Assisted reproductive technology (ART) incorporating in vitro fertilization (IVF) are key strategies to increase the possibility of conceiving for individuals who have experienced difficulties with natural conception. As a result of socioeconomic factors in both Western and Asian cultures, there is an increasing propensity for males and females to delay childbirth until an older age. Due to the natural process of decreased fertility with age, this has meant that increasing numbers of patients are seeking support from fertility specialists. In addition, with the global increase in endocrine disorders including obesity and metabolic syndrome, along with other ovarian pathologies that affect fertility, for instance endometriosis, adenomyosis and leiomyomata, patient management is challenged over and above the impact of increasing age. Historically, a subgroup of patients defined as poor prognosis according to the Bologna criteria (1) have proved very difficult to treat and have poor outcomes. They possess a combination of characteristics such as advanced maternal age (40 years and above), poor response to standard gonadotropin stimulation (less than four oocytes on collection), and/or a minimal ovarian reserve, as evident by a low anti-Mullerian hormone (AMH) level (less than 0.5–1.1 ng/ml; 3.7–8.1 nmol/l) or low antral follicle count (AFC; less than 5–7 follicles). However, these criteria do not include patients who produce poor quality embryos for unknown reasons and are equally considered poor prognosis. Nonetheless, ART clinics are faced with an increased demand for improved treatment, and one mechanism to address this, but which has proved highly controversial, has been to include off-label adjuvant therapy as a part of IVF management.

Two of the most common IVF adjuvant therapies are growth hormone (GH) and dehydroepiandrosterone (DHEA) supplementation (2). However, as a consequence of various investigations that have been either poorly designed or utilized small patient cohorts, along with a lack of sufficiently powered randomized controlled trials (RCTs) in this area, the true beneficial effects of GH and DHEA are actively debated and still remain clinically unclear (2). GH is an anabolic peptide hormone that functions to increase cell proliferation and growth, and it has been demonstrated to improve the sensitivity to gonadotropin stimulation and boost oocyte yield in several IVF studies (3–9). Recently, we (10) along with others (9, 11–15) found that GH promoted pregnancy rate and/or live birth chance by reducing miscarriage rates (13, 16, 17). It is hypothesized that GH reduces aneuploidies by enhancing embryo quality, and some evidence indicated that it may have a role in this respect (14), but the molecular mechanism is not understood. On the other hand, numerous other studies (5, 18–23), along with some meta-analyses and systematic reviews (7, 8), have demonstrated that GH has no clear positive effect on pregnancy or live birth outcomes in IVF patients. Similarly, DHEA, a multifunctional adrenal prohormone that acts as a precursor for testosterone and estradiol synthesis (24), has also been reported to promote enhanced pregnancy and live birth rates in poor prognosis IVF patients (25–34). In addition, DHEA has been suggested to increase fertilization rates and embryo quality (27, 32, 35, 36). Equally, other investigations including meta-analyses (15, 37–39) have demonstrated no clear benefit in terms of oocytes collected, embryo quality, or clinical pregnancy and live birth rates (30, 40–43). Thus, the use of these adjuvant therapies in IVF remains highly controversial and inconclusive (2).

These hormones are believed to modulate ovarian physiology, including oocyte and follicle maturation, and could have local effects on the endometrium during ovulation and implantation (37, 38, 44). To date, very little research, RCT or observational, has investigated the synergistic effects of GH and DHEA in combination in the IVF setting (45). Following on from our previous 2017 retrospective investigation where we found that GH in isolation enhanced clinical outcomes (10), we aimed to determine whether the addition of DHEA to treatment cycles resulted in any further beneficial outcomes. Consequently, in this study, we examined the effect of (+)GH alone, (+)DHEA alone, and (+)GH–DHEA in combination on poor prognosis IVF patients and primary outcomes included pregnancy and live birth rates. As a secondary aim, due to the controversy surrounding the use of IVF adjuvants and the definition of poor prognosis patients, we intended to identify significant variables that should be considered when designing future prospective studies in this research area. Therefore, we included a broader definition of “poor prognosis” IVF patients, which extended the Bologna criteria to those with repetitive implantation failure (RIF) and those who generated lower quality embryos.

