The effect of growth hormone on the euploid rate of blastocysts in patients with advanced maternal age: study protocol for a single-blinded randomized controlled trial.

This randomized controlled trial aims to evaluate the effect of GH supplementation on the euploidy rate of blastocysts in AMA patients. This is a prospective, single-blinded, single-center, open-labeled randomized study carried out in the Shanghai Ji Ai Genetics and IVF Institute. The flow chart of this study is shown in Fig.  1 . Reporting of the study results will follow the 2010 revised Consolidated Standards of Reporting Trials statement [ 20 ] and the updated guidelines, 2012 [ 21 ]. Fig. 1 Flowchart of the study. PGT-A Preimplantation genetic testing for aneuploidy, GH Growth hormone, ICSI Preimplantation genetic testing, ITT Intention to treat, PP Per protocol TLM Flowchart of the study. PGT-A Preimplantation genetic testing for aneuploidy, GH Growth hormone, ICSI Preimplantation genetic testing, ITT Intention to treat, PP Per protocol TLM The first patient was enrolled on August 1, 2022. The study is currently in the recruitment phase, and the estimated study completion date is December 31, 2026. All women aged ≥ 38 years with tubal infertility for assisted reproduction treatment at our hospital will receive genetic counseling by a genetic specialist due to advanced maternal age and voluntarily choose to undergo conventional IVF or PGT-A. Patients who intended for PGT-A will be recruited for this study if they fulfill the inclusion criteria and do not meet any exclusion criteria. The eligible women will be randomly assigned to either the GH treatment group or the control group after detailed explanations and signing of the informed consent form. Women aged ≥ 38 years undergoing PGT-A. BMI in the normal range (18.50–24.0 kg/m 2 ). Normal semen analysis for the male partner. Women aged ≥ 38 years undergoing PGT-A. BMI in the normal range (18.50–24.0 kg/m 2 ). Normal semen analysis for the male partner. Patients with endometriosis grade 3 or higher, untreated hydrosalpinx. Women with a uterine cavity abnormality, such as a uterine congenital malformation (uterus uni-cornate, bicornate, or duplex), untreated uterine septum, adenomyosis, submucous myoma, or endometrial polyp(s). Women who are indicated for PGT-SR or PGT-M. History of endocrine disorder, autoimmune diseases, or diagnosed thrombophilia. History of GH supplementation in the previous IVF treatment or taking other supplementary drugs used during stimulation; patients with absolute or relative contraindications to GH treatment, including active malignancy or history of cancer, diabetic retinopathy, diabetes mellitus, and chronic kidney disease. Patients with endometriosis grade 3 or higher, untreated hydrosalpinx. Women with a uterine cavity abnormality, such as a uterine congenital malformation (uterus uni-cornate, bicornate, or duplex), untreated uterine septum, adenomyosis, submucous myoma, or endometrial polyp(s). Women who are indicated for PGT-SR or PGT-M. History of endocrine disorder, autoimmune diseases, or diagnosed thrombophilia. History of GH supplementation in the previous IVF treatment or taking other supplementary drugs used during stimulation; patients with absolute or relative contraindications to GH treatment, including active malignancy or history of cancer, diabetic retinopathy, diabetes mellitus, and chronic kidney disease. Participants will be clearly informed by investigators at enrollment that they may withdraw from this RCT at any period without giving any reason and that withdrawal will not lead to any impact on the medical care they are receiving. The study procedure is described by following the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) checklist (supplemental material 1), and the participant timeline is shown in Fig.  2 . Fig. 2 SPIRIT schedule of enrolment, interventions, and assessments. PGT-A Preimplantation genetic testing for aneuploidy, ET Frozen embryo transfer, GH Growth hormone SPIRIT schedule of enrolment, interventions, and assessments. PGT-A Preimplantation genetic testing for aneuploidy, ET Frozen embryo transfer, GH Growth hormone The randomization will be performed by a study nurse who is not involved in the recruitment or clinical management of the participants. At the start of the study, the grouping results are generated using an online randomization program through the website https://www.randoms-online.com/ at a ratio of 1:1. The randomization results are stored in sequentially coded, sealed, and opaque envelopes prepared by the nurse. The couples will be given an envelope when they have signed the informed consent form