesterone more than 15 ng/mL (n = 54). No statistically significant difference was observed in the clinical pregnancy rate and biochemical pregnancies in group with progesterone less than or more than 15 ng/mL on the day of ET [Table 2 ].
Table 2: Pregnancy outcome in women undergoing fresh embryo transfer based on the serum progesterone levels on day of ET
In the frozen ET cycles, we did not find any difference in the age, BMI, and endometrial thickness between the two groups having progesterone less than 15 ng/mL (Group A2) and more than 15 ng/mL (Group B2) on the day of ET [Table 3 ].
Table 3: Demographic profile of women undergoing fresh embryo transfer
In the frozen ET cycle, out of the 143 women recruited 36 cases had progesterone less than 15 ng/mL on the day of ET which was incremented. No statistical significant difference was observed in the clinical pregnancy or biochemical pregnancies in the two groups [Table 4 ].
Table 4: Pregnancy outcome in women undergoing frozen embryo transfer based on the serum progesterone levels on day of ET
DISCUSSION
Serum progesterone assay has been proposed as a surrogate of endometrial competence and can help define a “fertile” luteal phase.[10 Progesterone induces a secretory transformation of the endometrium in the luteal phase which after adequate estrogen priming improves endometrial receptivity.[11 12
Luteal phase of IVF-ET cycles is important as it might be the reason for the discrepancy between fertilization and pregnancy rates. Supplementation with progesterone may rescue potentially failing corpora lutea, thereby making the peri-implantation period more amiable to the implanting embryo.[13
Liu et al. [14 progesterone concentrations during the perimplantation period of the early luteal phase than patients who were not pregnant or had an early miscarriage.
Further, Mitwally et al. [15 progesterone concentration during the luteal phase and the clinical pregnancy outcome.
According to Humaidan et al. ,[16 progesterone concentration showing a sharp decline in the early pregnancy loss from approximately 80% to 10% as the mid-luteal-phase progesterone concentration increases from approximately 40 nmol/l to approximately 80–100 nmol/L. Studies have shown that an early pregnancy losses (EPL) may result not only from chromosomally abnormal embryos, but also from malfunctions of the endometrium or both.
A study by Alsbjerg et al. [17 progesterone gel supplementation from 90 mg to 180 mg, resulting in a significant decrease in the EPL rate (67% vs. 44%, respectively; P = 0.014) and a significant increase in the delivery rate (9% vs. 21%, respectively; P = 0.002).
In a prospective cohort study conducted at our center during April 2016– December 2016, we analyzed the impact of midluteal serum progesterone levels on clinical pregnancy outcome. Patients with progesterone levels less than 15 ng/mL and more than 15 ng/mL during the midluteal phase in both fresh and frozen ET cycles were placed in two separate groups. In fresh ET cycles (n = 66), we observed that in the group with serum progesterone less than 15 ng/mL during the midluteal phase (n = 11) the pregnancy rate was 9% and in the group with progesterone levels more than 15 ng/mL (n = 55), the pregnancy rate was 40% (P = 0.049), thereby implying a negative effect of low midluteal serum progesterone levels on pregnancy outcome in fresh ET cycles. In the frozen ET cycles (n = 79), we found that in patients with progesterone levels less than 15 ng/mL (n = 21) and more than 15 ng/mL during the midluteal phase (n = 58) there was no statistical and clinical difference in the clinical pregnancy in patients with progesterone less than 15 ng/mL (47%) and those with progesterone >15 ng/mL (51.7%) in the midluteal phase (P = 0.74). However, in the frozen ET cycles we found a statistically significant increase in the biochemical pregnancies in the group with progesterone <15 ng/mL (P = 0.029) implying that low mid luteal progesterone levels in the frozen ET cycles might cause a detrimental effect on the pregnancy outcome. We thereby concluded that evaluation of serum progesterone in both fresh and frozen ET cycles may help identify patients who need additional LPS to improve the pregnancy outcome. Based on the findings of our study, we conducted this study and tried to analyze if measuring and incrementing progesterone early (before the midluteal phase) might improve our pregnancy outcomes.
In fresh ET cycles out of the 62 women recruited in the study eight patients had serum progesterone <15 ng/mL on the day of ET, which was incremented after the results of progesterone levels were obtained, whereas rest of the patients had progesterone more than 15 ng/mL (n = 54) and so they continued the standard LPS regimen. No statistically significant difference was observed in the clinical pregnancy rate and biochemical pregnancies in the group with progesterone less than or more than 15 ng/mL on the day of ET.
