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The role of progesterone elevation in IVF
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Review
The role of progesterone elevation in IVF ⁎
Panagiotis Drakopoulosa,b, , Annalisa Raccaa,c, Joaquín Errázuriza,d, Michel De Vosa,b, Herman Tournayea, Christophe Blockeela,b,e, Nicola Pluchinof, Samuel Santos-Ribeiroa,b,g a
Center for Reproductive Medicine, Universitair Ziekenhuis Brussel, Brussels, Belgium Faculty of Medicine and Pharmacy, Department of Surgical and Clinical Science, Vrije Universiteit Brussel, Belgium University of Genoa, Academic Unit of Obstetrics and Gynecology, IRCCS AOU San Martino, Italy d Centre for Reproductive Medicine, Clínica Alemana, Santiago, Chile e University of Zagreb-School of Medicine, Department of Obstetrics and Gynecology, Zagreb, Croatia f Division of Obstetrics and Gynecology, University Hospital of Geneva, Geneva, Switzerland g IVI-RMA, Lisbon, Portugal b c
A R T I C LE I N FO
A B S T R A C T
Keywords: In vitro fertilization Progesterone elevation Endometrial receptivity Embryo quality Implantation
Elevation of progesterone during the late follicular phase of stimulated in-vitro fertilization cycles is a frequent event, which negatively impacts the outcome. Over the years evidence has demonstrated a direct relationship between late-follicular elevated progesterone and endometrial receptivity. In this regard, elective cryopreservation of all good quality embryos and transfer in a subsequent frozen/thawed cycle is the most common strategy adopted by clinicians in case of elevated progesterone. Nonetheless, recent evidence suggests that elective cryopreservation might not entirely resolve the reduced pregnancy outcomes associated with the elevation of progesterone, considering that the increase may affect not only implantation, but also embryo quality.
1. Introduction Elevation of progesterone during the late follicular phase of stimulated in-vitro fertilization (IVF) cycles is a frequent event, occurring in 12–38% of cycles [1], as it cannot be completely prevented by the administration of gonadotropin-releasing hormone (GnRH) analogues. The incidence of late follicular phase elevated progesterone varies among studies and this marked variation could be explained by the method of progesterone assessment. Several cut-off levels for elevated progesterone in stimulated IVF cycles have been suggested, ranging from 0.8 to 2.0 ng/mL. Nonetheless, 1.50 ng/mL as a cut-off for elevated progesterone is most commonly used [1]. This threshold is supported by the presence of a marked difference in endometrial gene expression profile between infertile patients with a progesterone serum concentration above and below 1.50 ng/mL on the day of human chorionic gonadotrophin (hCG) administration [2]. Over the years, there has been a debate on whether elevated progesterone may hinder pregnancy rates following IVF treatment. The debate came to an end following the results of a recent meta-analysis including more than 60,000 IVF cycles, clearly demonstrating that in case of progesterone elevation, there is a negative impact on reproductive outcomes [3]. Based on these findings, most clinicians choose to freeze all embryos in cycles with elevated progesterone and ⁎
perform the transfer in a subsequent frozen/thawed cycle. However, it should be stated that the cut-off of progesterone elevation should be considered with caution, given the high variability of the assays used for progesterone measurement, making difficult the comparison of the results of different studies and therefore hindering the validity of the afore mentioned meta-analysis [4,5]. The mechanism by which the rise of progesterone during the follicular phase affects pregnancy rate is still not fully understood. Initially, it has been advocated that elevated levels of peripheral progesterone have no effect on oocyte/embryo quality, and that their detrimental effects are mainly mediated through the endometrium, leading to an asynchrony between the endometrium and the developing embryo. Nevertheless, recent evidence suggests that elevated progesterone levels may additionally be associated with a decrease in embryo quality and cumulative live birth rates [6–8]. In this review, evidence on the mechanism of premature progesterone elevation and its impact on reproductive outcomes of IVF treatment are summarized. 2. The effect of supraphysiologic circulating hormone levels on endometrial receptivity Although the duration of the follicular phase of the menstrual cycle
Corresponding author at: Centre for Reproductive Medicine, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Laarbeeklaan 101, 1090 Brussel, Belgium. E-mail address: [email protected] (P. Drakopoulos).
https://doi.org/10.1016/j.repbio.2019.02.003 Received 31 December 2018; Received in revised form 4 February 2019; Accepted 7 February 2019 1642-431X/ © 2019 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Panagiotis Drakopoulos, et al., Reproductive Biology, https://doi.org/10.1016/j.repbio.2019.02.003
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elucidated. A stimulated IVF cycle is not comparable to the natural cycle. In stimulated cycles, multiple follicular growth occurs, resulting in supraphysiological serum E2 concentrations during the follicular phase. Daily injections with recombinant follicle stimulating hormone (FSH) are necessary to prevent serum FSH levels from dropping below the threshold, which is required for follicle development. Consequently, the follicles are under constant FSH stimulus until the final oocyte maturation. Ovarian-stimulation-derived progesterone overproduction has traditionally been considered to be a consequence of CYP17-produced progesterone in the theca cells of the multiple developing follicles. This progesterone produced in the follicular phase is usually metabolized downstream by the LH-activity in the later stages of follicular development, as LH promotes the conversion of progesterone into androgens, which will be then transported and converted into E2 by the granulosa cells. However, recent evidence suggests that progesterone is probably produced directly in a dose-dependent manner by FSH activity in the granulosa cells [13]. Using in-vitro-study on human ovarian cortical samples, it was shown that exogenous FSH seems to stimulate the expression of 3β-hydroxysteriod dehydrogenoase (3β-HSD) and progesterone biosynthesis in granulosa cells in addition to its stimulatory effect on the expression of other steroidogenic enzymes [13]. FSH has a direct stimulatory effect on the activity of 3β-HSD and therefore may increase the conversion of pregnenolone to progesterone in a dose dependent manner. According to the two-cell–two-gonadotropin theory, pregnenolone and progesterone produced by granulosa cells should enter the theca cells in order to be converted into androgens. In case of ovarian stimulation, characterized by constantly elevated FSH-levels, the amount of precursor steroids produced may exceed the capacity of the ovary to efficiently convert them into estrogens. This may delay the conversion of progesterone into androgens in theca cells and lead to their accumulation and leakage into the systemic circulation, which could explain why FSH stimulation promotes progesterone output from granulosa cells. Besides enhanced FSH stimulation during IVF treatment, another factor that is associated with progesterone rise is the prolongation of the follicular phase by delaying hCG administration. Kolibianakis et al. [14] demonstrated that a prolonged exposure of the endometrium to high concentrations of progesterone could hinder pregnancy rates, if the follicular phase is prolonged by 2 days. In the same context, Kyrou et al. [15], showed that a cut-off for estradiol on the day of hCG administration of more than 1790.5 pg/mL, associated with more than 10 follicles of at least 11 mm in diameter, was highly likely to result in a progesterone rise of > 1.50 ng/mL. The association between high E2 and premature progesterone elevation could be explained by an excess of proliferating granulosa cells that may lead to an increased progesterone production. Given that the cause of premature progesterone rise could be the continuous FSH stimulation, a hypothesis that a step-down protocol, implying lower FSH doses at the end of stimulation, could potentially overcome the problem of progesterone rise was raised. The hypothesis was confirmed by a recent post-hoc data analysis, showing that patients who met the criteria for final oocyte maturation after receiving a single injection of corifolitropin alfa (a long acting gonadotropin characterized by a rapid absorption which results in peak concentrations of FSH two days after the injection and a progressive decrease thereafter) had a significantly lower incidence of premature progesterone elevation compared with patients receiving daily recombinant FSH-injections over the same duration of stimulation [16]. Thus, these findings support the idea that enhanced and persistent FSH stimulation towards the end of the follicular phase is the primary cause of premature progesterone rise. Another assumption was that premature progesterone elevation in stimulated cycles may be caused by the lack of LH/hCG activity in IVF protocols and that LH supplementation could potentially decrease the incidence of progesterone rise. Prior studies showed that LH/hCG
may be subject to both inter-cycle and inter-women variability, endogenous sexual hormones remain within relatively low levels prior to ovulation, specifically a median (5th and 95th percentiles) of 182.80 pg/mL (131.30 pg/mL – 388.28 pg/mL) for estradiol (E2) and 0.80 ng/mL (0.39 ng/mL – 1.30 ng/mL) for progesterone [9]. Meanwhile, these same hormones reach substantially higher levels with ovarian stimulation. These supraphysiologic hormones concentrations present during IVF have been strongly associated with a negative effect on endometrial receptivity (ER) [2], with the exceeding majority of biopsies performed on the day of oocyte retrieval demonstrating both histologic and gene expression patterns suggestive of endometrial advancement. Studies in oocyte donors have also shown that ovarian stimulation may accelerate the appearance of endometrial pinopodes and nucleolar channel systems ultrastructural formations which are closely related to ER and the window of implantation (WOI) which promote abnormal intracavity interleukin-1 (IL-1) expression, and cause abnormal placentation and embryonic growth [10]. To this extent, the monitoring of both the endometrial and ovarian responses to ovarian stimulation with transvaginal ultrasound and serum hormonal assessments have become important predictors of assisted reproductive technologies (ART) success. 