Methods: This study enrolled 105 patients with POR and 58 patients without POR (controls) who were diagnosed according to the Bologna criteria and underwent conventional IVF-ET. Poorovarianresponders were randomly assigned to two groups: the POR-GH group, which received pretreatment with GH 4 IU/d on day 2 of the previous menstrual cycle before IVF until the trigger day, and the POR-C group, which received no pretreatment. OS markers in follicular fluid (FF), ROS levels in granulosa cells (GCs), and the IVF outcomes of the groups were compared. Results: Endometrial thickness on trigger day, the number of cleaved embryos, the number of higher-quality embryos, and the rates of embryo formation, higher-quality embryo formation, implantation and clinical pregnancy were significantly increased in the POR-GH group compared with the POR-C group ( P < 0.05). Moreover, compared to those in the non-POR group, FF malondialdehyde (MDA), total oxidant status (TOS), oxidative stress index (OSI) and ROS levels in GCs were significantly higher, whereas superoxide dismutase (SOD) and the total antioxidant capacity (TAC) were significantly lower in the POR-C group ( P < 0.05). Furthermore, compared with those in the POR-C group, the FF TAC was significantly increased in the POR-GH group, and TOS, OSI and intracellular ROS levels were significantly reduced ( P < 0.05).
However, most of these interventions have achieved extremely limited advan- tages; consequently, the optimal protocol for POR is still unknown . The in- troduction of GnRH antagonists presented hope for POR. GnRH antagonists can induce pituitary suppression within a few hours without a “flare-up” effect, and the suppression can be released immediately after their discontinuation . In the present study, we investigated the effectiveness of the GnRH antagonist protocol in expected poorovarianresponders. We found that the duration of stimulation and the total doses of Gn in the GnRHa group were 11.51 ± 1.46 days and 3317.34 ± 775.29 IU, which were significantly more than those in the GnRHant group (9.70 ± 2.23 days and 2306.88 ± 757.22 IU, P < 0.05). These were in keep- ing with the results of previous studies. Several studies demonstrated a lower implantation rate and pregnancy rate in the GnRH-ant protocol, which limited its wide application . However, our study showed that, for expected POR (age ≥ 40 years), the implantation rate and clinical pregnancy rate of the GnRHant group were slightly higher than those in the GnRHa group (12.24% vs. 10.10%, P = 0.437; 23.36% vs. 23.03%, P = 1.000 ). In our previous study, we demonstrated that, for potentially high responders, the GnRHa protocol was superior to the GnRHant protocol in terms of normal fertilization rate, implantation rates, and clinical pregnancy rates . These findings showed that the GnRHant protocol may be an optimal protocol for expected poorovarianresponders, but not for potentially high responders.
GnRH antagonist may shorten the duration of stimu- lation, lower the total gonadotropin requirements, reduce patient’s cost, decrease cycle cancellation, and has a higher ongoing pregnancy and a better delivery rate in poor responder patients [14,15]. There is a new method to use GnRH antagonist before ovarian stimula- tion in order to lengthen the follicle phase and rescue more follicles. Doing so, more oocytes and zygotes were attained, implantation rate and ongoing pregnancy rates were improved . Addition of LH may be beneficial effect in poorovarianresponders [17,18], but other pro- spective and randomized trial show that the addition of rLH to the ovarian stimulation protocol produces no further benefit in older poor responder patients . Endogenous FSH in the preceding luteal phase can sti- mulate larger follicles and subsequently lead to a size discrepancy. A novel strategy for treating poor respon- ders is to give estradiol in the luteal phase before IVF hyperstimulation, the estradiol can suppress FSH in the preceding luteal phase and result in more coordinated cohort of follicles responding to the stimulation process. The luteal E 2 protocol may improve embryo quality and
Despite the enormous number of studies on poorovarianresponders published in the last 30 years, their man- agement remains one of the biggest challenges and most controversial topics in Assisted Reproduction with less than satisfactory progress. According to recent reviews, the incidence of this condition, which also has a genetic predisposition, ranges between 9% and 24% . A rate of 16.16% has been reported for “unexpected” PoorOvarianResponders (PORs) . There is no universally accepted definition, but most people refer to cases with less than 4 follicles and/or estradiol (E2) levels less than 500 pg/ml on the day of HCG administration. Accord- ing to the ESHRE consensus in a first real attempt to find a common definition of “poorresponders” , early detection of the problem with different methods of assessment of ovarian reserves, is of particular importance in order to select the most appropriate method of treatment. Today, the AFC (Andral Follicle Count) or AMH (Anti-Mul- lerian Hormone) measurement is recommended to predict ovarian response  , with the latter presenting as an excellent and equally promising test  . Moreover, time interval up to 12 months does not seem to mod- ify its predicting capacity . For scientific purposes however, according to the 2011 ESHRE consensus 3 , more than one criterion should be contemporaneously presented.
