Assessment of the luteal phase in stimulated and substituted cycles.

Csapo et al. (1972 and 1973) demonstrated the importance of progesterone during the first weeks of a pregnancy. In their initial study, the removal of the corpus luteum prior to seven weeks of gestation led to pregnancy loss (Csapo et al., 1972). However, the authors found that pregnancy could be maintained even after removal of the corpus luteum by external administration of progesterone (Csapo et al., 1973). Progesterone induces a secretory transformation of the endometrium in the luteal phase (Bourgain et al.,1990). By inducing this change after adequate estrogen priming, progesterone improves endometrial receptivity (Kolibianakis and Devroey, 2002). Endometrial receptivity is a self-limited period in which the endometrial epithelium acquires a functional and transient ovarian steroid-dependent status that allows blastocyst adhesion (Martin et al., 2002). Decreased endometrial receptivity is considered largely responsible for the low implantation rates in IVF (Paulson et al., 1990). Progesterone also promotes local vasodilatation and uterine musculature quiescence by inducing nitric oxide synthesis in the decidua (Bulletti and de Ziegler, 2005). Inadequate uterine contractility may lead to ectopic pregnancies, miscarriages, retrograde bleeding with dysmenorrhea and endometriosis (Bulletti and de Ziegler, 2005). The uterine-relaxing properties of progesterone were supported by a study of IVF embryo transfer outcomes by Fanchin et al. (2001). This study investigated the consequences of uterine contractions (UC) as visualized by ultrasound (US) during embryo transfer. Results indicated that a high frequency of uterine contractions on the day of embryo transfer hindered transfer outcome, possibly by expelling embryos out of the uterine cavity. A negative correlation between UC frequency and progesterone concentrations was detected underlining the benefits of progesterone in IVF (Fanchin et al., 2001). Currently available formulations of progesterone include oral, vaginal, rectal and intramuscular (i.m.) (Penzias, 2002; Chakmakjian, 1987). Progesterone administered orally is subjected to first-pass prehepatic and hepatic metabolism. This metabolic activity results in progesterone degradation to its 5α- and 5β- reduced metabolites (Penzias, 2002). Parenteral administration (vaginal, rectal and i.m.) of progesterone surpasses the metabolic consequences of orally administered progesterone (De Ziegler et al., 1995). Oral micronized progesterone was used for luteal support in IVF with poor results until the end of 1980s (Buvat J, et al., 1990). Devroey et al. (1989) and Bourgain et al. (1990) reported an absence of the secretory transformation of the endometrium in patients with premature ovarian failure who had been treated with oral micronised progesterone when compared to patients treated with intramuscular injections or vaginal micronised progesterone. This finding suggested that oral administration reduced the hormone’s bioavailibility. To overcome this problem, dydrogesterone (DG) was introduced to support the luteal phase of stimulated IVF cycles (Belaisch-Allart et al., 1987). DG, a retroprogesterone with good oral bioavailability, is a biologically active metabolite of progesterone and has an anti-estrogenic effect on the endometrium, achieving the desired secretory transformation (Whitehead, 1980; Chakravarty et al., 2005). Recently, Chakravarty et al. (2005) undertook a prospective, randomized study (n = 430) that compared the efficacy, safety and tolerability of oral DG with vaginal micronised progesterone as luteal phase support after in-vitro fertilization (IVF). Both DG and P were associated with similar rates of successful pregnancies (24.1% vs. 22.8%, respectively; P = NS). However, it has been demonstrated clearly that after sufficient estrogen endometrial priming, exogenous administered vaginal micronised progesterone is significantly more effective than oral dydrogesterone in creating an ‘in phase’ secretory endometrium. ( Fig. 1 , Fig. 2 , Fatemi et al., 2007). The oral DG might be sufficient for luteal supplementation in IVF cycles; however more large randomized controlled trails are needed, before a conclusion can be made. A number of publications have evaluated the rectal use of natural progesterone