5.2.1 Growth Hormone

Growth hormone (GH) is a polypeptide hormone secreted by somatotropic cells of the anterior pituitary gland that stimulates cell growth and proliferation. It was previously demonstrated that GH may play a crucial role in follicular development and oocyte maturation via regulating the synthesis of insulin growth factor-1 (IGF-1) in granulosa cells and increase follicular sensitivity to gonadotropins [2]. There are IGF-1 receptors within oocytes, granulosa, and theca cells. In women undergoing IVF, IGF-1 concentrations are directly related to the number of developing follicles due to the suppressive effect of IGF-1 on follicular apoptosis. GH itself is also required for follicular development and inhibition of follicular apoptosis.

Several studies have been conducted to evaluate the effect of GH supplementation in women with POR undergoing ART. The main success parameters considered in those studies are the number of oocytes retrieved and clinical pregnancy/live birth rates. One of the largest studies performed on the supplementation of GH was the LIGHT (live birth rate in vitro fertilization and growth hormone treatment) trial from Australia and New Zealand in 2016 [3]. When patients were prospectively randomized to study arm (12 IU/day GH beginning on the day of stimulation) or controls, although there were some improvements in response to ovarian stimulation, the authors did not notice any benefit with regard to live birth rate in the study arm. However, one should be cautious that this study has not been published as a full paper.

In spite of prospective trials, a recent retrospective study including 400 women in which 161 had been treated with GH (average of 1.5 IU per day), Keane et al. [4] reported that GH supplementation significantly increased clinical pregnancy rate by 3.42-fold (95% CI 1.82–6.44, p < 0.0005) and live birth rate by 6.16-fold (95% CI 2.83–13.39 p < 0.0005). When the data were scrutinized based on female age, the authors noticed that the effect of GH was mainly related to patient’s age. Whereas between 35 and 40 years, it was 4.50 more likely to get pregnant in GH cycles, it did not have a significant effect on the chance of clinical pregnancy in those aged <35 years or ≥40 years.

In a recent meta-analysis including only randomized controlled trials (RCTs), although none of the seven studies showed improvement in clinical pregnancy rates, clinical pregnancy and live birth rates were found to significantly increase (OR 2.13; 95% CI 1.06–4.28 and OR 2.96; 95% CI 1.17–7.52, respectively) after analyzing pooled data [5]. In another meta-analysis including all types of trials (n = 12 studies), although authors presented significantly higher numbers of retrieved oocytes (OR 1.94; 95% CI 1.19–2.69) and obtained more available embryos (OR 1.72; 95% CI 1.13–2.31) under the GH therapy, clinical pregnancy and live birth rates were similar with or without GH treatment [12]. The subgroup analysis indicated that clinical pregnancy and live birth rates were significantly increased when GH was co-treated with gonadotropin; however, there were no significant differences found as for the clinical pregnancy and live birth rates when it was supplemented during the preceding luteal phase of the cycle [6].

To sum up, considering all the studies performed to date, there is some evidence that supports an improvement with regard to the number of oocytes retrieved and clinical pregnancy/live birth rates when GH supplementation was preferred. However, for more definitive results, further studies with larger sample size are needed to confirm the effects of GH supplementation and to define the optimal dose and schema in women with POR undergoing ART.

5.2.2 Dehydroepiandrosterone

Dehydroepiandrosterone (DHEA) is an endogenous steroid and originates from the adrenal zona reticularis (%85) and ovarian theca cells (%15) [7]. It improves steroidogenesis as a precursor for estradiol and testosterone and enhances follicular development [8]. It can also boost the level of IGF-1, which in turn promotes folliculogenesis by enhancing the effect of gonadotropin and reducing follicular arrest [9]. Furthermore, DHEA might decrease the level of hypoxic inducible factor-1 when compared with the controls (0.50 ± 0.52 vs. 0.08 ± 0.29, p = 0.018), which has crucial role in immunological responses, homeostasis, vascularization, and anaerobic metabolism [10].