3. Progesterone in the natural menstrual cycle In a natural cycle, circulating progesterone remains low throughout the follicular phase until ovulation, specifically at a median of 0.30 ng/ mL (0.19 ng/mL – 0.51 ng/mL) [9]. Prior to ovulation, the main source of progesterone shifts from the adrenal cortex to the ovary and, at that same time, progesterone receptor mRNA and proteins become more abundant in the nuclei of the granulosa cells [11]. The luteinising hormone (LH) peak causes a slight but noticeable physiologic late-follicular phase progesterone peak which, apart from contributing for the timing of ovulation, may be essential for follicular development. Animal experiments have shown that blocking mid-cycle progesterone production is detrimental for oocyte maturation, oocyte fertilization competence and granulosa/theca luteinisation [11]. Meanwhile, Following adequate priming by E2, progesterone secreted by a recently luteinized follicle(s) will regulate an intricate sequence of molecular events in preparation for implantation. After ovulation, progesterone limits any further proliferation of the endometrium over the course of the next three days by directly restricting the function of E2 and the expression of E2-binding receptors, while stimulating further the development of both vascular and glandular structures. This selective growth of the vascular and glandular components within a confined structure causes coiling of the spiral vessels and increases glandular tortuosity. At the moment of implantation, the endometrium has become differentiated into two distinct layers: the basal layer (most adjacent to the myometrium, a layer which remains relatively unchanged throughout the menstrual cycle and is critical for endometrial regeneration following the cyclical sloughing events endured during menstruation) and the functional layer (constituted by the stratum spongiosum and an overlaying stratum compactum). These successive morphological changes which progesterone sets into motion follow a predictable day-by-day sequence of development which were the basis of the histological endometrial dating classification system developed by Noyes et al. [12]. This classic endometrial classification system remained, until recently, an unchallenged gold standard for the evaluation of luteal function, where endometrial samples with a developmental stage two-days apart from the expected were considered to be “out of phase”. 4. Mechanism and causes of progesterone elevation during ovarian stimulation Till now, the exact cause of progesterone elevation towards the end of the follicular phase of ovarian stimulation has not been fully 2
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single measurement by using ratios such as progesterone-to-follicle, progesterone-to-oocyte, progesterone-to-estradiol ratios [30,31]. However, such indexes have failed to perform superiorly, thus limiting their routine clinical practice [32]. Huang et al. [33] concluded in their retrospective analysis that the duration of progesterone elevation seemed to play a major role in how circulating progesterone affected pregnancy outcome. This approach was unique in the sense that it was the first to raise the question “how long can the endometrium be exposed to progesterone above a certain universal threshold?”. As progesterone receptors are present in endometrium in all phases of the menstrual cycle, namely during menses and the early follicular phase, it would be reasonable to assume that progesterone exposure might be detrimental even when occurring days before ovulation triggering. Furthermore, this concept of a hindering cumulative progesterone exposure is more in line with the current knowledge of how progesterone regulates endometrial development, as it is generally thought that the ovaries need to increase and maintain the secretion of progesterone above a certain level in order to initiate and sustain the development of a secretory endometrium. However, whether the threshold to trigger these endometrial changes is universal (e.g. 1.50 ng/mL, regardless of the patient’s baseline progesterone exposure) or relative (i.e. a difference between the amount of progesterone produced and the usual baseline circulating progesterone concentration encountered in each patient) is unknown. While being a potentially useful hypothesis, the approach performed by Huang et al. [33] failed to be confirmed in a recent and large retrospective study [34]. One of the reasons for these conflicting results may stem from the fact that the study elected a universal arbitrary cut-off of 1.00 ng/mL, which is frequently found at the end of the follicular phase of unstimulated cycles as well [9]. In the light of a potential detrimental effect of “high” progesterone on reproductive outcomes, “freeze only” seems to be the most common strategy by clinicians, as already mentioned. In a recent cost-effective study performed by Healy et al. [35] more than 7000 ART transfer cycles were evaluated. The main objective of the study was to define a threshold of progesterone on which the “freeze only” approach is costeffective. Based on these findings, “high” progesterone (from 1.50 ng/ mL to 2.0 ng/mL) seems to have a clinically significant negative effect and captures a good percentage of patients at higher risk for fresh transfer failure, thus making the "freeze only" strategy a cost-effective option. Nonetheless, elective cryopreservation might not be enough to solve the reduced pregnancy outcomes, considering that elevated progesterone may affect not only implantation, but also embryo quality, as discussed in the next section.
activity may indeed have a protective effect, preventing a possible premature progesterone rise, when comparing patients stimulated with recombinant FSH (rFSH) versus human menopausal gonadotrophin (hMG) [17]. Nevertheless, in a large prospective randomized control trial (RCT) including 749 patients treated with either rFSH or highpurified (HP) hMG, serum progesterone levels at the end of stimulation were similar [18]. 5. The effect of progesterone elevation on endometrial receptivity in ovarian stimulation cycles Fundamental evidence has accumulated over the years demonstrating a direct relationship between late-follicular elevated progesterone and ER. Specifically values of progesterone above 1.50 ng/mL have been associated with: a) a histological advancement which may extent beyond 3 days, b) an abnormal expression of the implantation regulating proteins vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), c) abnormalities in the gene expression which could be related to natural killer (NK) cell activity and, more recently, d) differences in epigenetic profiles [19]. Many research groups have attempted to assess whether these findings are translatable into hindered pregnancy outcomes in IVF. The majority of available studies have demonstrated that “high” progesterone levels may hinder IVF success [1,3]. However, these results were initially disputed by other researchers who found late-follicular progesterone elevation to be either irrelevant [20] or even beneficial in selected infertile populations [21]. In particular, it seems that high responders are not exempt from the detrimental effects of prematurely rising serum progesterone levels but the threshold interval where the detrimental effect begins is higher in the high responders compared with the low and normal responders [22]. Furthermore, a 2007 meta-analysis showed that “high” serum progesterone levels on the day of hCG administration did not significantly affect pregnancy rates [23]. However, the heterogeneity of the studies included in this meta-analysis may have biased the conclusions and updated meta-analyses by the same group eventually provided contradictory results [3]. This may due to the limited reproducibility when different progesterone assays are used, making the interpretation of the several thresholds critical [4]. Although the matter whether progesterone rise affects oocyte/embryo competence remains contentious as discussed later, some investigators consider that the issue of progesterone elevation may be, at least in part, resolved by elective embryo cryopreservation, given the previously-mentioned translational studies associating “high” progesterone levels to an abnormal endometrial genomic/epigenetic expression in the luteal phase. This has led many centres to change their clinical practice and to measure serum progesterone levels on the day of ovulation triggering and to adopt a “freeze-only” strategy when the threshold of 1.50 ng/mL is exceeded. Nonetheless, the everyday use of late-follicular elevated progesterone as an ART outcome predictor in current clinical practice has proven to be harder than originally expected, owing mostly to the fact that a) progesterone production may not affect pregnancy outcomes linearly [1,24] and b) it is frequently encountered in good-prognosis women with an otherwise healthy multi-follicular response [25]. Furthermore, previous studies have recommended caution in the use of progesterone elevation as an indication for all-embryo cryopreservation in high-responder women or the adaptation of the cut-off of “high” progesterone according to ovarian response [3]. Meanwhile, others have postulated that elevated progesterone may only effect cleavage stage embryo transfers [26], a hypothesis that has been refuted more recently by others [1,27,28]. Finally, the detrimental effect of circulating progesterone may be set into motion even when progesterone is below 1.50 ng/mL [29]. Altogether, these conflicting studies are a testament of the limitations of using a single progesterone assessment during IVF to decide whether to defer embryo transfer or not. Many investigators have attempted to enhance the predictive capacity of this
6. Progesterone and embryo quality Despite that the effects of late follicular progesterone rise on endometrial receptivity have been confirmed, evidence on whether increased progesterone may affect embryo quality is still limited. While some previous studies suggested that embryo quality is not affected by elevated progesterone [36], others postulated the opposite, namely that elevated serum progesterone on the day of hCG trigger may negatively influence the top embryo quality rate (Table 1). Specifically, Check et al. [37] and Melo et al. [36] evaluated pregnancy rates in oocyte donation cycles, as an indirect measure of the embryo quality and the investigators found no difference in the two study groups, using a cut-off of progesterone of 1.0 ng/mL and 1.2 ng/mL, respectively [36,37]. Similarly, Ubaldi et al. [38] compared 53 cases of elevated progesterone (more than 1.0 ng/mL) versus 1275 cases with lower progesterone values, in heterologous-ICSI cycle suppressed with GnRH agonist; no differences in embryo quality as well as in pregnancy outcomes were detected [38]. On the other hand, recent studies focused exclusively on embryo quality of infertile women: Huang et al. [6], evaluated cleavage stage embryos and Vanni et al. [7] blastocyst stage embryos; both studies showed a decrease in top embryo quality 3
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Table 1 Negative effect of progesterone elevation on embryo quality. Yes
Number of embryos/cycles included, day of transfer
Progesterone cut-off levels
No
Number of embryos/cycles included, day of transfer
Progesterone cut-off levels
Huang et al. [6] Vanni et al. [7] Racca et al. [8] Healy et al. [35]
2630 embryos, Day 3 986 cycles, Day 5 3400 cycles, Day 3 and 5 4350 embryos, Day 5 and 6
> 2.0 ng/ml > 1.49 ng/ml ≥1.5 ng/ml > 1.5 ng/ml
Melo et al. [36] Check et al. [37] Ubaldi et al. [38] Baldini et al. [41]
240 cycles, Day 5 270 cycles, Day unknown 24 cycles, Day 3 131 cycles, Day 3
≥1.2 ng/ml > 1 ng/mL ≥1.1 ng/ml ≥1.2 ng/ml
may even be associated with a decrease in embryo quality and cumulative live birth rates. In this regard, reduction of FSH-stimulation intensity towards the end of stimulation could result in a lower incidence of P elevation and therefore may be a feasible rescue strategy. Finally, studies comparing cumulative live birth rates in case of progesterone elevation or not in patients with a “freeze all strategy” and evidence derived from PGT-A cycles complicated by late follicular progesterone rise could shed more light on the role of progesterone in IVF.