and then allocated to the GH or the Control group according to the randomization results in the envelope. Given that growth hormone is delivered via subcutaneous injection, medical practitioners and patients will possess knowledge of the grouping information and the respective interventions involved. There will be no blinding of the treatment allocation to the doctors and participants in the study. To ensure the reality of this study, the embryologists performing the morphokinetic annotation and embryo quality evaluation, the genetic scientists performing PGT-A data analyses, and the independent statistician will be blinded to the group allocation. Conventional GnRH antagonist protocol for ovarian stimulation (OS) will be used for all patients either by using daily recombinant FSH (rFSH, Gonal F, Merck-Serono, Switzerland) or human menopausal gonadotropin (hMG, (human menopausal gonadotrophin, Lizhu Pharmaceutical Trading Co., China). Generally, rFSH or hMG will begin on day 2 or day 3 of the menstrual cycle if there are no ovarian cysts or follicles > 10 mm in diameter and serum oestradiol level is < 100pg/mL. The initial doses will be 225–300IU/day according to body mass index (BMI), number of antral follicles, and basal hormone levels. Gonadotrophin dosage adjustments will be based on the ovarian response at the discretion of the clinicians in charge. Women will receive an antagonist (cetrorelix 0.25 mg/day; Merck-Serono, Switzerland) once subcutaneously daily from day 6 of ovarian stimulation till the day of the ovulation trigger. Human chorionic gonadotrophin (hCG 2000 IU, Lizhu Pharmaceutical Trading Co., China) and GnRH agonist (decapeptyl 0.2 mg, Ferring Pharmaceuticals, Netherlands) will be given for triggering of final maturation when at least 3 follicles reach > 17 mm in diameter. Blood will be checked for serum oestradiol and progesterone levels on the day of ovulation trigger. Transvaginal-guided oocyte retrieval will be performed 36 h after the trigger. The retrieved cumulus-oocyte complexes will be placed immediately in a culture medium covered by lightweight paraffin oil and incubated in a humidified 37℃, 5%/6% CO2 incubator, and incubated for 2–4 h before injection. 2 IU recombinant human growth hormone (Jintropin, Changchun GeneScience Pharmaceutical Co. Ltd., China) will be injected subcutaneously daily from the second day of menstruation for the participants in the GH group. Antagonist treatment for ovarian stimulation begins on the 2–3 days of the next menstruation, and growth hormone will be continued until the day of egg retrieval, with a total duration of administration for about six weeks. Consistent with the risk minimization principle of the Helsinki Declaration, the control group will undergo the conventional antagonist protocol for ovarian stimulation as described above, without pretreatment injections as placebo. On the day of oocyte retrieval, semen samples will be collected via masturbation following a 3 to 7–day period of sexual abstinence. Preparation of the semen samples will be conducted using either the density gradient centrifugation method or the swim-up protocol. Fertilization will be achieved through ICSI, with the injection of a single spermatozoon occurring within four hours post-follicular aspiration. Zygotes will be incubated individually in a time-lapse incubator (EmbryoSlide, Vitrolife, Gothenburg, Sweden) utilizing G1 medium (days 0 to 3) and G2 medium (days 3 to 5 or 6) (Vitrolife), in an environment of 6% CO2 and 5% O2 at 37℃. On day 3, a laser incision will be made across the zona pellucida of each embryo (Hamilton-Thorne, USA), followed by a trophectoderm (TE) biopsy and cryopreservation at the blastocyst stage. Vitrification and warming of the blastocysts will be performed using a Cryotop device and vitrification kit (KITAZATO, Shizuoka, Japan). A series of images will be captured from seven focal planes at 15-minute intervals using a TLM machine (EmbryoScope, Vitrolife). Two trained embryologists, blinded to the ploidy status, will annotate precise kinetic timings, embryonic dysmorphisms, and irregular cleavage events via EmbryoViewer (Vitrolife). If discrepancies in timing exceed one hour, re-annotation will occur after discussion. The average morphokinetic timings will be employed for analysis. The following timings will be defined and measured post-mid-time of the ICSI microinjection procedure: time of second polar body emission (tPB2), appearance of two pronuclei (tPNa), pronuclei fading (tPNf), division time from two to eight blastomeres (t2 to t8), first fusion of two blastomere membranes (tC), transition to morula stage (tM), initiation of cavity