In spite of preclinical data supporting its utilization, the clinical relevance during OS in women with POR or DOR undergoing in vitro fertilization (IVF) cycle is still controversial. In a recent meta-analysis including 5 studies with a total of 910 patients, although the use of DHEA did not change the number of retrieved oocytes, there was a higher likelihood of pregnancy (OR 1.8, 95% CI 1.29–2.51) [11]. Notably, DHEA was used for 12 weeks with a dose of 75 mg/day before OS in most of the studies. When the authors analyzed the association between DHEA use and the likelihood of abortion, they reported low heterogeneity between studies (I2 = 0.0%), and the use of DHEA was associated with a significant reduction in the likelihood of abortion (OR 0.25, CI 0.07–0.95; p = 0.045) [11].

Unlike other studies, Chern et al. investigated the effect of DHEA whose DHEA-sulphate level was less than 180 μg/dL [12] among poor ovarian responders. The authors reported that DHEA therapy might be beneficial in women with lower DHEA-sulphate level and raise the possibility of attaining more than three oocytes [12]. Another recent prospective case-control study [13] revealed that there were statistically significant differences between the groups with and without DHEA supplementation for oocyte yield (6.35 ± 2.41 vs. 3.98 ± 3.2), Grade I embryos generated (55% vs. 30%), positive pregnancy rate (21/34 vs. 10/28), and live birth rate (18/34 vs. 4/28). The beneficial effect was obvious especially in women <30 years old [13].

In conclusion, one should assume that DHEA treatment might enhance clinical pregnancy and live birth rates according to limited data. However, lack of large-scale RCTs, unknown long-term health risks, and cosmetic concerns are the main factors that limit its utilization in clinical practice.

5.2.3 Transdermal Testosterone

The rationale behind the testosterone intervention is based on the fact that androgens appear to play an important role in early follicular development and granulosa cell proliferation as well as in increasing the number of small antral follicles [14]. In a meta-analysis published in 2012, three RCTs were included to evaluate the effects of transdermal testosterone on ovarian stimulation outcomes in women with POR [15]. When a total of 113 women treated with transdermal testosterone were compared with a total of 112 controls, both clinical pregnancy (RR 2.07, 95% CI 1.13–3.78) and live birth (RR, 1.91, 95% CI 1.01–3.63) rates were significantly improved. However, a following RCT including 50 women with POR failed to note any significant difference with regard to live birth rate when patients were stratified to transdermal testosterone pretreated and no pretreatment groups (7.7% versus 8.3%, respectively) [16].

In summary, although available data support utilization of testosterone during OS, more research with larger sample size is needed in the future to confirm the efficacy and optimal treatment schema in women with POR.

5.2.4 Letrozole

Letrozole is a nonsteroidal competitive inhibitor of the aromatase enzyme system and inhibits the conversion of androgens to estrogens. It is commonly used in women with polycystic ovary syndrome for ovulation induction and in women with hormone-dependent cancers such as breast cancer to decrease the blood level of estrogen during ovarian stimulation for fertility preservation [17, 18]. The biological rationale for the possible augmentative benefit of aromatase inhibitors in ovarian stimulation is the positive effect of intraovarian androgens on development of small follicles and proliferation of granulosa and theca cells to augment follicular sensitivity to FSH [19].

Initial attempt to evaluate the efficacy of letrozole was done by comparing micro-dose flare-up (MF) protocol. In a retrospective case-control study including 1383 consecutive cycles predicted to have or with a history of poor ovarian response, MF protocol was used in 673 patients (1026 cycles), and the antagonist/letrozole (AL) protocol was used in the remaining 212 patients (357 cycles) [20]. Whereas the total gonadotropin consumption, duration of stimulation, estradiol level on the day of hCG administration, number of oocytes retrieved were significantly lower, and the rate of at least one top-quality embryo transferred were higher with the AL protocol, the clinical pregnancy rates were comparable between the two groups [20].