formation rate as progesterone increased [6,7]. Therefore, the advantages of prolonging ovarian stimulation to maximize the number of collected oocytes might eventually be hindered by a decrease in top quality embryos for transfer, if increasing progesterone levels are detected. In the same context, Healy et al. demonstrated a negative effect of elevated serum progesterone on slow growing blastocysts (day 6) compared to day 5 blastocysts, in terms of live birth rate, while Simon et al. recently showed that progesterone concentrations above 1 ng// mL were significantly negatively correlated to the percentage of top quality embryos and to the implantation rates [39]. Given the paucity of evidence and the fact that all published studies were mainly focused on pregnancy rates in fresh or in the first frozen-thawed embryo transfer rather than on cumulative pregnancy rate, Racca et al. [8,40] was the first to evaluate at the same time progesterone levels, embryo quality and live birth rates. In that study, the main outcomes were cumulative live birth rates and embryo utilization rates, calculated as the total number of embryos transferred and cryopreserved divided by the total number of fertilized oocytes. The results showed that for both cleavage and blastocyst stage embryos, the utilization rates were lower for the elevated progesterone group (progesterone > 1.50 ng/mL). Moreover, cumulative live birth rates were not significantly higher in the elevated progesterone group, as it may have been expected, due to the higher number of number of oocytes retrieved; a potential explanation could be that patients in the “high” progesterone group may have had a higher consumption of embryos necessary to achieve a live birth. On the other hand, given that endometrial receptivity in the presence of “high” progesterone is known to be impaired, fresh transfer of the top quality embryos/blastocysts in patients with elevated progesterone might have been the main cause of the observed decreased cumulative outcomes in the study by Racca et al. [8] and the fact that frozen cycles may provide higher pregnancy rates in case of progesterone rise the day of hCG administration [41]. In this regard, relevant clinical insight on the effect of progesterone on embryo quality in terms of cumulative live birth rates should be derived from “freeze only” cycles—where no fresh embryo transfer is performed in the presence of progesterone elevation and the whole cohort of embryos is cryopreserved. Such a study comparing the effectiveness of a "freeze only" strategy in the presence of elevated progesterone to other “freeze only” cycles to eliminate the bias in endometrial receptivity- could provide robust evidence regarding the role of progesterone in embryo quality. Furthermore, a retrospective or prospective study in Preimplantation Genetic Testing for Aneuploidy (PGT-A) patients could further elucidate whether elevated progesterone may affect aneuploidy rates. To the best of our knowledge, such a study has not been conducted yet.
Conflict of interest None. Authors’ contributions P.D, A.R, J.E and S.S-R are responsible for the concept design and wrote the manuscript, P.D, M.D-V, H.T, C.B, N.P and S.S-R contributed to the editing of the manuscript. Acknowledgement No Acknowledgement. References [1] Bosch E, Labarta E, Crespo J, Simon C, Remohi J, Jenkins J, et al. Circulating progesterone levels and ongoing pregnancy rates in controlled ovarian stimulation cycles for in vitro fertilization: analysis of over 4000 cycles. Hum Reprod 2010;25. https://doi.org/10.1093/humrep/deq125. 2092–100. [2] Labarta E, Martinez-Conejero JA, Alama P, Horcajadas JA, Pellicer A, et al. Endometrial receptivity is affected in women with high circulating progesterone levels at the end of the follicular phase: a functional genomics analysis. Hum Reprod 2011;26. https://doi.org/10.1093/humrep/der126. 1813–25. [3] Venetis CA, Kolibianakis EM, Bosdou JK, Tarlatzis BC. Progesterone elevation and probability of pregnancy after IVF: a systematic review and meta-analysis of over 60 000 cycles. Hum Reprod Update 2013;19. https://doi.org/10.1093/humupd/ dmt014. 433–57. [4] Lawrenz B, Sibal J, Garrido N, Abu E, Jean A, Melado L, et al. Inter-assay variation and reproducibility of progesterone measurements during ovarian stimulation for IVF. PLoS One 2018;13:e0206098https://doi.org/10.1371/journal.pone.0206098. [5] Patton PE, Lim JY, Hickok LR, Kettel LM, Larson JM, Pau KYF. Precision of progesterone measurements with the use of automated immunoassay analyzers and the impact on clinical decisions for in vitro fertilization. Fertil Steril 2014;101. https:// doi.org/10.1016/j.fertnstert.2014.02.037. 1629–36. [6] Huang B, Ren X, Wu L, Zhu L, Xu B, Li Y, et al. Elevated progesterone levels on the day of oocyte maturation may affect top quality embryo IVF cycles. PLoS One 2016;11. https://doi.org/10.1371/journal.pone.0145895. e0145895–8. [7] Vanni VS, Somigliana E, Reschini M, Pagliardini L, Marotta E, Faulisi S, et al. Top quality blastocyst formation rates in relation to progesterone levels on the day of oocyte maturation in GnRH antagonist IVF/ICSI cycles. PLoS One 2017;12:e0176482https://doi.org/10.1371/journal.pone.0176482. [8] Racca A, Santos-Ribeiro S, De Munck N, Mackens S, Drakopoulos P, et al. Impact of late-follicular phase elevated serum progesterone on cumulative live birth rates: is there a deleterious effect on embryo quality? Hum Reprod 2018;33. https://doi. org/10.1093/humrep/dey031. 860–8. [9] Stricker R, Eberhart R, Chevailler M-C, Quinn FA, Bischof P, Stricker R. Establishment of detailed reference values for luteinizing hormone, follicle stimulating hormone, estradiol, and progesterone during different phases of the menstrual cycle on the Abbott ARCHITECT® analyzer. Clin Chem Lab Med (CCLM) 2019;44:631. https://doi.org/10.1515/CCLM.2006.160. n.d. [10] Mainigi MA, Olalere D, Burd I, Sapienza C, Bartolomei M, Coutifaris C. Peri-implantation hormonal milieu: elucidating mechanisms of abnormal placentation and fetal growth1. Biol Reprod 2014;90:1975. https://doi.org/10.1095/biolreprod.113. 110411.