formation (tSB), hatched blastocyst (tHB), duration of the third cell cycle (cc3 = t5 - t3), second synchrony (s2 = t4 - t3), and third synchrony (s3 = t8 - t5). Moreover, dysmorphisms will be assessed, including multinucleation at the 2-cell stage (MN2), fragmentation at the 2-cell stage (> 25% cytoplasmic fragments, Frag-2), and uneven cleavage at the 2-cell/4-cell stage (over 25% variation in blastomere size, uneven-2/uneven-4). Observations will also include direct cleavage (single blastomeres dividing from one to three cells within 5 h) and reverse cleavage (cleavage of reabsorbed blastomeres). The KIDScore is an AI-driven embryo scoring program that is generated through the EmbryoViewer software and relies on kinetic parameters associated with embryo implantation potential [ 22 ]. A kappa value that can test the inter-rater reliability of qualitative parameters is adopted for categorical variables. The kappa values for MN2, Frag-2, direct cleavage, uneven-2, uneven-4, and reverse cleavage will be 0.97, 0.91, 0.95, 0.84, 0.88, and 1.00, respectively. Blastocysts are evaluated according to Gardner’s classification system, which provides a comprehensive grading framework [ 23 ]. This grading system allocates three distinct quality scores to each blastocyst based on the following criteria: (1) the developmental stage of the blastocyst, encompassing its degree of expansion and hatching status; (2) the score assigned to the inner cell mass; and (3) the score of the trophectoderm. Blastocysts that receive a score of B or higher for either the inner cell mass or the trophectoderm are classified as viable for clinical use. Following our genetic laboratory protocols, biopsies were assessed for ploidy status utilizing the NGS platform [ 24 , 25 ]. Specifically, the whole genome DNA from the trophectoderm (TE) biopsies was amplified, randomly fragmented, and prepared into a library using the pre-implantation genetic screening for aneuploidy kit (Berry Genomics Corp., Beijing, China). The library was then purified, pooled, denatured, and sequenced on an Illumina NextSeq CN500. Sequencing reads were aligned to the human genome (hg19), with a size threshold of ≥ 3 Mb for identifying copy number variations (CNVs) and ≥ 5 Mb for mosaic CNVs. Only euploid embryos are recommended for transfer. The prioritization of embryo transfer will be primarily based on chromosomal status, followed by morphological grading. The timing of the transfer will be determined by the clinicians based on the participants’ conditions. Following national assisted reproductive technology (ART) practice guidelines, a single blastocyst will be transferred under ultrasound guidance using a soft embryo transfer catheter. Endometrial preparation may utilize either hormone replacement or natural cycles. A blastocyst transfer occurs when endometrial thickness reaches 8 mm following hormone replacement treatment. Luteal support is administered per the center’s standard operating procedures. In natural cycles, oral dydrogesterone (Duphaston) is prescribed at a dosage of 10 mg three times daily, commencing on the day of ovulation and continuing until serum hCG testing or menstruation. In hormone replacement cycles, vaginal progesterone gel (Crinone, 90 mg daily) or progesterone injections (40 mg daily) are used, in conjunction with oral dydrogesterone 20 mg daily, and maintained until serum hCG testing. Embryo implantation was defined as positive serum β-HCG levels 14 days after embryo transfer. Clinical pregnancy was defined as visualization of the gestational sac on ultrasonography at 7 weeks of pregnancy. Ongoing pregnancy was confirmed when a pulsating fetal pole was present at 12 weeks of gestation. Miscarriage was defined as invisualization of the gestational sac at 7 weeks of pregnancy or pregnancy loss after 7 weeks of pregnancy. At 12 weeks of gestation, first-trimester pregnancy complications (miscarriage, ectopic pregnancy, and gestational trophoblastic neoplasia) will be documented in the case report form (CRF) for the first pregnancy follow-up time point. Antenatal care will be referred for these women when the ongoing pregnancy is beyond 12 weeks. Informed consent for the collection of pregnancy and delivery data will be obtained from participants at the start of the study. Participants will be contacted post-delivery via phone to gather information on pregnancy outcomes, including delivery, miscarriage, week of gestation, newborn gender, number of infants born, birth weights, and any obstetric complications. The primary outcome is the euploidy rate of blastocysts. The secondary outcome indexes include pregnancy outcomes after transfer, as well as