Other than case-control studies, a systematic review has been published recently including 4 studies and 223 women. The authors reported that letrozole supplementation does not improve the number of oocytes retrieved or clinical pregnancy rates (OR 1.28; 95% CI 0.60–2.73) [5]. In a following RCT [21], which had not been included in previous meta-analysis, the authors aimed to investigate whether IVF outcomes would differ between patients with POR according to Bologna criteria who received three different gonadotropin doses with or without the addition of letrozole during ovulation stimulation. Whereas 31 patients were treated with 450 IU gonadotropins, another cohort of 31 women was treated with 300 IU gonadotropins. The last group comprising 33 patients was treated with 150 IU gonadotropins in combination with letrozole. The authors reported that the total dose of gonadotropin was significantly less when letrozole was supplemented, but number of retrieved oocytes, implantation, and ongoing pregnancy rates were comparable among all groups [21]. It is not clear if mild stimulation group benefited from letrozole or the low-dose gonadotropin use per se to have comparable outcomes to high-dose gonadotropin groups. The data strongly suggested that increasing the gonadotropin doses in women with POR did not improve the outcome -in assisted reproductive technologies.

5.2.5 Luteinizing Hormone

Luteinizing hormone (LH) has crucial role during the early folliculogenesis by increasing FSH-receptor expression in granulosa cells, acting synergistically with IGF-1, and increasing recruitment of preantral and antral follicles. During the late follicular phase, its function is mandatory for optimization of steroidogenesis, regulation of final folliculogenesis, and oocyte maturation.

Based on theoretical advantages, there have been various conditions in which LH supplementation was tested. In patients with hypo-response, as defined by unexpected poor ovarian response in spite of apparently normal ovarian reserve, LH supplementation from stimulation days 7–10 might be more efficient with regard to number of oocytes retrieved, implantation and pregnancy rates when compared with increasing FSH dose only [22–24]. In a randomized, open-label, controlled trial performed in two age subgroups, impact of LH administration on GnRH antagonist cycles was evaluated [25]. In women ≤35 years old, they were randomized either FSH (225 IU/day) or FSH (150 IU/day) + LH (75 IU/day) arms [25]. With a similar design, women with an age of 36–39 years old were randomized as (300 IU/day) or FSH (225 IU/day) + LH (75 IU/day) arms. Whereas there was no significant difference between the two protocols in terms of implantation and pregnancy rates in women ≤35 years old, there was statistically higher implantation and ongoing pregnancy rate per cycle in LH supplemented group for women 36–39 years old [25]. Besides those particular cohorts of patients, unfortunately, available data (n = 7 studies) does not support adding LH to FSH with the aim of improving success rates in patients with POR or DOR when compared with women treated with FSH alone [26].

5.2.6 Melatonin

Melatonin is a hormone secreted by the pineal gland that aids sleep process as well as serves as an antioxidant [27]. It was reported that melatonin levels are higher in follicular fluid of preovulatory follicles suggesting that it has a potential role in follicular development and maturation [28]. Melatonin levels of the patients were also positively correlated with antral follicle count and serum anti-Müllerian hormone concentrations [29].

Although there are several studies aiming to assess the role of melatonin treatment in animal models, there is paucity of data for its role in women with POR or DOR during an IVF cycle. In a double-blinded RCT [28], the authors investigated the effect of melatonin on the outcome of ART cycles in women with diminished ovarian reserve by administering 3 mg daily from the fifth day of the preceding cycle before OS. Although higher serum estradiol levels on the trigger day and better-quality embryos were obtained compared to controls, overall pregnancy rate did not differ from the control group [28]. In spite of those encouraging results, we need more data to obtain conclusive results for the efficacy of melatonin supplementation.

5.3.1 Coenzyme Q10

The underlying molecular mechanisms of ovarian aging are not fully understood; however, one of the hypothesis is a breakage of DNA strand due to increased oxidative stress [30]. Since mitochondria and nucleus have their own DNA, the accumulation of reactive oxygen species may lead to oxidative damage in both of them. In this respect, coenzyme Q10 (CoQ10) might be a potential source of adjuvant to enhance OS outcome and pregnancy rate in patients with diminished ovarian reserve given to its antioxidant feature. Together with its antioxidative property, CoQ10 also plays a role in the production of cellular energy and adenosine triphosphate.