7. Conclusion In conclusion, the elevation of progesterone levels during the late follicular phase in stimulated IVF cycles is a frequent event. The causes of premature progesterone elevation are still unclear, with recent studies pointing towards enhanced FSH-stimulation, as a key factor for premature progesterone rise. “High” progesterone levels seem to mainly affect the endometrium and the window of implantation, probably leading to an asynchrony between the endometrium and the developing embryo. Moreover, new evidence suggests that elevated progesterone 4
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[11] Hild-Petito S, Stouffer RL, Brenner RM. Immunocytochemical localization of estradiol and progesterone receptors in the monkey ovary throughout the menstrual cycle. Endocrinology 1988;123. https://doi.org/10.1210/endo-123-6-2896. 2896–905. [12] Noyes RW, Hertig AT, JRAJOO. Dating the endometrial biopsy. n.d. 1975. https:// doi.org/10.1016/S0002-9378(16)33500-1. [13] Oktem O, Akin N, Bildik G, Yakin K, Alper E, Balaban B, et al. FSH Stimulation promotes progesterone synthesis and output from human granulosa cells without luteinization. Hum Reprod 2017;32. https://doi.org/10.1093/humrep/dex010. 643–52. [14] Kolibianakis EM, Venetis CA, Bosdou JK, Zepiridis L, Chatzimeletiou K, Makedos A, et al. Corifollitropin alfa compared with follitropin beta in poor responders undergoing ICSI: a randomized controlled trial. Hum Reprod 2015;30. https://doi. org/10.1093/humrep/deu301. 432–40. [15] Kyrou D, Popovic-Todorovic B, Fatemi HM, Bourgain C, Haentjens P, et al. Does the estradiol level on the day of human chorionic gonadotrophin administration have an impact on pregnancy rates in patients treated with rec-FSH/GnRH antagonist? Hum Reprod 2009;24. https://doi.org/10.1093/humrep/dep290. 2902–9. [16] Lawrenz B, Beligotti F, Engelmann N, Gates D, Fatemi HM. Impact of gonadotropin type on progesterone elevation during ovarian stimulation in GnRH antagonist cycles. Hum Reprod 2016;31. https://doi.org/10.1093/humrep/dew213. 2554–60. [17] Smitz J, Andersen AN, Devroey P, Arce JC, for the MERIT Group. Endocrine profile in serum and follicular fluid differs after ovarian stimulation with HP-hMG or recombinant FSH in IVF patients. Hum Reprod 2006;22. https://doi.org/10.1093/ humrep/del445. 676–87. [18] Devroey P, Pellicer A, Nyboe Andersen A, Arce J-C. A randomized assessor-blind trial comparing highly purified hMG and recombinant FSH in a GnRH antagonist cycle with compulsory single-blastocyst transfer. Fertil Steril 2012;97. https://doi. org/10.1016/j.fertnstert.2011.12.016. 561–71. [19] Xiong Y, Wang J, Liu L, Chen X, Xu H, Li TC, et al. Effects of high progesterone level on the day of human chorionic gonadotrophin administration in in vitro fertilization cycles on epigenetic modification of endometrium in the peri-implantation period. Fertil Steril 2017;108. https://doi.org/10.1016/j.fertnstert.2017.06.004. 269–276.e1. [20] Martínez F, Coroleu B, Clua E, Reproductive RT. Serum progesterone concentrations on the day of HCG administration cannot predict pregnancy in assisted reproduction cycles. Rbmojournalcom 2004. https://doi.org/10.1016/S1472-6483(10) 60514-7. n.d. [21] Doldi N. Elevated serum progesterone on the day of HCG administration in IVF is associated with a higher pregnancy rate in polycystic ovary syndrome. Hum Reprod 1999;14. https://doi.org/10.1093/humrep/14.3.601. 601–5. [22] Oktem O, Yakin K, Oguz SY, Isiklar A, Balaban B, Urman B. High responders are not exempt from detrimental effects of prematurely rising progesterone levels in fresh embryo transfer cycles. Reprod Biomed Online 2018:1–10. https://doi.org/10. 1016/j.rbmo.2018.11.008. [23] Venetis CA, Kolibianakis EM, Papanikolaou E, Bontis J, Devroey P, Tarlatzis BC. Is progesterone elevation on the day of human chorionic gonadotrophin administration associated with the probability of pregnancy in in vitro fertilization? A systematic review and meta-analysis. Hum Reprod Update 2007;13. https://doi.org/ 10.1093/humupd/dmm007. 343–55. [24] Santos-Ribeiro S, Polyzos NP, Haentjens P, Smitz J, Camus M, Tournaye H, et al. Live birth rates after IVF are reduced by both low and high progesterone levels on the day of human chorionic gonadotrophin administration. Hum Reprod 2014;29. https://doi.org/10.1093/humrep/deu151. 1698–705. [25] Younis JS. Elevated P level on the day of hCG administration is related to FSH dose: is it the whole truth? Hum Reprod 2011;26. https://doi.org/10.1093/humrep/ deq355. 498–9. [26] Papanikolaou EG, Kolibianakis Pozzobon C, Tank P, Tournaye H, Bourgain C, et al. Progesterone rise on the day of human chorionic gonadotropin administration impairs pregnancy outcome in day 3 single-embryo transfer, while has no effect on day 5 single blastocyst transfer. Fertil Steril 2009;91. https://doi.org/10.1016/j. fertnstert.2006.12.064. 949–52. [27] Yang S, Pang T, Li R, Yang R, Zhen X, Chen X, et al. The individualized choice of
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
5
embryo transfer timing for patients with elevated serum progesterone level on the HCG day in IVF/ICSI cycles: a prospective randomized clinical study. Gynecol Endocrinol 2015;31. https://doi.org/10.3109/09513590.2014.995620. 355–8. Corti L, Papaleo E, Pagliardini L, Rabellotti E, Molgora M, et al. Fresh blastocyst transfer as a clinical approach to overcome the detrimental effect of progesterone elevation at hCG triggering: a strategy in the context of the Italian law. Eur J Obstet Gynecol Reprod Biol 2013;171. https://doi.org/10.1016/j.ejogrb.2013.08.017. 73–7. Roque M, Valle M, Guimarães F, Sampaio M, Geber S. Freeze-all policy: fresh vs. frozen-thawed embryo transfer. Fertil Steril 2015;103. https://doi.org/10.1016/j. fertnstert.2015.01.045. 1190–3. Wu Z, Li R, Ma Y, Deng B, Zhang X, Meng Y, et al. Effect of HCG-day serum progesterone and oestradiol concentrations on pregnancy outcomes in GnRH agonist cycles. Reprod Biomed Online 2012;24. https://doi.org/10.1016/j.rbmo.2012.02. 003. 511–20. Lai T-H, Lee F-K, Lin T-K, Horng S-G, Chen S-C, Chen Y-H, et al. An increased serum progesterone-to-estradiol ratio on the day of human chorionic gonadotropin administration does not have a negative impact on clinical pregnancy rate in women with normal ovarian reserve treated with a long gonadotropin releasing hormone agonist protocol. Fertil Steril 2009;92. https://doi.org/10.1016/j.fertnstert.2008. 06.036. 508–14. Martínez F, Rodríguez I, Devesa M, Buxaderas R, Gómez MJ, Coroleu B. Should progesterone on the human chorionic gonadotropin day still be measured? Fertil Steril 2016;105:86–92. https://doi.org/10.1016/j.fertnstert.2015.09.008. Huang CC, Lien YR, Chen HF, Chen MJ, Shieh CJ, Yao YL, et al. The duration of preovulatory serum progesterone elevation before hCG administration affects the outcome of IVF/ICSI cycles. Hum Reprod 2012;27. https://doi.org/10.1093/ humrep/des141. 2036–45. Santos-Ribeiro S, Racca A, Roelens C, De Munck N, Mackens S, et al. Evaluating the benefit of measuring serum progesterone prior to the administration of hCG: effect of the duration of late-follicular elevated progesterone following ovarian stimulation on fresh embryo transfer live birth rates. Reprod Biomed Online 2018:1–8. https://doi.org/10.1016/j.rbmo.2018.11.016. Healy MW, Yamasaki M, Patounakis G, Richter KS, Devine K, DeCherney AH, et al. The slow growing embryo and premature progesterone elevation: compounding factors for embryo-endometrial asynchrony. Hum Reprod 2017;32. https://doi.org/ 10.1093/humrep/dew296. 362–7. Melo MAB, Meseguer M, Garrido N, Bosch E, Pellicer A, Remohi J. The significance of premature luteinization in an oocyte-donation programme. Hum Reprod 2006;21. https://doi.org/10.1093/humrep/dei474. 1503–7. Check JH, Hourani C, Choe JK, Callan C, Adelson HG. Pregnancy rates in donors versus recipients according to the serum progesterone level at the time of human chorionic gonadotropin in a shared oocyte program. Presented at the 1993 Annual Pacific Coast Fertility Society Meeting 1993. https://doi.org/10.1016/S00150282(16)56514-5. Fertility and Sterility 1994;61:262–4. Ubaldi F, Albano C, Peukert M, Riethmuller-Winzen H, Camus M, Smitz J, et al. Endocrinology: subtle progesterone rise after the administration of the gonadotrophin-releasing hormone antagonist Cetrorelix in intracytoplasmic sperm injection cycles. Hum Reprod 1996;11. https://doi.org/10.1093/oxfordjournals. humrep.a019409. 1405–7. Simon C, Moreau J, Gatimel N, Cohade C, Parinaud J, Leandri R. Impact of estradiol and progesterone levels during the late follicular stage on the outcome of GnRH antagonist protocols. Gynecol Endocrinol 2019;16:1–4. https://doi.org/10.1080/ 09513590.2018.1538346. Bu Z, Zhao F, Wang K, Guo Y, Su Y, Zhai J, et al. Serum progesterone elevation adversely affects cumulative live birth rate in different ovarian responders during in vitro fertilization and embryo transfer: a large retrospective study. PLoS One 2014;9:e100011https://doi.org/10.1371/journal.pone.0100011. Baldini D, Savoia MV, Sciancalepore AG, Malvasi A, Vizziello D, Beck R, et al. High progesterone levels on the day of HCG administration do not affect the embryo quality and the reproductive outcomes of frozen embryo transfers. La Clinica Terapeutica 2018;169. e91–5.
