At present, the "long" GnRH-agonist regimen with relative high doses of exogenous gonadotropins is probably the most frequently used stimulation protocol; this regimen has become a comfortable routine over the years in many IVF clinics. In the last years, the availability of GnRH antagonists has allowed the clinical development of "mild" ovarian stimulation protocols involving subtle interference with single dominant follicle selection. Some studies have compared the success rate of "mild" vs. standard ovarian stimulation regimens, either in women with normal ovarian reserve or in subjects with poor ovarian reserve.
Women with normal ovarian reserve
Three prospective, randomized controlled trials compared the effectiveness of "mild" stimulation regimen with the conventional "long" GnRH agonist protocol with an early follicular phase FSH start.
The single centre RCT by Hohmann et al. [
18] included 142 patients with good reproductive prognosis who were randomly distributed into three groups: (A) (n= 45) treated with the GnRH-agonist Triptorelin and, after down-regulation, with a fixed daily dose of 150 IU rFSH; (B) (n= 48) and (C) (n= 49) treated with rFSH initiated on cycle day 2 (group B) or 5 (group C) and with the GnRH-antagonist Cetrorelix starting when the largest follicle reached 14 mm diameter. Group C showed a shorter duration of stimulation, reflected in a significantly lower total dose of exogenous rFSH; in group C, more cycles were cancelled due to insufficient response and less oocytes were harvested, but better quality embryos were finally obtained. Fewer cycles in group C were characterized by a total fertilization failure or by abnormal embryo development. Nevertheless, because of the higher number of oocytes retrieved in group A and B and to the better availability of embryos among which selecting the best, no significant difference in the quality of the best transferred embryo was observed among the three groups. After stronger ovarian stimulation (groups A and B), only 7% of the patients who retrieved less than 5 oocytes conceived, whereas after "mild" stimulation 67% of these patients conceived. Overall, no differences were found among the three groups as far as the pregnancy rate per started cycle was concerned.
The results of this study suggest that a low number of retrieved oocytes after "mild" ovarian stimulation could have a different meaning from a low number of retrieved oocytes after a conventional stimulation. It was shown that the ideal number of oocytes after a conventional "long" protocol is 13 and that when the number of retrieved eggs is much lower (or even much higher), the pregnancy rate is compromised [
19]. A poor oocyte yield after classical ovarian stimulation likely reflects a poor ovarian responsiveness to FSH, that is associated with poor IVF outcome, whereas a low number of oocytes after "mild" stimulation probably represents a normal response, a smoother selection of follicles (and oocytes) more likely to finally result in high quality embryos and in a pregnancy [
20]. A low overall dose of exogenous FSH probably stimulates only the most mature follicles having optimal receptor endowment, and allows a sort of "quality selection" among follicles (and finally among oocytes) avoiding to force poor quality follicles to grow anyway [
20].
Another RCT by Heijnen et al. [
21] included 404 regularly cycling, normal BMI patients and almost 800 consecutive IVF cycles. In this study, one group was given "mild" ovarian stimulation with GnRH antagonist co-treatment combined with selected single embryo-transfer (SSET), whereas the other received a standard ovarian stimulation with the GnRH agonist "long" protocol combined with the transfer of two embryos. The "mild" treatment group was characterized by a lower duration of stimulation, a lower total FSH dose and by less retrieved oocytes. The pregnancy rate per cycle was significantly lower in the "mild" stimulation group (17.6% vs 28.6%, p < 0.0001); however, the "mild" stimulation protocol, easier to stand and cheaper for patients, reduced the rate of treatment discontinuation and induced some patients to undergo shortly a second IVF attempt. This attitude resulted in a cumulative live birth rate after 1 year of IVF treatments that was comparable in the two groups (43.4% with the mild protocol, 44.7% with the standard regimen), and the twinning rate was significantly lower in the "mild" stimulation-SSET transfer group (0.5% vs. 13.1%, p < 0.0001) [
21]. According to this study, a reduced chances of birth per cycle in the "mild" regimen might be compensated by the increased number of IVF attempts in a set time. In fact, the overall discomfort to patients, evaluated with the hospital anxiety scale, the depression scale, the somatic Hopkins' subscale and the subjective sleep quality scale, were lower in the group assigned to the "mild" stimulation. Interestingly, also the economical costs of the treatment were significantly lower with the mild stimulation/SSET compared with a standard treatment involving conventional stimulation; it must be remarked, however, that the money saving was mainly due to the almost complete absence of twin pregnancies in the "mild" group rather then to the cost of IVF procedure itself [
21]. Interestingly, a prediction model aimed to estimate the chance of ongoing pregnancy after "mild" stimulation and SSET has been prepared using multivariate logistic regression analysis; this model is claimed to be helpful to optimize results identifying patients for which the "mild" stimulation plus SSET strategy can be appropriate [
22].