CoQ10 supplementation was previously tested in normal-responder women to improve cycle outcomes. The available data suggested that CoQ10 concentration within the follicular fluid was positively correlated with embryo quality and pregnancy rate [31, 32]. However, particularly for low-prognosis young women, there is only one RCT investigating the effect of CoQ10 on ovarian response and embryo quality [33]. In this trial, a total of 186 consecutive young patients (age < 35) with low ovarian reserve parameters were randomized to CoQ10 pretreatment for 60 days before IVF cycle or to no pretreatment arm. Although baseline demographic and clinical characteristics were comparable between the two groups, CoQ10 pretreatment arm required significantly lower gonadotropin dose with gained increased number of oocytes and high-quality embryos [33]. Although there was numerically higher clinical pregnancy and live births per embryo transfer and initiated cycle in favor of CoQ10 group, it did not reach statistical significance [33].

Although CoQ10 appears to be a promising supplement particularly for young women but with low ovarian reserve markers, further research is required with larger sample size to conclude its efficacy. Timing, duration, and dose of CoQ10 also need attention to be defined for future investigations.

5.3.2 Vitamin D

Previous studies demonstrated that vitamin D might play a role in ovarian steroidogenesis, but the mechanism underlying the relationship between deficiency and reproduction is still unclear [34]. Those assumptions are mainly due to the fact that gonadal function may be influenced when there is obvious vitamin D deficiency, as observed by the expression of vitamin D receptor mRNA in human ovaries, mixed ovarian cell cultures, and granulosa cell cultures [35]. Although there is no study showing the effects of vitamin D supplementation on ovarian stimulation outcomes particularly in women with POR, the association between vitamin D and ovarian reserve was investigated in some studies with conflicting results. According to the largest cross-sectional study including 388 premenopausal women with regular menstrual cycles, a relationship exists between circulating 25-hydroxyvitamin D (25 OH-D) and AMH in women ≥40 years old. The authors emphasized that 25 OH-D deficiency might be associated with lower ovarian reserve in late reproductive-aged women [36]. In contrast, a prospective cross-sectional study including 283 consecutive infertile women younger than 42 years old revealed that the mean AMH (3.9 ± 3.8 ng/mL vs. 4.3 ± 4.8 ng/mL) and AFC (13.9 ± 13.3 vs. 12.7 ± 11.4) levels did not differ significantly between patients with 25 OH-D deficiency or not [35]. In multiple linear regression analysis, after adjusting for potential confounders (age, body mass index, smoking status, infertility cause, and season of blood sampling), the regression slope in all participants for total 25 OH-D predicting log10 AMH was 0.006 (standard error = 0.07, P = 0.9).

To sum up, we can underline the need for further research for the possible effect of vitamin D on ovarian reserve and fertility not only for poor responders but also for the whole infertile population.

5.3.3 Weight Loss and IVF

Understanding the correlation between female body mass index (BMI) and IVF outcomes is very important since approximately half of reproductive-aged women in the USA is overweight [37]. In the context of Nurses’ Health Study-3 (2010–2014), in which 1950 women has been prospectively followed and attempting pregnancy, the authors tested whether a weight change since age 18 years, current BMI, and BMI at age 18 years were associated with fecundity [38]. The authors noticed that for every 5-kg increase in body weight from age 18 years, current duration of pregnancy attempt increased by 5% (95% CI, 3–7%). Compared with women who maintained weight, the adjusted median current duration was 0.5 months shorter in those who lost weight, 0.3 months longer for those who gained 4–9.9 kg and 10–19.9 kg, and 1.4 months longer for those who gained 20 kg or more (p trend <0.001). The adjusted time ratio (95% CI) for a 5-kg/m² increase in current BMI was 1.08 (95% CI, 1.04–1.12). Overall, gaining weight in adulthood, being overweight or obese in adulthood, and being underweight at age 18 years were associated with a modest reduction in fecundity [38].

Other than natural conception, studies investigating the potential impact of elevated BMI on fertility treatment outcomes are conflicting. Although some studies are reporting no significant adverse effect of elevated BMI on IVF outcomes [39, 40], there are other studies associating elevated BMI with fewer retrieved oocytes and reduced rates of pregnancy and live birth [41]. In the most recent study, from 2018, 51,198 women who initiated their first autologous IVF cycle in 13 different fertility centers were examined for the impact of BMI on IVF cycle outcomes [42]. Women who are overweight (BMI = 25–29.9 kg/m²) or obese (BMI >30 kg/m²) experienced greater odds of cycle cancellation, fewer retrieved oocytes, fewer usable embryos, and lower odds of ongoing clinical pregnancy. In concordance, a meta-analysis including 33 studies including 47,967 treatment cycles [43], overweight or obese women undergoing IVF were observed to have a lower relative risk of clinical pregnancy (RR = 0.90, P < 0.0001) and live birth (RR = 0.84, P = 0.0002), with a higher miscarriage rate (RR = 1.31, P < 0.0001).