Contents lists available at ScienceDirect
Reproductive Biology journal homepage: www.elsevier.com/locate/repbio
Review
The role of progesterone elevation in IVF ⁎
Panagiotis Drakopoulosa,b, , Annalisa Raccaa,c, Joaquín Errázuriza,d, Michel De Vosa,b, Herman Tournayea, Christophe Blockeela,b,e, Nicola Pluchinof, Samuel Santos-Ribeiroa,b,g a
Center for Reproductive Medicine, Universitair Ziekenhuis Brussel, Brussels, Belgium Faculty of Medicine and Pharmacy, Department of Surgical and Clinical Science, Vrije Universiteit Brussel, Belgium University of Genoa, Academic Unit of Obstetrics and Gynecology, IRCCS AOU San Martino, Italy d Centre for Reproductive Medicine, Clínica Alemana, Santiago, Chile e University of Zagreb-School of Medicine, Department of Obstetrics and Gynecology, Zagreb, Croatia f Division of Obstetrics and Gynecology, University Hospital of Geneva, Geneva, Switzerland g IVI-RMA, Lisbon, Portugal b c
A R T I C LE I N FO
A B S T R A C T
Keywords: In vitro fertilization Progesterone elevation Endometrial receptivity Embryo quality Implantation
Elevation of progesterone during the late follicular phase of stimulated in-vitro fertilization cycles is a frequent event, which negatively impacts the outcome. Over the years evidence has demonstrated a direct relationship between late-follicular elevated progesterone and endometrial receptivity. In this regard, elective cryopreservation of all good quality embryos and transfer in a subsequent frozen/thawed cycle is the most common strategy adopted by clinicians in case of elevated progesterone. Nonetheless, recent evidence suggests that elective cryopreservation might not entirely resolve the reduced pregnancy outcomes associated with the elevation of progesterone, considering that the increase may affect not only implantation, but also embryo quality.
1. Introduction Elevation of progesterone during the late follicular phase of stimulated in-vitro fertilization (IVF) cycles is a frequent event, occurring in 12–38% of cycles [1], as it cannot be completely prevented by the administration of gonadotropin-releasing hormone (GnRH) analogues. The incidence of late follicular phase elevated progesterone varies among studies and this marked variation could be explained by the method of progesterone assessment. Several cut-off levels for elevated progesterone in stimulated IVF cycles have been suggested, ranging from 0.8 to 2.0 ng/mL. Nonetheless, 1.50 ng/mL as a cut-off for elevated progesterone is most commonly used [1]. This threshold is supported by the presence of a marked difference in endometrial gene expression profile between infertile patients with a progesterone serum concentration above and below 1.50 ng/mL on the day of human chorionic gonadotrophin (hCG) administration [2]. Over the years, there has been a debate on whether elevated progesterone may hinder pregnancy rates following IVF treatment. The debate came to an end following the results of a recent meta-analysis including more than 60,000 IVF cycles, clearly demonstrating that in case of progesterone elevation, there is a negative impact on reproductive outcomes [3]. Based on these findings, most clinicians choose to freeze all embryos in cycles with elevated progesterone and ⁎
perform the transfer in a subsequent frozen/thawed cycle. However, it should be stated that the cut-off of progesterone elevation should be considered with caution, given the high variability of the assays used for progesterone measurement, making difficult the comparison of the results of different studies and therefore hindering the validity of the afore mentioned meta-analysis [4,5]. The mechanism by which the rise of progesterone during the follicular phase affects pregnancy rate is still not fully understood. Initially, it has been advocated that elevated levels of peripheral progesterone have no effect on oocyte/embryo quality, and that their detrimental effects are mainly mediated through the endometrium, leading to an asynchrony between the endometrium and the developing embryo. Nevertheless, recent evidence suggests that elevated progesterone levels may additionally be associated with a decrease in embryo quality and cumulative live birth rates [6–8]. In this review, evidence on the mechanism of premature progesterone elevation and its impact on reproductive outcomes of IVF treatment are summarized. 2. The effect of supraphysiologic circulating hormone levels on endometrial receptivity Although the duration of the follicular phase of the menstrual cycle
Corresponding author at: Centre for Reproductive Medicine, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Laarbeeklaan 101, 1090 Brussel, Belgium. E-mail address: [email protected] (P. Drakopoulos).
https://doi.org/10.1016/j.repbio.2019.02.003 Received 31 December 2018; Received in revised form 4 February 2019; Accepted 7 February 2019 1642-431X/ © 2019 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Panagiotis Drakopoulos, et al., Reproductive Biology, https://doi.org/10.1016/j.repbio.2019.02.003
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elucidated. A stimulated IVF cycle is not comparable to the natural cycle. In stimulated cycles, multiple follicular growth occurs, resulting in supraphysiological serum E2 concentrations during the follicular phase. Daily injections with recombinant follicle stimulating hormone (FSH) are necessary to prevent serum FSH levels from dropping below the threshold, which is required for follicle development. Consequently, the follicles are under constant FSH stimulus until the final oocyte maturation. Ovarian-stimulation-derived progesterone overproduction has traditionally been considered to be a consequence of CYP17-produced progesterone in the theca cells of the multiple developing follicles. This progesterone produced in the follicular phase is usually metabolized downstream by the LH-activity in the later stages of follicular development, as LH promotes the conversion of progesterone into androgens, which will be then transported and converted into E2 by the granulosa cells. However, recent evidence suggests that progesterone is probably produced directly in a dose-dependent manner by FSH activity in the granulosa cells [13]. Using in-vitro-study on human ovarian cortical samples, it was shown that exogenous FSH seems to stimulate the expression of 3β-hydroxysteriod dehydrogenoase (3β-HSD) and progesterone biosynthesis in granulosa cells in addition to its stimulatory effect on the expression of other steroidogenic enzymes [13]. FSH has a direct stimulatory effect on the activity of 3β-HSD and therefore may increase the conversion of pregnenolone to progesterone in a dose dependent manner. According to the two-cell–two-gonadotropin theory, pregnenolone and progesterone produced by granulosa cells should enter the theca cells in order to be converted into androgens. In case of ovarian stimulation, characterized by constantly elevated FSH-levels, the amount of precursor steroids produced may exceed the capacity of the ovary to efficiently convert them into estrogens. This may delay the conversion of progesterone into androgens in theca cells and lead to their accumulation and leakage into the systemic circulation, which could explain why FSH stimulation promotes progesterone output from granulosa cells. Besides enhanced FSH stimulation during IVF treatment, another factor that is associated with progesterone rise is the prolongation of the follicular phase by delaying hCG administration. Kolibianakis et al. [14] demonstrated that a prolonged exposure of the endometrium to high concentrations of progesterone could hinder pregnancy rates, if the follicular phase is prolonged by 2 days. In the same context, Kyrou et al. [15], showed that a cut-off for estradiol on the day of hCG administration of more than 1790.5 pg/mL, associated with more than 10 follicles of at least 11 mm in diameter, was highly likely to result in a progesterone rise of > 1.50 ng/mL. The association between high E2 and premature progesterone elevation could be explained by an excess of proliferating granulosa cells that may lead to an increased progesterone production. Given that the cause of premature progesterone rise could be the continuous FSH stimulation, a hypothesis that a step-down protocol, implying lower FSH doses at the end of stimulation, could potentially overcome the problem of progesterone rise was raised. The hypothesis was confirmed by a recent post-hoc data analysis, showing that patients who met the criteria for final oocyte maturation after receiving a single injection of corifolitropin alfa (a long acting gonadotropin characterized by a rapid absorption which results in peak concentrations of FSH two days after the injection and a progressive decrease thereafter) had a significantly lower incidence of premature progesterone elevation compared with patients receiving daily recombinant FSH-injections over the same duration of stimulation [16]. Thus, these findings support the idea that enhanced and persistent FSH stimulation towards the end of the follicular phase is the primary cause of premature progesterone rise. Another assumption was that premature progesterone elevation in stimulated cycles may be caused by the lack of LH/hCG activity in IVF protocols and that LH supplementation could potentially decrease the incidence of progesterone rise. Prior studies showed that LH/hCG
may be subject to both inter-cycle and inter-women variability, endogenous sexual hormones remain within relatively low levels prior to ovulation, specifically a median (5th and 95th percentiles) of 182.80 pg/mL (131.30 pg/mL – 388.28 pg/mL) for estradiol (E2) and 0.80 ng/mL (0.39 ng/mL – 1.30 ng/mL) for progesterone [9]. Meanwhile, these same hormones reach substantially higher levels with ovarian stimulation. These supraphysiologic hormones concentrations present during IVF have been strongly associated with a negative effect on endometrial receptivity (ER) [2], with the exceeding majority of biopsies performed on the day of oocyte retrieval demonstrating both histologic and gene expression patterns suggestive of endometrial advancement. Studies in oocyte donors have also shown that ovarian stimulation may accelerate the appearance of endometrial pinopodes and nucleolar channel systems ultrastructural formations which are closely related to ER and the window of implantation (WOI) which promote abnormal intracavity interleukin-1 (IL-1) expression, and cause abnormal placentation and embryonic growth [10]. To this extent, the monitoring of both the endometrial and ovarian responses to ovarian stimulation with transvaginal ultrasound and serum hormonal assessments have become important predictors of assisted reproductive technologies (ART) success. 