This retrospective study echoed our previous study (10) and showed that patient age, the quality of transferred embryos, and the utilization of GH (with or without DHEA) were significant predictors of clinical pregnancy and live birth rates in IVF patients categorized as poor prognosis. Importantly, following on from our previous study which focused solely on GH (10), the current investigation demonstrated that DHEA administration alone did not significantly influence primary outcomes in comparison to (−)Adj, and it did not alter nor enhance the positive effects from GH therapy when administered in combination. Generally, addition of DHEA to GH cycles led to a decrease in ORs, but the rates and ORs were still more beneficial in comparison to (−)Adj or (+)DHEA therapy alone. Furthermore, DHEA did not affect other secondary parameters such as number of oocytes retrieved or fertilization rates as previously reported (27, 32, 35, 36). Contrary to other reports (25–34), these findings question the use of DHEA as an adjuvant in IVF, but reiterate the potential beneficial effects of GH.

While we acknowledge that the (+)DHEA alone group contained the smallest number of cases, the evidence suggested that DHEA did not convey any beneficial effect as an IVF adjuvant, including with GH, and these data build upon the only other report to investigate the impact of DHEA and GH combinations in IVF (45). In their study of 85 women receiving 183 IVF cycles, Haydardedeoğlu et al. demonstrated that DHEA for 12 weeks, with at least 4 weeks of transdermal testosterone and a late-luteal administration of GH in the previous cycle prior to stimulation, resulted in greater clinical pregnancy and live birth rates, along with more follicles, oocytes retrieved, and higher fertilization rate. There was no difference reported in the morphological quality of developing embryos (45). In our analysis, we also observed differences in pregnancy and live birth rates (45). We also found no significant change in the number of oocytes retrieved nor the fertilization rate. The number of zygotes with two pronuclei developing was lower with (+)GH–DHEA, but not significant, although it may reflect the significantly increased age of the women undertaking these combination adjuvant cycles in our study. Similar to the aforementioned study, we also observed no significant difference in embryo quality as assessed by the morphological analysis. However, they used a complicated treatment strategy that involved three different adjuvant agents, and the treatment group also consisted of only 37 cycles (45). Therefore, our combination study may indeed be considered superior to the study by Haydardedeoğlu et al., until the data become available for the combined GH–DHEA trial NCT02766764, which is currently recruiting.

Importantly, the (+)GH results in this study also reflect our earlier work (13) and comparable data derived from an RCT by Tesarik et al. (16), which suggested that GH lowered miscarriage rate leading to more live births. There are some notable differences between the studies such as patients in the study by Tesarik et al. being older with a mean age of 42 years and the researchers transferring three to four embryos. In terms of the ORs for clinical pregnancy and live births in this study, similar ORs were observed in several meta-analyses (12, 15, 17). However, other recent meta-analyses indicated that GH had no significant influence on live birth, with lower ORs (7, 8), but a slight increase in pregnancy rate (8).

The main aim of this study is to retrospectively examine in one of the largest adjuvant cohorts, the impact of adjuvant therapy on clinical IVF outcomes. Another crucial aim is to identify critical confounding variables and participant inclusion characteristics outside of the Bologna criteria that could be considered when designing future IVF adjuvant RCTs. The most important confounding variables we identified were patient age and quality of the transferred embryo, which is supported by our previous report (10). In this analysis, we showed that additional patient characteristics such as AMH, BMI, number of infertility factors, previous IVF attempts, or mean number of embryos transferred did not have an independent effect on clinical pregnancy or live birth chance and may be related to our specific rFSH dosing algorithms (48, 54).

The patients in our analysis were categorized as poor prognosis, with low AFC (≤8 follicles) and significantly reduced serum AMH level. Those treated with (+)GH, (+)DHEA, and (+)GH–DHEA were older on average with a lower AMH in comparison to those (−)Adj. In spite of this perceived very poor ovarian reserve and advanced maternal age, we showed that when (+)GH was added to cycles, with or without DHEA, the oocyte and embryo utilization rates, along with pregnancy and live births rates, were greater. We also investigated the effect of (+)GH on patients with different AFC gradings, but neither AFC nor the presence or absence of GH or DHEA significantly altered clinical pregnancy or live birth chance in different AFC groupings. Notably, because patient ovarian reserve has not been described in any other IVF study utilizing GH (17), direct comparison of our findings is restricted. However, AFC as a specific marker of ovarian reserve was insignificant following adjustment for patient age. In relation to age, we did not observe any benefit in patients older than 41 years. Taken together, these findings have implications for these specific parameters within the Bologna criteria. Therefore, newer adjuvant studies should probably focus on women 40 years or below, who generate lower quality embryos with standard stimulation and with RIF, rather than those defined by the Bologna criteria.