The third RCT comparing the "mild" protocol and the "long" classical protocol was performed by Baart et al [
23] on 111 patients who were randomized into two groups, one (n = 67) receiving rFSH 150 IU/day from day five of the cycle plus GnRH-antagonist in a flexible schedule, the other (n = 44) receiving rFSH 225 IU/die after two weeks of pituitary down-regulation by GnRH-agonist. In this study, couples with severe male factor were excluded. The ongoing pregnancy rate per started cycle was 21% in the "mild" group and 18% in the control group, a difference not statistically significant. Interestingly, in this study embryos were biopsied at the 8-cells stage and 1 or 2 blastomeres were then analyzed by fluorescent in-situ hybridization (FISH) considering 10 chromosomes: despite "mild" stimulation obtained significantly fewer oocytes and embryos, both regimens finally generated the same number (1.8/cycle) of chromosomically normal embryos. This observation suggests that the reduced pharmacological interference with ovarian physiology could generate oocytes of better genetical quality. Indeed, some data in the mouse model show that exogenous disturbances in the signals regulating folliculogenesis may alter the late stage of oocyte growth, increasing the risk of altered chromosome segregation in subsequent meiotic divisions: an increased incidence of chromosomal abnormalities, in fact, was observed in oocytes after exposure to high doses of gonadotropins during in vitro maturation of mouse oocytes [
23,
24]. Mild stimulation approaches, aiming at a more physiological response, might be able to improve the genetical quality of oocyte and embryos in humans. This hypothesis, however, needs to be validated by further trials including a higher number of patients and embryos, and possibly using techniques (e.g. CGH) able to study the whole set of chromosomes on a single blastomere.
Taken together, these three RCTs comparing the "mild" with the classical stimulation regimen included 592 first IVF attempts, among which 313 were performed with the "mild" stimulation protocol and 279 with the classical "long" regimen. Although individually these trials found comparable results in terms of IVF effectiveness, pooling data together the ongoing pregnancy rate per started cycle sorts out to be 15% in the "mild" group and 29% in the classical group, a difference that suggests a well definite trend toward a lower IVF effectiveness when the "mild" strategy is applied. This suggestion is further reinforced by the fact that the three RCTs did not consider freeze-thaw cycles, and the chance of obtaining surplus embryos/oocytes to freeze is obviously much lower in "mildly" stimulated cycles than in the classical ovarian stimulation cycles. Having frozen embryos/oocytes to transfer in a subsequent cycle can probably increase the overall IVF pregnancy chance per oocyte pick-up by approximately 10-15%: thus, the gap between the two competitors could probably be wider considering freeze/thawing cycles, and could possibly reach statistical significance. New RCTs including freeze-thaw cycles in the comparison between "mild" and classical stimulation regimens are definitely needed to get a higher level of evidence about the respective effectiveness of the two strategies.
A factor limiting the effectiveness of "mild" strategy in terms of pregnancy rate per cycle is likely to be the relatively high rate of cycle cancellation due to mono- or bi-follicular response (around 15-20%) when gonadotropins are started on day 5 of the cycle. When such a response is observed, a valuable option is to stop stimulation and start it again the next month starting earlier with medications (on day 2-4). Ovarian aging and high BMI have been identified as relevant variables to predict the risk of insufficient response to "mild" stimulation, and a predictive model have been developed in order to minimize the need of cancelling the cycle [
25].
On the other side, classical ovarian stimulation is claimed to be a factor able to impair endometrial maturation and consequently embryo implantation chance. Some studies indeed showed that the gene expression profiling of the endometrium in conventionally stimulated cycles is extremely different from the one that can be observed during a natural cycle [
26,
27]: interestingly, the endometrial gene expression pattern is more similar to the natural one in cycles with GnRH-antagonists than in cycles with GnRH-agonists [
28]. Furthermore, classical ovarian stimulation regimens are associated with about ten-folds supra-physiological circulating estradiol levels that have a well documented negative impact on the developmental and implantation potential of human embryos [
24,
29]. Some data suggest that the best endometrial receptivity to embryo implantation may be found in natural cycle, but mild stimulations have a lower impact on endometrial quality than classical regimens [
30,
31]. The "mild" stimulation regimen is associated with significantly lower peak estradiol levels, and possibly can impact on the endometriom more softly than classical regimens: the "endometrial factor" can probably represent one scored point in favour of "mild" stimulation.
Women with poor ovarian reserve
Ovarian stimulation for women with a poor ovarian reserve is probably one of the most frustrating aspects of IVF procedure: most of the treatments proposed to enhance oocyte yield and pregnancy rates in "poor" responders have failed to show any convincing evidence [
32].