in women undergoing IVF/ICSI (Chakmakijan et al., 1987; Ioannidis et al., 2005). Chakmakijan et al. (1987) studied the bioavailability of micronized progesterone (P) by measuring sequential serum P concentrations after a single bolus of 50-200 mg P given sublingually, orally (capsule and tablet), vaginally and rectally (suppositories) during the follicular phase of a group of normally menstruating women. When compared to other routes of P administration, rectal application resulted in serum concentrations during the first eight hours twice as high as other forms. However, to the best of our knowledge, there are no prospective randomized trials to compare the rectal administration of progesterone with other administration routes for IVF. The intravaginal route of progesterone supplementation in IVF has gained wide application as a first choice luteal support regimen, mainly due to patient comfort and effectiveness (Levine et al., 2000). Following intravaginal administration of progesterone, high uterine progesterone concentrations with low peripheral serum values are observed, due to counter-current exchange in progesterone transport between anatomically close blood vessels (Cicinelli et al., 2000) and due to the uterine first pass effect, where liver metabolisation is absent (De Ziegler et al., 1995). There is increasing evidence in the literature that vaginal P is at least as effective as i.m. P at providing luteal support in induced cycles (Simunic et al., 2007). In the latest meta-analysis by Nosarka et al. (2005), vaginal and intramuscular progesterone had comparable implantation and clinical pregnancy rates. In Europe, there are two different forms of intravaginal progesterone on the market, natural micronised progesterone (Utrogestan® Laboratories Besins International, Paris, France) and Crinone ®8% (Fleet Laboratories Ltd., Watford, United Kingdom), a controlled and sustained-release vaginal gel. Utrogestan ® 100 mg capsules are administered vaginally three times two capsules daily (600 mg/d) whereas Crinone 8% is administered vaginally once a day, i.e. 90 mg, (Simunic et al., 2007; Ludwig et al., 2002). To establish the minimal effective dose of vaginal micronized progesterone, Chanson et al. (1996) conducted a small (n = 40) prospective randomized study comparing two different dose regimens (400 mg versus 600 mg each day). No differences in clinical pregnancy rates were noted. However, further prospective randomized trials are essential to define the necessary dose of vaginal micronized progesterone for luteal phase support in IVF. In a prospective, randomized study Ludwig et al. (2002) compared vaginal Crinone 8% with vaginal Utrogestan® for luteal phase support. Clinical pregnancy rates, clinical abortion rates until 12 weeks of gestation and ongoing pregnancy rates were comparable between the two groups (Ludwig et al., 2002). Simunic (2007) and Ludwig (2002) evaluated the tolerability and acceptability of both preparations from patients’ point of view. Crinone® 8% gel proved more tolerable than Utrogestan® vaginal capsules because of a lower number of side effects (Simunic et al., 2007; Ludwig et al., 2002). I.M.. progesterone supplementation is given as an injection of natural progesterone-in-oil (Costabile et al., 2001). In 1985, Leeton et al. first demonstrated the extension of the luteal phase of stimulated IVF cycles treated with 50 mg i.m. progesterone. The doses of i.m. progesterone used for luteal phase support vary between 25 and 100mg per day without any significant difference concerning the outcome (Pritts and Atwood, 2002). This route of administration is often associated with a number of side effects, including painful injections and a rash (Lightman et al., 1999), causing a lack of enthusiasm for this treatment modality (Costabile et al., 2001). Injections of Progesterone in oil can also lead to inflammatory reactions and abscess formation (Propst et al., 2001). In addition, several case reports have been published in which patients receiving i.m. progesterone for luteal supplementation have developed acute eosinophilic pneumonia (Bouckaert et al., 2004; Veysman et al., 2006). This drug-induced disease shows that the use of i.m. progesterone can also be associated with a severe morbidity in otherwise healthy young patients (Bouckaert et al., 2004). In an