Although there is enough number of studies to conclude against obesity with respect to IVF outcome, it is interesting to note that there are no conclusive data that indicate if weight reduction can rectify success rates. In a multicenter randomized controlled trial including 317 women, intensive weight reduction prior to IVF revealed that the live birth rate was 29.6% (45/152) in the weight reduction and IVF group and 27.5% (42/153) in the IVF-only group [44]. The difference was not statistically significant (difference 2.2%, 95% CI, 12.9 to −8.6, P = 0.77). The mean (SD) weight change was −9.44 (6.57) kg in the weight reduction and IVF group as compared to +1.19 (1.95) kg in the IVF-only group, being highly significant (p < 0.0001). Notably, significantly more live births were achieved through spontaneous pregnancies in the weight reduction and IVF group, at 10.5%, as compared to the IVF-only group, at 2.6% (P = 0.009). Miscarriage rates and gonadotropin dose used for IVF stimulation did not differ between groups (29.6% vs. 27.5%) [44].

Since there is no direct assessment of patients with high BMI and POR, we can assume that obesity might further decrease the chance of live birth based on available studies conducted among the whole population. However, as is in the case of whole population, there are no data suggesting that losing weight during or immediately before IVF makes any benefit with regard to live birth rate beyond some improvements on ovarian stimulation parameters.

5.3.4 Dietary Context and IVF

The dietary context and its effect on fertility treatment have been a matter of debate between physicians and patients. However, there are only a few prospective and longitudinal studies looking into this issue. In the context of EARTH (Environment and Reproductive Health) study, which is an ongoing prospective cohort started in 2006 aiming at identifying determinants of fertility, the effect of pretreatment whole-grain intake on IVF outcomes were assessed [45]. The authors reported that higher pretreatment whole-grain intake was associated with higher probability implantation and live birth. The adjusted percentage of cycles resulting in live birth for women in the highest quartile of whole-grain intake (>52.4 g/day) was 53% (95% CI, 41–65%) compared with 35% (95% CI, 25–46%) for women in the lowest quartile (<21.4 g/day) [45].

Other than grains, EARTH study group has also investigated association between protein intake and fertility. In two consecutive studies, the authors reported that whereas women with higher pretreatment intakes of fish had a higher probability of live birth following an ART cycle [46], a higher dairy protein intake (≥5.24% of energy) was associated with lower AFC among women presenting for infertility treatment [47]. The authors also confirmed that serum polyunsaturated fatty acids concentrations, including omega-3 but not omega-6 were positively associated with probability of live birth among women undergoing ART [48]. For the folate and vitamin B-12 intake, the authors noticed that women in the highest quartile of serum folate (>26.3 ng/mL) had 1.62 (95% CI, 0.99–2.65) times the probability of live birth compared with women in the lowest quartile (<16.6 ng/mL) [49]. Women in the highest quartile of serum vitamin B-12 (>701 pg/mL) had 2.04 (95%; CI, 1.14–3.62) times the probability of live birth compared with women in the lowest quartile (<439 pg/mL). Of interest, women with serum folate and vitamin B-12 concentrations greater than the median had 1.92 (95% CI, 1.12–3.29) times the probability of live birth compared with women with folate and vitamin B-12 concentrations less than or equal to the median. This translated into an adjusted difference in live birth rates of 26% (95% CI, 10–48%; p = 0.02) [49].

The optimal dietary setting that should be warranted during fertility treatment needs further evaluation with RCTs, because available studies suggesting positive effects of various products on treatment outcome have not been validated. Until that time, specifically for patients with POR and/or DOR, physicians might offer general recommendations for the ingredients of the diet based on aforementioned studies and findings.