embryo developmental and morphokinetic parameters. Live birth rate of the first frozen embryo transfer: deliveries ≥22 weeks gestation with heartbeat and breath. Cumulative live birth rate: cumulative live birth of the euploid blastocysts obtained within 6 months of randomization. Ongoing pregnancy: a viable pregnancy beyond 12 weeks' gestation of the first frozen embryo transfer. Clinical pregnancy rate: the presence of intrauterine gestational sac by transvaginal ultrasound at 6 gestational weeks of the first frozen embryo transfer. Number of euploid blastocysts in an OS cycle. The probability of obtaining euploid blastocysts in an OS cycle. KIDScore of euploid embryos: an AI-driven embryo score generated through the EmbryoViewer software which relies on kinetic parameters and is associated with embryo implantation potential. Live birth rate of the first frozen embryo transfer: deliveries ≥22 weeks gestation with heartbeat and breath. Cumulative live birth rate: cumulative live birth of the euploid blastocysts obtained within 6 months of randomization. Ongoing pregnancy: a viable pregnancy beyond 12 weeks' gestation of the first frozen embryo transfer. Clinical pregnancy rate: the presence of intrauterine gestational sac by transvaginal ultrasound at 6 gestational weeks of the first frozen embryo transfer. Number of euploid blastocysts in an OS cycle. The probability of obtaining euploid blastocysts in an OS cycle. KIDScore of euploid embryos: an AI-driven embryo score generated through the EmbryoViewer software which relies on kinetic parameters and is associated with embryo implantation potential. Treatment-related data, including baseline information, details of ovarian hyperstimulation, as well as developmental and morphodynamic parameters of the embryos, will be collected on the day of embryo frozen. Results from PGT-A will be gathered one month following oocyte retrieval. Information regarding the frozen embryo transfer (FET) cycle will be documented on the day of the transfer. Follow-up data on all pregnancies resulting from FET will be monitored in accordance with the study protocol, continuing until a live birth occurs. Participant information forms will be developed for data entry. Quality control measures will be implemented at two levels: investigators will verify the accuracy of data entry, and an independent investigator will regularly oversee data monitoring and validation throughout the study. Furthermore, data will be backed up daily to an additional computer located in close proximity to the server. To enhance adherence to the intervention protocols, the investigators will maintain a proper scientific research attitude and be available to answer participants’ questions, which will help increase compliance. To protect participants’ confidentiality, their personal information will be removed during collection, sharing, and storage. All cycles of COH assigned to participants will be identified by a consistent patient identification number. According to previous research conducted at our hospital, the embryo euploidy rate for women with AMA is approximately 20%. Based on existing retrospective studies, including data from our center, a meaningful increase of 15% after growth hormone treatment has been identified [ 13 , 17 ]. This suggests that the embryo euploid rate could rise from 20% to 35%. For this study, we established a 1:1 inclusion ratio, with a power of 90%, significance level α = 0.05 and β = 0.1. Consequently, we calculated a required sample size of 181 participants for each group. To account for potential loss to follow-up, we expect to include a total of 400 participants in this study. Data will be analyzed using both intention-to-treat and per-protocol approaches. The demographic characteristics of the two groups will be compared. Continuous variables will be presented as mean ± SD for parametric data and as the median and interquartile range (IQR) for non-parametric data. Statistical comparisons will be conducted using Student’s t-test, Mann-Whitney U test, or χ 2 test as appropriate. All statistical analyses will be carried out using SPSS software version 26.0, and a p-value of less than 0.05 will be deemed statistically significant. No interim analysis will be conducted during the study period. GH is commonly used in patients undergoing IVF with diminished ovarian reserve or AMA. It has been shown to benefit ovarian stimulation and embryo transfer outcomes by recent studies, while the impact of GH on the ploidy status of the embryos from patients with AMA has not been proved by RCTs, and the mechanisms of the underlying effect of GH on IVF outcomes remain to be investigated. Meanwhile, there