open-label trial in 1184 women from 16 U.S. American centers Levine evaluated the clinical and ongoing pregnancy rates in IVF cycles involving vaginal and i.m. progesterone. Vaginal and i.m. progesterone were found to have comparable clinical (35.05% V.S. 35.2%, respectively) and ongoing pregnancy rates (30.2% and 33.64%, respectively) (Levine, 2000). A meta-analysis published in 2002 by Pritts and Atwood included five prospective randomized trails comparing i.m. administration of progesterone with vaginal. A total of 891 cycles were evaluated in those studies. Clinical pregnancy rate and delivery rate were significantly higher when i.m. progesterone was used (RR clinical pregnancy rate/ET 1.33 (95% CI:1.02-1.75, Delivery rate 2.06 (95% CI:1.48-2.88)). The two most important hormones produced by the corpus luteum are progesterone and estradiol (Fatemi et al., 2007). The role of progesterone for luteal support in stimulated cycles is well established (Fatemi et al., 2007). However, it has not yet been clearly demonstrated whether additional supplementation of E2 in stimulated IVF cycles may be beneficial (Fatemi et al., 2007). In a prospective randomized study, Smitz et al. evaluated the possible benefit of adding estradiol valerate 6 mg per os daily to the vaginal micronised progesterone (600 mg daily) given as luteal supplementation in 378 women treated with a gonadotrophin releasing-hormone agonist and human menopausal gonadotrophins for in IVF (Smitz et al., 1993). The clinical pregnancy rate was similar between the two groups (29.2% with the estradiol co-treatment and 29.5% with progesterone only treatment). Also Lewin et al., (1994) in a prospectively randomized study, could not find any advantage in the addition of 2 mg estradiol valerate to Progesterone as luteal phase support of long GnRH agonist and hMG-induced IVF-ET cycles in one hundred patients (clinical pregnancy rate 26.5% versus 28% with and without estradiol co-treatment, respectively). A meta-analysis by Pritts and Atwood (2002) suggested that addition of estrogen to progesterone might improve the implantation rates. However, the authors referred to only one study confirming the beneficial effect of estradiol in the luteal phase (Farhi et al., 2000). Any beneficial effect of adding E2 to progesterone might depend upon its dosage. Lukaszuk et al (2005), in a prospective, randomized study, recently evaluated the effect of different E2 supplementation doses (0, 2, or 6 mg) during the luteal phase on implantation and pregnancy rates in women undergoing intracytoplasmic sperm injection (ICSI) in agonist cycles (n = 231). Significantly higher pregnancy rates (PR) were recorded in those who received low dose E2 supplementation compared with no estradiol substitution (PR 23.1% vs. 32.8%). The best pregnancy results were found in the group with high dose E2 supplementation (PR 51.3%). It was shown that the addition of a high dose of E2 to daily progesterone supplementation significantly improved the probability of pregnancy in women treated with a long GnRH analogue protocol for COH. Farhi et al. (2000), in a prospective, randomized study, evaluated the effect of adding E2 to progestin supplementation during the luteal phase in 271 patients undergoing IVF who had E2 levels of higher than 2500 pg/dL at the day of hCG administration. All patients received progesterone supplementation at a dosage of 150 mg/d starting on the day after the oocyte retrieval (OR). Patients were randomized into two groups: those receiving 2 mg of E2 (Estrophem; Novo Nordisk, Bagsvaerd, Denmark), given orally, starting on day 7 after ET; and those receiving no exogenous E2 supplementation during the luteal phase. It was shown that for those patients who had been treated with the long GnRH agonist protocol for COH, the addition of E2 to the progestin support regimen had a beneficial effect on pregnancy and implantation rates (39.6%, and 25.6% with and without estradiol co-treatment respectively; P < .0.05). However, such an effect could not be shown for patients with a short, GnRH agonist protocol. Different studies were conducted to examine whether the probability of pregnancy is increased by adding estrogen to progesterone for luteal phase support in patients treated by IVF. However, the currently available evidence as published in meta-analysis by