As an adjuvant, DHEA may increase the serum AMH level, the E 2 level on the day of hCG admin- istration and decrease the FSH level on cycle Day 2 [15, 16]. So, DHEA pretreatment could result in a higher number of retrieved oocytes, fertilized oocytes, overall number of embryos, number of grade I embryos  and the ongo- ing pregnancy rate . The miscarriage rate after DHEA administration was not only lower than in the average IVF patients but also com- parable to that reported in normally fertile pop- ulations. Low miscarriage rate was statistically impossible to be achieved in DOR patients with- out the assumption of DHEA effect on embryo ploidy . However, a recent study found that supplementation with DHEA had significant relationship with poor prognosis in women undergoing ovarian stimulation for IVF, no sig- nificant benefit could be found regarding the gonadotropin requirements, duration of stimu- lation oocyte, embryo yield and pregnancy rate . Due to the controversial results and the lack of large-scale data analysis supporting its effectiveness and safety, the widespread use of DHEA cannot be currently recommended . So we reviewed all randomized controlled trials (RCTs) and aimed to assess whether pre- DHEA treatment could improve the clinical results of IVF in poorovarianresponders. Materials and methods
Poorovarian response is a major challenge in in-vitro fertilisation-embryo transfer (IVF-ET). Because many women today are postponing pregnancy and the average age of IVF patients has increased correspondingly, poor response is not a rare occurrence in ovarian stimulation and has become part of the daily clinical management for clinicians in IVF centres . According to our clin- ical experience, few poorresponders would like to give up an initiated cycle with most wanting to know the chance of conception during the next cycle if they were not pregnant. Appropriate counseling would help patients decide whether to continue or stop IVF treatment to avoid both unnecessary economic losses and health risks. Many hormonal and ultrasound markers, such as age, basal serum follicle stimulating hormone (FSH) level, serum anti-Mullerian hormone levels and the number of antral follicles, have been developed to predict the cycle success or identify patients at high risk of poor response before ovulation induction [2-4]. However, there is still no ideal and consistent predictive marker of IVF outcome for poorovarianresponders.
Poorovarianresponders (PORs) are the women who re- spond suboptimally to ovarian stimulation with gonado- tropins. While a variety of definitions exist for POR , the ESHRE consensus group standardized the definition of POR and established the Bologna criteria in 2011 . It is a great challenge for PORs to reach live birth in in vitro fertilization (IVF) cycles [3, 4]. No certain protocol or sin- gle intervention is accepted as an effective method to overcome the poor prognosis of PORs . Therefore, mul- tiple strategies, including various IVF protocols , the use of adjuvant supplements [7, 8] and accumulation of vitrified oocytes or embryos  have been attempted for PORs undergoing IVF cycles. However, the optimal man- agement for PORs remains an unsolved problem.