There are significant strengths associated with this study including the use of a large data set (n = 626 cycles), a low number of transferred embryos per cycle (average 1.5/cycle), and incorporation of several potential confounding variables such as age, AFC, AMH, and transferred embryo quality. However, like all retrospective analyses and most GH and DHEA studies in the IVF research space, it has significant limitations that should be cautiously considered when interpreting the findings. The potential positive effects of GH and the disappointing effects of DHEA are strictly associative rather than causative, as this study was not designed as an intervention RCT. In addition, given the retrospective design, the study is also subject to significant patient selection bias and the process by which the control group [i.e., (−)Adj] was selected must be carefully understood. Justifiably, it was decided to generate this group using treatment cycles free from adjuvant intervention, but to only include these adjuvant-free cycles from women who received adjuvant therapy during their entire treatment history. This meant that the group comprised only women considered poor prognosis, being offered adjuvant therapy due to one or more of the five reasons outlined in Section “Materials and Methods.” In an attempt to randomize cases and to counter any other perceived selection bias, we chose to analyze only the very first IVF cycle with fresh embryo transfer for each women during the study period, regardless of whether there was an adjuvant intervention. This process also prevented any confounding that may be interpreted from including multiple treatment cycles for individual women.

In addition, there is a significant heterogeneity in terms of poor prognosis factors and combination thereof in the adjuvant treatment groups. While the adjuvant groups, especially GH, tended to have a higher proportion of patients with these factors, the heterogeneity is an important limitation of this study. Future studies must focus on more homogenous groups, but as indicated here, these could incorporate factors outside of the traditional Bologna criteria and possibly have a more nuanced approach like that of the POSEIDON study (55). Other factors that may be important but not considered in the current analysis was patient socioeconomic status and parity. Socioeconomic status and affordability could be critical confounders as patients were required to pay for IVF adjuvants. Taken together, while not as robustly designed and as powerful as a prospective RCT, the current study design limited any perceived bias and is one of the largest GH studies in IVF research to date.

Overall, we have shown for the first time in a large data set, and adjusting for various confounding variables, that (+)GH alone or in combination with DHEA, significantly increased the chance of clinical pregnancy and live birth outcomes in poor prognosis IVF patients, who tended to be significantly older (1.5–2.0 years) and had a lower average serum AMH value. This new retrospective adjuvant IVF study with GH and DHEA alone and in combination is the first to include aspects of analysis such as AFC, AMH, BMI, and embryo quality assessment and has provided further evidence to indicate the potential beneficial effects of GH supplementation in IVF treatment. It also demonstrated that DHEA did not appear to have a significant influence on primary IVF outcomes. Although the study has certain limitations in that it is observational and retrospective in nature, bias was minimized as best as possible, and the data suggested that GH supplementation may provide more live births, mainly in younger women and questions the use of adjuvant therapy in women older than 41 years. Finally, the data indicate that future GH and DHEA IVF RCTs should strictly account for transferred embryo quality and expectedly age, while possibly focusing on younger women with RIF or those generating a greater proportion of low-quality embryos.

Our clinic is accredited with the Reproductive Technology Accreditation Committee, a National body, as well as the Reproductive Technology Council of Western Australia, a State body. These agencies monitor all activities according to respective Codes of Practice. Specific ethics approval was not required for this study as all procedures and blood tests were embraced by routine approved clinical protocols. However, retrospective analysis and reporting of the data was approved under Curtin University Ethics Committee approval no. RD_25-10. In addition, as a part of our documentation system, written informed-consent was obtained from each participant who accepted the use of adjuvants, and they were required to pay for these adjuvants over and above the IVF treatment charges.

The present work was designed by JY, KK, and SD. Data extraction and analysis were performed by KK, PH, SD, and JY. Patient recruitment was undertaken by PR, GB, and SS. The initial manuscript draft was prepared by KK and subsequently revised by JY, SD, and PH. All the authors approved the final submitted version.

All authors have nothing to disclose and approve the submitted copy of the manuscript. KK is employed as a Research Fellow at Curtin University and a portion of this position is financially supported by PIVET Medical Centre. PH, PR, GB, and SS provide consultancy services to PIVET Medical Centre. SD is a Curtin University Professor and an adjunct Research Consultant at PIVET Medical Centre. JY is the salaried Medical Director of PIVET Medical Centre and an adjunct Clinical Professor at Curtin University.