3. Progesterone in the natural menstrual cycle In a natural cycle, circulating progesterone remains low throughout the follicular phase until ovulation, specifically at a median of 0.30 ng/ mL (0.19 ng/mL – 0.51 ng/mL) [9]. Prior to ovulation, the main source of progesterone shifts from the adrenal cortex to the ovary and, at that same time, progesterone receptor mRNA and proteins become more abundant in the nuclei of the granulosa cells [11]. The luteinising hormone (LH) peak causes a slight but noticeable physiologic late-follicular phase progesterone peak which, apart from contributing for the timing of ovulation, may be essential for follicular development. Animal experiments have shown that blocking mid-cycle progesterone production is detrimental for oocyte maturation, oocyte fertilization competence and granulosa/theca luteinisation [11]. Meanwhile, Following adequate priming by E2, progesterone secreted by a recently luteinized follicle(s) will regulate an intricate sequence of molecular events in preparation for implantation. After ovulation, progesterone limits any further proliferation of the endometrium over the course of the next three days by directly restricting the function of E2 and the expression of E2-binding receptors, while stimulating further the development of both vascular and glandular structures. This selective growth of the vascular and glandular components within a confined structure causes coiling of the spiral vessels and increases glandular tortuosity. At the moment of implantation, the endometrium has become differentiated into two distinct layers: the basal layer (most adjacent to the myometrium, a layer which remains relatively unchanged throughout the menstrual cycle and is critical for endometrial regeneration following the cyclical sloughing events endured during menstruation) and the functional layer (constituted by the stratum spongiosum and an overlaying stratum compactum). These successive morphological changes which progesterone sets into motion follow a predictable day-by-day sequence of development which were the basis of the histological endometrial dating classification system developed by Noyes et al. [12]. This classic endometrial classification system remained, until recently, an unchallenged gold standard for the evaluation of luteal function, where endometrial samples with a developmental stage two-days apart from the expected were considered to be “out of phase”. 4. Mechanism and causes of progesterone elevation during ovarian stimulation Till now, the exact cause of progesterone elevation towards the end of the follicular phase of ovarian stimulation has not been fully 2
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single measurement by using ratios such as progesterone-to-follicle, progesterone-to-oocyte, progesterone-to-estradiol ratios [30,31]. However, such indexes have failed to perform superiorly, thus limiting their routine clinical practice [32]. Huang et al. [33] concluded in their retrospective analysis that the duration of progesterone elevation seemed to play a major role in how circulating progesterone affected pregnancy outcome. This approach was unique in the sense that it was the first to raise the question “how long can the endometrium be exposed to progesterone above a certain universal threshold?”. As progesterone receptors are present in endometrium in all phases of the menstrual cycle, namely during menses and the early follicular phase, it would be reasonable to assume that progesterone exposure might be detrimental even when occurring days before ovulation triggering. Furthermore, this concept of a hindering cumulative progesterone exposure is more in line with the current knowledge of how progesterone regulates endometrial development, as it is generally thought that the ovaries need to increase and maintain the secretion of progesterone above a certain level in order to initiate and sustain the development of a secretory endometrium. However, whether the threshold to trigger these endometrial changes is universal (e.g. 1.50 ng/mL, regardless of the patient’s baseline progesterone exposure) or relative (i.e. a difference between the amount of progesterone produced and the usual baseline circulating progesterone concentration encountered in each patient) is unknown. While being a potentially useful hypothesis, the approach performed by Huang et al. [33] failed to be confirmed in a recent and large retrospective study [34]. One of the reasons for these conflicting results may stem from the fact that the study elected a universal arbitrary cut-off of 1.00 ng/mL, which is frequently found at the end of the follicular phase of unstimulated cycles as well [9]. In the light of a potential detrimental effect of “high” progesterone on reproductive outcomes, “freeze only” seems to be the most common strategy by clinicians, as already mentioned. In a recent cost-effective study performed by Healy et al. [35] more than 7000 ART transfer cycles were evaluated. The main objective of the study was to define a threshold of progesterone on which the “freeze only” approach is costeffective. Based on these findings, “high” progesterone (from 1.50 ng/ mL to 2.0 ng/mL) seems to have a clinically significant negative effect and captures a good percentage of patients at higher risk for fresh transfer failure, thus making the "freeze only" strategy a cost-effective option. Nonetheless, elective cryopreservation might not be enough to solve the reduced pregnancy outcomes, considering that elevated progesterone may affect not only implantation, but also embryo quality, as discussed in the next section.
activity may indeed have a protective effect, preventing a possible premature progesterone rise, when comparing patients stimulated with recombinant FSH (rFSH) versus human menopausal gonadotrophin (hMG) [17]. Nevertheless, in a large prospective randomized control trial (RCT) including 749 patients treated with either rFSH or highpurified (HP) hMG, serum progesterone levels at the end of stimulation were similar [18]. 5. The effect of progesterone elevation on endometrial receptivity in ovarian stimulation cycles Fundamental evidence has accumulated over the years demonstrating a direct relationship between late-follicular elevated progesterone and ER. Specifically values of progesterone above 1.50 ng/mL have been associated with: a) a histological advancement which may extent beyond 3 days, b) an abnormal expression of the implantation regulating proteins vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), c) abnormalities in the gene expression which could be related to natural killer (NK) cell activity and, more recently, d) differences in epigenetic profiles [19]. Many research groups have attempted to assess whether these findings are translatable into hindered pregnancy outcomes in IVF. The majority of available studies have demonstrated that “high” progesterone levels may hinder IVF success [1,3]. However, these results were initially disputed by other researchers who found late-follicular progesterone elevation to be either irrelevant [20] or even beneficial in selected infertile populations [21]. In particular, it seems that high responders are not exempt from the detrimental effects of prematurely rising serum progesterone levels but the threshold interval where the detrimental effect begins is higher in the high responders compared with the low and normal responders [22]. Furthermore, a 2007 meta-analysis showed that “high” serum progesterone levels on the day of hCG administration did not significantly affect pregnancy rates [23]. However, the heterogeneity of the studies included in this meta-analysis may have biased the conclusions and updated meta-analyses by the same group eventually provided contradictory results [3]. This may due to the limited reproducibility when different progesterone assays are used, making the interpretation of the several thresholds critical [4]. Although the matter whether progesterone rise affects oocyte/embryo competence remains contentious as discussed later, some investigators consider that the issue of progesterone elevation may be, at least in part, resolved by elective embryo cryopreservation, given the previously-mentioned translational studies associating “high” progesterone levels to an abnormal endometrial genomic/epigenetic expression in the luteal phase. This has led many centres to change their clinical practice and to measure serum progesterone levels on the day of ovulation triggering and to adopt a “freeze-only” strategy when the threshold of 1.50 ng/mL is exceeded. Nonetheless, the everyday use of late-follicular elevated progesterone as an ART outcome predictor in current clinical practice has proven to be harder than originally expected, owing mostly to the fact that a) progesterone production may not affect pregnancy outcomes linearly [1,24] and b) it is frequently encountered in good-prognosis women with an otherwise healthy multi-follicular response [25]. Furthermore, previous studies have recommended caution in the use of progesterone elevation as an indication for all-embryo cryopreservation in high-responder women or the adaptation of the cut-off of “high” progesterone according to ovarian response [3]. Meanwhile, others have postulated that elevated progesterone may only effect cleavage stage embryo transfers [26], a hypothesis that has been refuted more recently by others [1,27,28]. Finally, the detrimental effect of circulating progesterone may be set into motion even when progesterone is below 1.50 ng/mL [29]. Altogether, these conflicting studies are a testament of the limitations of using a single progesterone assessment during IVF to decide whether to defer embryo transfer or not. Many investigators have attempted to enhance the predictive capacity of this
6. Progesterone and embryo quality Despite that the effects of late follicular progesterone rise on endometrial receptivity have been confirmed, evidence on whether increased progesterone may affect embryo quality is still limited. While some previous studies suggested that embryo quality is not affected by elevated progesterone [36], others postulated the opposite, namely that elevated serum progesterone on the day of hCG trigger may negatively influence the top embryo quality rate (Table 1). Specifically, Check et al. [37] and Melo et al. [36] evaluated pregnancy rates in oocyte donation cycles, as an indirect measure of the embryo quality and the investigators found no difference in the two study groups, using a cut-off of progesterone of 1.0 ng/mL and 1.2 ng/mL, respectively [36,37]. Similarly, Ubaldi et al. [38] compared 53 cases of elevated progesterone (more than 1.0 ng/mL) versus 1275 cases with lower progesterone values, in heterologous-ICSI cycle suppressed with GnRH agonist; no differences in embryo quality as well as in pregnancy outcomes were detected [38]. On the other hand, recent studies focused exclusively on embryo quality of infertile women: Huang et al. [6], evaluated cleavage stage embryos and Vanni et al. [7] blastocyst stage embryos; both studies showed a decrease in top embryo quality 3
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Table 1 Negative effect of progesterone elevation on embryo quality. Yes
Number of embryos/cycles included, day of transfer
Progesterone cut-off levels
No
Number of embryos/cycles included, day of transfer
Progesterone cut-off levels
Huang et al. [6] Vanni et al. [7] Racca et al. [8] Healy et al. [35]
2630 embryos, Day 3 986 cycles, Day 5 3400 cycles, Day 3 and 5 4350 embryos, Day 5 and 6
> 2.0 ng/ml > 1.49 ng/ml ≥1.5 ng/ml > 1.5 ng/ml
Melo et al. [36] Check et al. [37] Ubaldi et al. [38] Baldini et al. [41]
240 cycles, Day 5 270 cycles, Day unknown 24 cycles, Day 3 131 cycles, Day 3
≥1.2 ng/ml > 1 ng/mL ≥1.1 ng/ml ≥1.2 ng/ml
may even be associated with a decrease in embryo quality and cumulative live birth rates. In this regard, reduction of FSH-stimulation intensity towards the end of stimulation could result in a lower incidence of P elevation and therefore may be a feasible rescue strategy. Finally, studies comparing cumulative live birth rates in case of progesterone elevation or not in patients with a “freeze all strategy” and evidence derived from PGT-A cycles complicated by late follicular progesterone rise could shed more light on the role of progesterone in IVF.