Prior randomized controlled trials (RCTs) com- pared microdose flare-up to the GnRH-ant proto- cols in POR and they offered varied results. The reason of these may be due to the lack of a uni- form definition of poorresponders. This makes it difficult to compare treatment outcomes and de- velop and assess protocols for prevention and management. Demirol et al. (8) showed that using flare-up protocols when compared with using an- tagonist yielded significantly higher mature oo- cytes, higher IR in flare protocols with similar CPR. Kahraman et al. (9) found higher peak E2 level in flare protocols, apart from the fact that no statistically significant differences were noted in cycle outcomes or CPR. A meta-analysis in 2006 (18) reported higher number of retrieved oocytes in the GnRH-a protocols when the meta-analysis was applied to the four trials that had used GnRH- ant vs. flare-up protocols. Also, it was reported that there was no difference between GnRH-ant and GnRH-a (long and flare-up protocols) with respect to CCR and PR. Similarly, in our study, significantly higher numbers of oocytes retrieved and higher implantation rates in microdose flare- up were found compared to GnRH-ant protocol
Initiating ovarian stimulation in the early follicular phase is essential for fresh transfer and for the endomet- rium to be receptive during that cycle. However, due to advances in the field of cryobiology [7, 8], this is no lon- ger necessary. Ovarian stimulation can conclude with elective freezing of oocytes or embryos, with similar re- sults to those of a fresh embryo transfer  even in pa- tients with poor response . In fact, one of the proposed strategies in patients with poor response is the accumulation of oocytes for subsequent fertilization . Previous studies demonstrated the appearance of more than one wave of follicular growth within a cycle, sug- gesting the presence of obtainable follicles during the lu- teal phase . Although ovarian stimulation during the luteal phase was reserved for women with cancer in whom oncological treatment could not be delayed [13– 15], in recent years, luteal phase stimulation has been identified as an adequate method of obtaining a suffi- cient number of competent oocytes . This offers the possibility of collecting oocytes twice in the same cycle in order to obtain the highest number of eggs in the shortest period of time [17–19]. Results of double stimu- lation in poorresponders suggest a better response in the second stimulation during the luteal phase, but this effect could be explained by priming of stimulation in the follicular phase . Thus, the efficacy of ovarian stimulation in the luteal phase of women with POR compared with conventional protocols is yet to be deter- mined [20–22].
Several observational and non-randomized controlled studies have suggested that flushing results in a higher number of retrieved oocytes [3, 4, 11, 12]. However, in the last decade, additional randomized controlled trials (RCTs) [13–15] have failed to demonstrate that follicular flushing could improve the number of retrieved oocytes and mature oocytes and pregnancy outcomes when compared with direct aspiration. In addition, flushing was obviously associated with an increase in the dur- ation of the retrieval procedure and the use of anesthetic agents . Currently, clinicians have almost reached a consensus that in unselected patients or normal ovarianresponders, follicular flushing has very limited benefit and should not be encouraged in routine treatment, though in other groups of patients, such as PORs and those undergoing natural cycle or minimal stimulation IVF, evidence is lacking [17–19].
On the other hand, the result of studies with positive findings should be assessed with caution. In Garcia-Velasco study, despite the higher number of oocytes retrieved and marked improvement in implantation rate in the letrozole-added group, no significant differences were found regarding cycle cancellation, fertilization, and pregnancy rates between the compared groups (19). In fact, there are cyclic differences in a cohort of recruitable follicles and variability in ovarian response to stimulation programs. One episode of poor response to a stimulation protocol will repeat in the second attempt, with the same protocol in only 46-62.4% of cases (38, 39). In light of this observation, the improvement in cycle outcome with adding letrozole might be related to the variability in ovarian response not to drug effect.