Similarly in our frozen ET cycle patients out of the 143 women recruited 36 cases had progesterone less than 15 ng/mL on the day of ET, which was incremented and again we did not find any difference in the clinical pregnancy rate or biochemical pregnancies in the two groups stressing the importance of measuring and incrementing progesterone early in the cycle.
Ellenbogen et al. [18 progesterone concentrations were significantly higher (15.3 ± 4.9 ng/mL vs. 12.9 ± 4.9 ng/mL, P < 0.002) in patients who became pregnant compared with those who did not. In pregnant patients, 53% had mid-luteal progesterone exceeding 15 ng/mL vs. 31% in nonpregnant patients (P < 0.01).
The same group did a prospective RCT in 2017 to establish whether increasing progesterone dosage for patients with low levels at mid-luteal phase may improve pregnancy rates. They included a total of 146 patients undergoing IVF treatment and measured serum progesterone levels 7 days after ET and increased the progesterone support in patients with serum progesterone levels less than 15 ng/mL. However, they found that mid-luteal intervention by increasing progesterone support did not improve cycle outcomes. The probable reason for the same could be the delayed increase in the dose of progesterone supplementation. This study also suggested that assessing progesterone levels earlier in the implantation window may be beneficial for adjustment of the optimal dosage. The limitation of this study was also that they recruited women undergoing different stimulation protocols; however, in our study all women underwent the GnRH antagonist protocol.[19
A study done by Alvarenz et al. [20 progesterone levels were measured the day prior to euploid FET. Serum progesterone levels more than 10.6 ng/mL were considered as adequate and euploid FET was performed on the following day, whereas Pl levels < 10.6 ng/mL was considered as low, iLPS was added in the form of daily Psc injections, and a new P analysis was performed on the following day. FET was only performed on the same day when a restored P > 10.6 ng/mL was achieved (98.2% of cases), thereby suggesting an “opportunity window” for improved ongoing pregnancy rates and miscarriage rates in cases of inadequate P levels the day previous to FET. The same group did a similar study even in women undergoing NC FET and found mean serum P levels on the day before FET were significantly higher in patients who had a live birth compared to those who did not (14.5 ± 7.0 vs. 12.0 ± 6.6 ng/mL, 95% CI [0.83; 4.12]).[21
Lambarta et al. [22 progesterone 400 mg bd. Patients with serum P < 9.2 ng/mL on the day of ET had a significantly lower OPR suggesting a minimum threshold of serum P values on the day of ET that needs to be reached in artificial endometrial preparation cycles to optimize pregnancy outcome. The same group did a study in women undergoing FET in self and donor oocyte cycles (n = 1150) and found that women with serum P levels <8.8 ng/mL had a significantly lower ongoing pregnancy rate (36.6% vs. 54.4%) and live birth rate (35.5% vs. 52.0%) when using micronized vaginal progesterone in a dose of 400 mg bd as LPS. One-third of their patients showed inadequate levels of serum P which impacted the success of the ART cycle, thereby they suggested monitoring P levels in the mid-luteal phase is recommended when using MVP for dose adjustments to optimize pregnancy outcomes.[23
The interest on the impact of serum P levels during window of implantation has emerged in the last years. There seems to be a relationship between low serum P levels and poorer outcome, in terms of ongoing pregnancy or live birth rate. We incremented our progesterone levels early in the cycle aiming to have adequate progesterone levels during the window of implantation as increasing it later in the cycle might not be of much benefit as the window of implantation (WOI) would have already closed.
Another interesting finding in our study was the statistically significant lower levels of progesterone on the day of trigger in the patient group with low progesterone levels (<15 ng/mL) on the day of ET in fresh cycles. A study done by Santos et al. [24 P values (<0.5 ng/mL) results in lower live birth rates and the reason for the same as proposed by them could be luteinization defect. They suggested that a more intense or earlier LPS could counter-balance the negative influence of low Progesterone on the live birth rates.
CONCLUSION
Measuring serum progesterone levels on the day of ET and incrementing if levels of progesterone are less than 15 ng/mL to obtain optimal progesterone levels during the WOI in both fresh and frozen ET cycles might help in improving our pregnancy outcomes.
Financial support and sponsorship
Not applicable.
Conflicts of interest
There are no conflicts of interest.
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