formation rate as progesterone increased [6,7]. Therefore, the advantages of prolonging ovarian stimulation to maximize the number of collected oocytes might eventually be hindered by a decrease in top quality embryos for transfer, if increasing progesterone levels are detected. In the same context, Healy et al. demonstrated a negative effect of elevated serum progesterone on slow growing blastocysts (day 6) compared to day 5 blastocysts, in terms of live birth rate, while Simon et al. recently showed that progesterone concentrations above 1 ng// mL were significantly negatively correlated to the percentage of top quality embryos and to the implantation rates [39]. Given the paucity of evidence and the fact that all published studies were mainly focused on pregnancy rates in fresh or in the first frozen-thawed embryo transfer rather than on cumulative pregnancy rate, Racca et al. [8,40] was the first to evaluate at the same time progesterone levels, embryo quality and live birth rates. In that study, the main outcomes were cumulative live birth rates and embryo utilization rates, calculated as the total number of embryos transferred and cryopreserved divided by the total number of fertilized oocytes. The results showed that for both cleavage and blastocyst stage embryos, the utilization rates were lower for the elevated progesterone group (progesterone > 1.50 ng/mL). Moreover, cumulative live birth rates were not significantly higher in the elevated progesterone group, as it may have been expected, due to the higher number of number of oocytes retrieved; a potential explanation could be that patients in the “high” progesterone group may have had a higher consumption of embryos necessary to achieve a live birth. On the other hand, given that endometrial receptivity in the presence of “high” progesterone is known to be impaired, fresh transfer of the top quality embryos/blastocysts in patients with elevated progesterone might have been the main cause of the observed decreased cumulative outcomes in the study by Racca et al. [8] and the fact that frozen cycles may provide higher pregnancy rates in case of progesterone rise the day of hCG administration [41]. In this regard, relevant clinical insight on the effect of progesterone on embryo quality in terms of cumulative live birth rates should be derived from “freeze only” cycles—where no fresh embryo transfer is performed in the presence of progesterone elevation and the whole cohort of embryos is cryopreserved. Such a study comparing the effectiveness of a "freeze only" strategy in the presence of elevated progesterone to other “freeze only” cycles to eliminate the bias in endometrial receptivity- could provide robust evidence regarding the role of progesterone in embryo quality. Furthermore, a retrospective or prospective study in Preimplantation Genetic Testing for Aneuploidy (PGT-A) patients could further elucidate whether elevated progesterone may affect aneuploidy rates. To the best of our knowledge, such a study has not been conducted yet.
Conflict of interest None. Authors’ contributions P.D, A.R, J.E and S.S-R are responsible for the concept design and wrote the manuscript, P.D, M.D-V, H.T, C.B, N.P and S.S-R contributed to the editing of the manuscript. Acknowledgement No Acknowledgement. References [1] Bosch E, Labarta E, Crespo J, Simon C, Remohi J, Jenkins J, et al. Circulating progesterone levels and ongoing pregnancy rates in controlled ovarian stimulation cycles for in vitro fertilization: analysis of over 4000 cycles. Hum Reprod 2010;25. https://doi.org/10.1093/humrep/deq125. 2092–100. [2] Labarta E, Martinez-Conejero JA, Alama P, Horcajadas JA, Pellicer A, et al. Endometrial receptivity is affected in women with high circulating progesterone levels at the end of the follicular phase: a functional genomics analysis. Hum Reprod 2011;26. https://doi.org/10.1093/humrep/der126. 1813–25. [3] Venetis CA, Kolibianakis EM, Bosdou JK, Tarlatzis BC. Progesterone elevation and probability of pregnancy after IVF: a systematic review and meta-analysis of over 60 000 cycles. Hum Reprod Update 2013;19. https://doi.org/10.1093/humupd/ dmt014. 433–57. [4] Lawrenz B, Sibal J, Garrido N, Abu E, Jean A, Melado L, et al. Inter-assay variation and reproducibility of progesterone measurements during ovarian stimulation for IVF. PLoS One 2018;13:e0206098https://doi.org/10.1371/journal.pone.0206098. [5] Patton PE, Lim JY, Hickok LR, Kettel LM, Larson JM, Pau KYF. Precision of progesterone measurements with the use of automated immunoassay analyzers and the impact on clinical decisions for in vitro fertilization. Fertil Steril 2014;101. https:// doi.org/10.1016/j.fertnstert.2014.02.037. 1629–36. [6] Huang B, Ren X, Wu L, Zhu L, Xu B, Li Y, et al. Elevated progesterone levels on the day of oocyte maturation may affect top quality embryo IVF cycles. PLoS One 2016;11. https://doi.org/10.1371/journal.pone.0145895. e0145895–8. [7] Vanni VS, Somigliana E, Reschini M, Pagliardini L, Marotta E, Faulisi S, et al. Top quality blastocyst formation rates in relation to progesterone levels on the day of oocyte maturation in GnRH antagonist IVF/ICSI cycles. PLoS One 2017;12:e0176482https://doi.org/10.1371/journal.pone.0176482. [8] Racca A, Santos-Ribeiro S, De Munck N, Mackens S, Drakopoulos P, et al. Impact of late-follicular phase elevated serum progesterone on cumulative live birth rates: is there a deleterious effect on embryo quality? Hum Reprod 2018;33. https://doi. org/10.1093/humrep/dey031. 860–8. [9] Stricker R, Eberhart R, Chevailler M-C, Quinn FA, Bischof P, Stricker R. Establishment of detailed reference values for luteinizing hormone, follicle stimulating hormone, estradiol, and progesterone during different phases of the menstrual cycle on the Abbott ARCHITECT® analyzer. Clin Chem Lab Med (CCLM) 2019;44:631. https://doi.org/10.1515/CCLM.2006.160. n.d. [10] Mainigi MA, Olalere D, Burd I, Sapienza C, Bartolomei M, Coutifaris C. Peri-implantation hormonal milieu: elucidating mechanisms of abnormal placentation and fetal growth1. Biol Reprod 2014;90:1975. https://doi.org/10.1095/biolreprod.113. 110411.