Kolibianakis et al., 2008 suggests that the addition of estrogen to progesterone for luteal phase support does not increase the probability of pregnancy in IVF in both GnRH agonist and antagonist cycles. Since it was found that the corpus luteum can be rescued by the administration of hCG, this treatment has become the standard care for luteal support since the late 1980s (52). By stimulating the corpora lutea, hCG is an indirect form of luteal support. It is known to generate an increase in estradiol and progesterone concentrations thus rescuing the failing corpora lutea in stimulated IVF cycles (Fatemi et al., 2007). Administration of hCG has also been shown to increase the concentrations of placental protein 14, integrin and relaxin (luteal peptide hormone) which has been shown to increase at the time of implantation (Fatemi et al., 2007). In the meta-analysis published by Pritts and Atwood in 2002, hCG was shown to be equally effective as progesterone for luteal phase support with respect to pregnancy rates. The disadvantage of using hCG for luteal support stems from its potential for increasing hyperstimulation rates when compared with other treatments or no treatment at all. Significant increases in hyperstimulation rates have been confirmed in several studies (Fatemi et al., 2007). With regard to ovarian hyperstimulation syndrome (OHSS), one should therefore be cautious with the administration of hCG for luteal supplementation in stimulated IVF cycles (Fatemi et al., 2007). Luteal support with hCG should be avoided if estradiol levels are above 2500-2700 pg/ml on the day of hCG administration (Fatemi et al., 2007) and if the number of follicles is above 10 (Fatemi et al., 2007). GnRH agonist was recently suggested as a novel luteal-phase support that may act upon pituitary gonadotrophs, the endometrium and the embryo itself (Tesarik, 2006). It has been hypothesized that GnRH agonist may support the corpus luteum by stimulating the secretion of LH by pituitary gonadotroph cells or by acting directly on the endometrium through the locally expressed GnRH receptors (Pirard et al., 2005). In a prospective randomized study, Tesarik et al. (2006) evaluated the effect of GnRH agonist (0.1 mg triptorelin) administration in the luteal phase on outcomes in both GnRH agonist (n = 300) and GnRH antagonist (n = 300) ovarian stimulation protocols. They were randomly assigned to receive a single injection of GnRH agonist (study group) or placebo (control group) 6 days after ICSI. The pregnancy rates were enhanced for both protocols, in long GnRH agonist protocol the clinical implantation rate were 29.8% (97/325) vs. 18.2% (60/330) respectively (P < 0.05). Ongoing pregnancy rates were 46.8% (66/141) vs. 38.0% (54/142) respectively (P = NS). In patients treated with the GnRH antagonist protocol, clinical implantation rates were 27.1% (86/317) vs. 17.4% (57/328) respectively (P < 0.05) and ongoing pregnancy rates were 44.8% (65/145) vs. 31.9% (46/144) respectively (P < 0.05). Luteal-phase GnRH agonist administration additionally increased the luteal-phase serum HCG, estradiol and progesterone concentrations in both ovarian stimulation regimens. It was postulated that the beneficial effect may have resulted from a combination of effects on the embryo and on the corpus luteum. Despite these initial encouraging results, it is too early to adopt this treatment wholesale. With regard to safety, great concern exists about possible adverse effects on oocytes and, more importantly, on embryos (Lambalk and Homburg, 2006). To establish a potential positive role of GnRH agonist administration in the luteal phase of stimulated IVF cycles, further large prospective trials are needed. Until recently, there were no studies to either support or contest the generally accepted practice of prolonging progesterone supplementation during early pregnancy. Schmidt et al. (2001) was the first to publish a retrospective study to compare the delivery rate with IVF or ICSI in women who received progesterone supplementation with those who did not during the first weeks of pregnancy. For three weeks following a positive hCG test, 200 pregnant women received progesterone and 200 pregnant women received none (study group). The results showed no difference in the delivery rate. Of the 200 pregnancies in the study group, 126 (63%) ended