There is no one pituitary downregulation proto- col which is best suited for all poorresponders. Traditional GnRH agonist flare and long luteal phase protocols do not appear to be advantageous. Reduction of GnRH agonist doses, "stop" proto- cols, and microdose GnRH agonist flare regimes all appear to improve outcomes, although the pro- portional benefit of one approach over another has not been convincingly established. GnRH anta- gonists improve outcome in poorresponders, al- though, in general, pregnancy rates appear to be lower in comparison with microdose GnRH ago- nist flare regimens. Prediction of decreased re- sponse by a thorough assessment of ovarian re- serve prior to cycle initiation allows selection of an appropriate COH protocol tailored for each individual patient.
smoking,genetic factor and iatrogenic causes (7-9). Incidence of PORs has been reported in 9-24% (10, 11). A number of studies have indicated that they need more gonadotropin for stimulation (12). Several interventions were used to improve effectiveness of applied technique and the pregnancy outcomein this group, but there is no adequate evidence which intervention is appropriate (13-15). The most common protocol used for PORs is gonadotropin- releasing hormone (GnRH) agonist (16, 17). Minimal ovarian stimulation is anther protocolthat consists of low dose gonadotropin overlapping clomiphene or letrozole. The main advantages of this protocol are as follows: (i) cost effectiveness, (ii) shorter duration of stimulation, (iii) reduced gonadotropin requirements and (iv) patient-friendly method (3, 18-21). However, in some studies, minimal stimulation protocol was not considered as a cost effective and better method in compared with the standard protocols, and it was not also recommended (16, 20, 21). Clomiphene citrate overlapping gonadotropin with GnRH antagonist has been alsoapplied for PORs (22). In a study by Saadat et al., they have shown that clomiphene citrate causesan increase in endogenous follicle stimulating hormone (FSH) level(23) that leads toa low dose of gonadotropin and shorter duration of stimulation as compared with GnRH agonist (19, 22, 24). Therefore, clomiphene combined with gonadotropin offer an advantage for PROs (25). For the purpose of minimum effect on the endometrium, it has been suggested that the protocol starts on the second day of cycle (26). In a study by Ubaldi et al., they have showed that the GnRH antagonist prevents premature luteinizing hormone (LH) surge and ovulation advantages has to compare the GnRH agonist (9). The objective of this study was to compare the pregnancy outcomes achieved by in vitro fertilization (IVF) between conventional antagonist protocoland minimal stimulation protocol including clomiphene citrate overlapping with gonadotropin in PORs.
At first, the definition of poor responder has been a confounding matter, in that different criteria were used by reserchers. In any case, all the studies showed how pregnancy rates in poorovarianresponders remained substantially low . In 2001 the definition of poor re- sponders has been uniformed with the introduction of Bologna criteria , but still all the studies in patients fulfilling the Bologna criteria resulted in very low preg- nancy rates, irrespective of patients’ age and type of ovarian stimulation protocol [4, 7].
poorresponders and have reported controversial results (4). Previous studies regarding this field were different in methodology including the dosage, time of LZ addition, and the initiation time and dosage of gonadotropins (13). Yang and colleagues in a recent study compared the impacts of COH and IVF outcomes of the following three GnRH antagonist protocols: (i) use LZ (5 mg) for five days sequentially overlapping with gonadotropin, (ii) applying LZ (7.5 mg) for three days sequentially with gonadotropin, and (iii) the standard high-dose gonadotropin in a GnRH antagonist protocol in poorovarianresponders (4). They concluded that adding LZ with antagonist protocol is an affordable and preferable protocol” (4). In a recent meta-analysis study, Song and co-workers concluded that the clinical pregnancy rate may be lower with the antagonist/LZ protocol than micro-dose GnRH agonist flare-up protocol for treating poorresponders undergoing IVF/ICSI, but large-scale randomized controlled trials are required to evaluate the antagonist/LZ protocol (14).
protocols which decrease OHSS to an absolute mini- mum without compromising live birth rates [6, 7]. How- ever, the clinical management of patients with poorovarian reserve, so called poorovarianresponders (PORs) still remains a clinical challenge. This was fur- ther complicated by the fact that only until recently there was no general agreement about the diagnosis of POR. Thus, Polyzos and Devroey (2011) reported the use of as many as 41 different POR definitions in a total of 47 randomized controlled trials (RCT), which ham- pered the clinical value of interstudy comparison and meta-analysis in this heterogenous group of patients . In their title the authors provocatively asked whether there was any “light at the end of the tunnel for the POR patient”. Subsequently, in 2011, an ESHRE consen- sus group  took the effort to try to standardize the definition of POR, establishing the so called ESHRE Bol- ogna criteria (Table 1).