The standard approach to women estimated to be poor responders is based on starting the "long" protocol with a daily dose of approximately 300 FSH IU/die [
33]; the starting FSH dose used in any subsequent cycle is then adjusted (up to 600 FSH IU/die) according to the individual response in the first cycle [
34]. The strategy of performing an upward dose adjustment in women with poor ovarian reserve, however, has not shown any consistent benefit. In previous poor responders, in fact, the IVF outcome of those assigned to a starting dose of 225 FSH UI/day vs. those receiving 450 UI/day was shown to be similar, despite the latter obtained more oocytes [
35]. Also other studies showed that predicted poor responders had no benefit from increasing the FSH starting dose [
36,
37]. High gonadotropin doses may indeed lower the cycle cancellation rate, but have been observed to reduce the likelihood of clinical pregnancy and live birth rate and to increase the risk of spontaneous miscarriage [
38]. A negative effect of high dose regimens on the endometrial quality has been claimed to be responsible for the poor outcome of this regimen [
39], although probably even factors linked to embryo quality itself play a relevant role. High doses of FSH recruit "resistant" follicles rescuing them from atresia, but the oocytes that they host are of poor quality and usually do not result in the generation of good quality embryos [
40,
41]. Since the economical cost of gonadotropins is one of the major expenditures in IVF treatment, the huge increase in drug cost linked to high-dose gonadotropin regimens appears to be a nonsense if not paralleled by a significant improvement in clinical outcome.
A combination of Clomiphene citrate (CC) plus gonadotropins and GnRH antagonists has been proposed as a "mild" stimulation alternative for poor responders. CC is known to act as an anti- estrogen on the central nervous system, increasing the pulse frequency of endogenous FSH and LH and giving a moderate gonadotropin stimulus to the ovary [
42]. CC has been used for over thirty-five years to induce ovulation in WHO type II anovulatory women, and is still appreciated for its oral administration and low price; the combination with gonadotropins may counterbalance its undesired anti-estrogenic effect on the endometrium and at the same time may reduce the amount of gonadotropins required, thanks to the combined synergistic effect on the ovary.
The level of evidence supporting the use of the "mild" stimulation protocol with CC/Gn/GnRH antagonist in patients with poor ovarian reserve is rather poor, as properly designed studies on an appropriate number of patients are still unavailable. The first report describing the use of CC/Gn/GnRH antagonists in poor responders included only eighteen patients; compared to their response in a previous standard GnRH-agonist cycle, light improvements in cycle cancellation rate, oocyte yield and gonadotropin requirement were observed [
43]. Unfortunately, neither the number of patients, nor the study design allowed to get an acceptable level of evidence.
Takahashi et al. [
44] studied 40 poor responders with a story of multiple IVF failures with the "long" protocol: treating them with CC/FSH/GnRH antagonist he obtained an ovarian response comparable to the previous ones, but a significantly higher blastocyst development rate and a very good (41.2%) ongoing pregnancy rate. Again, the study design was not very informative and the patient number was too little.
D'Amato et al [
45] compared the combination of CC/FSH/GnRH antagonist to a long GnRH agonist protocol enrolling 145 women with a prior poor response; a significantly lower cancellation rate, higher peak oestradiol level, more retrieved oocytes and higher pregnancy and implantation rates were achieved with the antagonist protocol. In this study, however, high FSH amounts and not a "mild" gonadotropin stimulation were used: the observations, once again, are only indicative of the possibility of using this kind of stimulation at lower doses in poor responders.
Some other informations may be deduced from studies that compared the outcome of CC/Gn/GnRH antagonist treatment with a standard "long" protocol in patients with normal ovarian reserve: pregnancy rates comparable to the standard stimulation regimens were obtained by the "mild" strategy, with a significant reduction in the total dose of gonadotropin needed and of the economical costs [
46‐
48]. These results appear to be encouraging, although it remains to be proven that they can be replicated even with patients with poor ovarian reserve.
Interestingly, it was shown that when in CC/Gn/GnRH antagonist cycles the circulating level of LH is less than one-third at the time of hCG than it was at the beginning of stimulation, both the pregnancy and implantation rates are significantly reduced [
49]: this observation suggests to chose medications containing LH or hCG rather than FSH alone to be associated with CC in this kind of protocol.
Overall, the published results suggest that in patients with poor ovarian reserve the choice of a mild stimulation protocol instead of a classical, high dose regimen, could be particularly indicated. Although these patients have a very low risk of OHSS even using high doses, the quality of both their oocytes and their endometrium could likely to be better when a smoother stimulation approach is used. Further research, anyway, is needed to add scientific evidence to this hypothesis.