in live birth, 46 (23%) were biochemical, 5 (2.5%) were ectopic and 23 (11.5%) ended in abortion. In the control group, 128 pregnancies (64%) ended in a live birth, 35 (18%) were biochemical, 7 (3.5%) were ectopic, and 30 (15%) ended in abortion. Subsequently, a prospective randomized controlled trial was conducted. Nyboe Andersen et al., (2002) evaluated whether the prolongation of luteal support during early pregnancy had any influences on the delivery rate after IVF. In this study, luteal phase support was administered in the form of 200 mg vaginal progesterone three times daily (600 mg/d) during 14 days from the day ET until the day of a positive HCG test. The study group (n = 150) withdrew vaginal progesterone from the day of positive HCG. The control group (n = 153) continued administration of vaginal progesterone during the next 3 weeks of pregnancy. 118 (78.7%) patients delivered in the study group given no progesterone versus 126 (82.4%) in the control group who continued with progesterone. The difference was not significant. Results indicated that prolongation of progesterone supplementation in early pregnancy had no influence on the miscarriage rate, and thus no effect on the delivery rate. It would appear that the increase in endogenous HCG level during early pregnancy makes up for any possible lack of endogenous LH that has been caused by stimulated IVF cycles. First trimester progesterone supplementation in IVF may support early pregnancy through 7 weeks by delaying a miscarriage but it does not improve live birth rates (Proctor et al., 2006). The cause of luteal phase defect in stimulated IVF cycles seems to be related to the supra- physiologic levels of steroids. Luteal phase support with HCG or progesterone after assisted reproduction results in an increased pregnancy rate (Fatemi, et al., 2007). HCG is associated with a greater risk of OHSS. Luteal support with hCG should be avoided if E2 >2700pg/ml (Fatemi, et al., 2007) and if the number of follicles is >10 (Fatemi, et al., 2007). Natural micronised progesterone is not efficient if taken orally (Fatemi, et al., 2007). Vaginal and intra muscular progesterone seem to have comparable implantation and clinical pregnancy rates and delivery rates (Fatemi, et al., 2007). The addition of oral E2 to the progestin for luteal phase support still seems not to be beneficial (Kolibianakis et al., 2008). The length of luteal phase support in stimulated IVF cycles does not need to exceed 14 days from the day of transfer (day 3 post OR) until the day of a positive HCG test (Nyboe Anderson et al., 2002). In the coming years, IVF stimulation may evolve into a more physiologic process – a milder stimulation – with the significant fringe benefit of reducing or eliminating the current luteal phase defect. It appears that the cause of luteal phase defect in IVF is related to the supraphysiological levels of steroids, it would be interesting to find out which is the threshold, where the luteal phase defect initiates. Further more it should be more specified whether it is the progesterone, E2 or both causing the luteal phase defect in stimulated cycles. Therefore a progesterone antagonist could be administered in oocyte donors and the luteal endocrine profile of those patients should be evaluated. Also the combined use of an anti-estrogen, i.e. an aromatase inhibitor and a progesterone antagonist in oocyte donors should be further evaluated. CC occupies the hypothalamic estrogen receptors for several weeks (Dickey et al., 1996). The long term receptor occupancy might lead to higher luteal LH concentrations, correcting the luteal phase defect observed in stimulated IVF cycles (Van Steirteghem et al., 1988). It would be interesting to evaluate, whether there is a luteal phase defect in cycles stimulated with clomiphene citrate/ recombinant FSH and gonadotropin-releasing hormone antagonist, despite the significantly higher LH levels measured in the luteal phase of these cycles (Tavaniotou et al., 2002). Furthermore the administration of very low dose of HCG for luteal phase support in stimulated IVF cycles without the co-administration of P and E2 should be evaluated. Last but not least, further genetic research of endometrium should be performed, to find out why anno 2009 still we have such a low ongoing pregnancy rates after IVF/ICSI.