Our data support those reported in the literature and showing that, so far, no specific treatment improves the outcome in poorresponders treated with IVF. Despite the consensus of Bologna, today there is no one clear definition of poor responder. In 2003 Toner et al.  reported that female age impacts oocyte quality and that both AMH and AFC profile affect the number of re- trieved. To mitigate the negative effect of age, prophylac- tic oocyte cryopreservation can be proposed after ovarian stimulation for future use. This perspective is supported by advances made on vitrification . How- ever to reach pregnancy it will need between 12 to 15 oocytes  and even 22 to 50 depending on the woman’s age . Today in France this perspective is not allowed but in the meantime we can offer mild stimulation to collect lower number of oocytes with bet- ter quality and giving acceptable number of embryo for vitrification . This perspective is in line with the Baart et al. study  who found that that lower doses of gonadotropins are associated with embryos with lower aneuploidy rates and the Otsuki et al.  analysis which showed that high oestradiol levels can produce oocyte with vacuolated cytoplasm and reduce the chances of pregnancy. For poorresponders, multiple mild stimula- tions followed by vitrification can increase the number of suitable embryo for transfer after thawing and im- prove clinical pregnancy. Our study showed that the im- provement of pregnancy rate between protocol P1 and P2 or P3 is related to the number of suitable good em- bryos for transfer. In addition sequential transfer after vitrification on substituted or lightly stimulated cycles could improve implantation via better endometrial re- ceptivity and ongoing pregnancy rate [39–41].
decrease in fertility age has long been recognised from demographic and epidemiological studies especially in women aged between 30 and 35 years, which is followed by an accelerated decline among women aged over 35 years. An important finding in our study was the fact that DHEA has better prognosis in poor responder women who were <35 years old compared to those more ≥35 years old. DHEA treatment in this specific group allowed for more number of day-2 or -3 embryos to be obtained. The average of number of day-2 or -3 embryos obtained were two in non DHEA group and three in those with DHEA treatment group. DHEA is also said to improve oocytes quality thus yielding better embryos without aneuploidy and thereby reducing the risk of abortion. 25 On the other hand, Jirgi et al., 26 did not find any
In this trial, the oocyte/embryo results reflected the ef- ficacy of the different treatment strategy.Oocyte retrieval in natural cycle is individualized based on the occur- rence of a spontaneous LH surge; therefore, frequent monitoring and a 7-day-per-week working schedule are unavoidable for natural cycle IVF. A large studyof 1048 patients who underwent modified natural-cycle IVF demonstrated a 66% oocyte retrieval rate per scheduled retrieval and confirmed that the use of GnRH antago- nists did not substantially improve the outcome .Our trial demonstrated an efficacy of 71.57% for the retrieval rate and 38.24% for the embryo transfer rate in natural cycle IVF patients, which is in accordance with previous reports [9 – 12]; the MPA group demonstrated superior oocyte and embryo outcomes. Notably, the higher num- ber of retrieved oocytes and embryos may be associated with the administration of hMG during the late follicular phase of the MPA treatment. However, the rate of successfully retrieved, mature oocytes and cleaved em- bryos was comparable between the two groups, and the Table 2 IVF Outcomes of natural cycle and P-primed minimal stimulation in poorresponders
All patients included in this analysis had a standard methotrexate treatment with a regimen of oral 7.5 mg weekly, and with the dosage increasing to 20 mg weekly after 4 weeks, in combination with folic acid (1 mg daily). No patients received any other disease-modifying antirheumatic drug (DMARD), and no researchers’ in- volvement in the patient care was practiced. A good MTX response was noted for patients who were receiv- ing MTX and had a disease activity score based on 28 joint counts (DAS28) of ≤2.5 at 6 months. Poor re- sponders were defined as patients who were also receiv- ing MTX but had a DAS28 of >2.5 [21,22].