7. Conclusion In conclusion, the elevation of progesterone levels during the late follicular phase in stimulated IVF cycles is a frequent event. The causes of premature progesterone elevation are still unclear, with recent studies pointing towards enhanced FSH-stimulation, as a key factor for premature progesterone rise. “High” progesterone levels seem to mainly affect the endometrium and the window of implantation, probably leading to an asynchrony between the endometrium and the developing embryo. Moreover, new evidence suggests that elevated progesterone 4
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[11] Hild-Petito S, Stouffer RL, Brenner RM. Immunocytochemical localization of estradiol and progesterone receptors in the monkey ovary throughout the menstrual cycle. Endocrinology 1988;123. https://doi.org/10.1210/endo-123-6-2896. 2896–905. [12] Noyes RW, Hertig AT, JRAJOO. Dating the endometrial biopsy. n.d. 1975. https:// doi.org/10.1016/S0002-9378(16)33500-1. [13] Oktem O, Akin N, Bildik G, Yakin K, Alper E, Balaban B, et al. FSH Stimulation promotes progesterone synthesis and output from human granulosa cells without luteinization. Hum Reprod 2017;32. https://doi.org/10.1093/humrep/dex010. 643–52. [14] Kolibianakis EM, Venetis CA, Bosdou JK, Zepiridis L, Chatzimeletiou K, Makedos A, et al. Corifollitropin alfa compared with follitropin beta in poor responders undergoing ICSI: a randomized controlled trial. Hum Reprod 2015;30. https://doi. org/10.1093/humrep/deu301. 432–40. [15] Kyrou D, Popovic-Todorovic B, Fatemi HM, Bourgain C, Haentjens P, et al. Does the estradiol level on the day of human chorionic gonadotrophin administration have an impact on pregnancy rates in patients treated with rec-FSH/GnRH antagonist? Hum Reprod 2009;24. https://doi.org/10.1093/humrep/dep290. 2902–9. [16] Lawrenz B, Beligotti F, Engelmann N, Gates D, Fatemi HM. Impact of gonadotropin type on progesterone elevation during ovarian stimulation in GnRH antagonist cycles. Hum Reprod 2016;31. https://doi.org/10.1093/humrep/dew213. 2554–60. [17] Smitz J, Andersen AN, Devroey P, Arce JC, for the MERIT Group. Endocrine profile in serum and follicular fluid differs after ovarian stimulation with HP-hMG or recombinant FSH in IVF patients. Hum Reprod 2006;22. https://doi.org/10.1093/ humrep/del445. 676–87. [18] Devroey P, Pellicer A, Nyboe Andersen A, Arce J-C. A randomized assessor-blind trial comparing highly purified hMG and recombinant FSH in a GnRH antagonist cycle with compulsory single-blastocyst transfer. Fertil Steril 2012;97. https://doi. org/10.1016/j.fertnstert.2011.12.016. 561–71. [19] Xiong Y, Wang J, Liu L, Chen X, Xu H, Li TC, et al. Effects of high progesterone level on the day of human chorionic gonadotrophin administration in in vitro fertilization cycles on epigenetic modification of endometrium in the peri-implantation period. Fertil Steril 2017;108. https://doi.org/10.1016/j.fertnstert.2017.06.004. 269–276.e1. [20] Martínez F, Coroleu B, Clua E, Reproductive RT. Serum progesterone concentrations on the day of HCG administration cannot predict pregnancy in assisted reproduction cycles. Rbmojournalcom 2004. https://doi.org/10.1016/S1472-6483(10) 60514-7. n.d. [21] Doldi N. Elevated serum progesterone on the day of HCG administration in IVF is associated with a higher pregnancy rate in polycystic ovary syndrome. Hum Reprod 1999;14. https://doi.org/10.1093/humrep/14.3.601. 601–5. [22] Oktem O, Yakin K, Oguz SY, Isiklar A, Balaban B, Urman B. High responders are not exempt from detrimental effects of prematurely rising progesterone levels in fresh embryo transfer cycles. Reprod Biomed Online 2018:1–10. https://doi.org/10. 1016/j.rbmo.2018.11.008. [23] Venetis CA, Kolibianakis EM, Papanikolaou E, Bontis J, Devroey P, Tarlatzis BC. Is progesterone elevation on the day of human chorionic gonadotrophin administration associated with the probability of pregnancy in in vitro fertilization? A systematic review and meta-analysis. Hum Reprod Update 2007;13. https://doi.org/ 10.1093/humupd/dmm007. 343–55. [24] Santos-Ribeiro S, Polyzos NP, Haentjens P, Smitz J, Camus M, Tournaye H, et al. Live birth rates after IVF are reduced by both low and high progesterone levels on the day of human chorionic gonadotrophin administration. Hum Reprod 2014;29. https://doi.org/10.1093/humrep/deu151. 1698–705. [25] Younis JS. Elevated P level on the day of hCG administration is related to FSH dose: is it the whole truth? Hum Reprod 2011;26. https://doi.org/10.1093/humrep/ deq355. 498–9. [26] Papanikolaou EG, Kolibianakis Pozzobon C, Tank P, Tournaye H, Bourgain C, et al. Progesterone rise on the day of human chorionic gonadotropin administration impairs pregnancy outcome in day 3 single-embryo transfer, while has no effect on day 5 single blastocyst transfer. Fertil Steril 2009;91. https://doi.org/10.1016/j. fertnstert.2006.12.064. 949–52. [27] Yang S, Pang T, Li R, Yang R, Zhen X, Chen X, et al. The individualized choice of
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
5
embryo transfer timing for patients with elevated serum progesterone level on the HCG day in IVF/ICSI cycles: a prospective randomized clinical study. Gynecol Endocrinol 2015;31. https://doi.org/10.3109/09513590.2014.995620. 355–8. Corti L, Papaleo E, Pagliardini L, Rabellotti E, Molgora M, et al. Fresh blastocyst transfer as a clinical approach to overcome the detrimental effect of progesterone elevation at hCG triggering: a strategy in the context of the Italian law. Eur J Obstet Gynecol Reprod Biol 2013;171. https://doi.org/10.1016/j.ejogrb.2013.08.017. 73–7. Roque M, Valle M, Guimarães F, Sampaio M, Geber S. Freeze-all policy: fresh vs. frozen-thawed embryo transfer. Fertil Steril 2015;103. https://doi.org/10.1016/j. fertnstert.2015.01.045. 1190–3. Wu Z, Li R, Ma Y, Deng B, Zhang X, Meng Y, et al. Effect of HCG-day serum progesterone and oestradiol concentrations on pregnancy outcomes in GnRH agonist cycles. Reprod Biomed Online 2012;24. https://doi.org/10.1016/j.rbmo.2012.02. 003. 511–20. Lai T-H, Lee F-K, Lin T-K, Horng S-G, Chen S-C, Chen Y-H, et al. An increased serum progesterone-to-estradiol ratio on the day of human chorionic gonadotropin administration does not have a negative impact on clinical pregnancy rate in women with normal ovarian reserve treated with a long gonadotropin releasing hormone agonist protocol. Fertil Steril 2009;92. https://doi.org/10.1016/j.fertnstert.2008. 06.036. 508–14. Martínez F, Rodríguez I, Devesa M, Buxaderas R, Gómez MJ, Coroleu B. Should progesterone on the human chorionic gonadotropin day still be measured? Fertil Steril 2016;105:86–92. https://doi.org/10.1016/j.fertnstert.2015.09.008. Huang CC, Lien YR, Chen HF, Chen MJ, Shieh CJ, Yao YL, et al. The duration of preovulatory serum progesterone elevation before hCG administration affects the outcome of IVF/ICSI cycles. Hum Reprod 2012;27. https://doi.org/10.1093/ humrep/des141. 2036–45. Santos-Ribeiro S, Racca A, Roelens C, De Munck N, Mackens S, et al. Evaluating the benefit of measuring serum progesterone prior to the administration of hCG: effect of the duration of late-follicular elevated progesterone following ovarian stimulation on fresh embryo transfer live birth rates. Reprod Biomed Online 2018:1–8. https://doi.org/10.1016/j.rbmo.2018.11.016. Healy MW, Yamasaki M, Patounakis G, Richter KS, Devine K, DeCherney AH, et al. The slow growing embryo and premature progesterone elevation: compounding factors for embryo-endometrial asynchrony. Hum Reprod 2017;32. https://doi.org/ 10.1093/humrep/dew296. 362–7. Melo MAB, Meseguer M, Garrido N, Bosch E, Pellicer A, Remohi J. The significance of premature luteinization in an oocyte-donation programme. Hum Reprod 2006;21. https://doi.org/10.1093/humrep/dei474. 1503–7. Check JH, Hourani C, Choe JK, Callan C, Adelson HG. Pregnancy rates in donors versus recipients according to the serum progesterone level at the time of human chorionic gonadotropin in a shared oocyte program. Presented at the 1993 Annual Pacific Coast Fertility Society Meeting 1993. https://doi.org/10.1016/S00150282(16)56514-5. Fertility and Sterility 1994;61:262–4. Ubaldi F, Albano C, Peukert M, Riethmuller-Winzen H, Camus M, Smitz J, et al. Endocrinology: subtle progesterone rise after the administration of the gonadotrophin-releasing hormone antagonist Cetrorelix in intracytoplasmic sperm injection cycles. Hum Reprod 1996;11. https://doi.org/10.1093/oxfordjournals. humrep.a019409. 1405–7. Simon C, Moreau J, Gatimel N, Cohade C, Parinaud J, Leandri R. Impact of estradiol and progesterone levels during the late follicular stage on the outcome of GnRH antagonist protocols. Gynecol Endocrinol 2019;16:1–4. https://doi.org/10.1080/ 09513590.2018.1538346. Bu Z, Zhao F, Wang K, Guo Y, Su Y, Zhai J, et al. Serum progesterone elevation adversely affects cumulative live birth rate in different ovarian responders during in vitro fertilization and embryo transfer: a large retrospective study. PLoS One 2014;9:e100011https://doi.org/10.1371/journal.pone.0100011. Baldini D, Savoia MV, Sciancalepore AG, Malvasi A, Vizziello D, Beck R, et al. High progesterone levels on the day of HCG administration do not affect the embryo quality and the reproductive outcomes of frozen embryo transfers. La Clinica Terapeutica 2018;169. e91–5.