is currently no consensus on the timing and dosage of growth hormone therapy. Most of the studies in the past adopted short-term applications of GH for about 3–14 days or a single injection, with doses ranging from 1 to 12 IU daily to 12–18 IU every other day [ 26 , 27 ]. However, in theory, it takes about three months for presinus follicles to develop into mature follicles. A study by Yovich et al. found that serum levels of IGF-1, which is crucial for follicle growth and a marker of oocyte quality, increased significantly after four weeks of GH supplementation in women with a poor prognosis for IVF [ 28 ]. Therefore, most studies since 2017 suggest that GH should be administered for 6 weeks or more with a small daily dose of 1-4IU [ 29 – 31 ]. Based on the dosage and mode of supplementation used in previous studies, and with reference to the treatment experience of growth hormone deficiency in different age groups and IGF-1 levels, it is generally considered that 2 IU of growth hormone supplementation per day starting one menstrual cycle prior to ovulation is the most commonly used and cost-effective dose. In this RCT, the timing and dosage of growth hormone will be implemented accordingly. Regarding adverse events (AEs), the clinical practice guidelines for growth hormone (GH) treatment in adults and children indicate that most adverse effects are related to the metabolic impact of GH and are dose-dependent (32, 33). Since a low starting dose of 2 IU for a limited duration of 6 weeks is used, no adverse effects have been reported in previous studies involving GH use in patients with AMA undergoing IVF. To ensure the safety of the treatment, patients will be regularly monitored for glucose levels, thyroid function, and potential side effects (including edema, joint pain, and carpal tunnel syndrome) throughout the entire treatment and follow-up process according to these guidelines. Any discomfort or exacerbation of existing discomfort will be recorded, regardless of whether it is related to the study intervention. The decision to continue therapy or participate in a trial based on adverse events is at the discretion of the investigator and the participants involved. Neither participants nor the public was involved in the study design, development of the research question, or implementation of this study.

With the progressive delay in the age of marriage and childbearing among women in contemporary society, there has been a notable rise in the proportion of women with advanced maternal age (AMA) preparing for pregnancy [ 1 ]. This demographic shift is accompanied by a concomitant increase in the incidence of embryo aneuploidy, which significantly elevates the risk of adverse pregnancy outcomes, including infertility, miscarriage, and congenital anomalies, when compared to younger cohorts. Studies have indicated that women aged 24 to 35 years exhibit the highest rates of embryo euploidy, maintaining a stable level throughout this period [ 2 ]. After the age of 35, however, the likelihood of achieving euploid embryos declines linearly: it decreases to approximately 45% at age 35, less than 35% by age 38, and falls to below 20% for women aged 43 years and older [ 2 ]. In the context of assisted reproductive technology, the implementation of preimplantation genetic testing for aneuploidy (PGT-A) has been shown to enhance the pregnancy and live birth rates in women with AMA by facilitating the selection of euploid embryos for transfer [ 3 , 4 ]. This practice is supported by relevant clinical guidelines or expert consensus across Europe, America, and China, which designate women with AMA as appropriate candidates for PGT-A [ 5 – 7 ]. However, it is worth noting that PGT-A is only a screening technique, and it cannot fundamentally decrease the incidence of aneuploidy in oocytes or increase the possibility of obtaining euploid embryos in the population of women with AMA. Growth hormone (GH), a polypeptide hormone secreted by the pituitary gland, plays a critical role in promoting cell division and growth in vivo through two principal pathways: direct binding to specific receptors on target cells or by stimulating the hepatic secretion of insulin-like growth factor (IGF) [ 8 ]. Within the realm of assisted reproduction, a seminal study conducted by Weall et al. in 2015 for the first time identified the presence of growth hormone receptors on human oocytes, noting that women with AMA exhibited significantly lower receptor expression than their younger counterparts [ 9 ]. Meanwhile, the research team observed that GH receptor expression was upregulated on oocytes, leading to a substantial increase in the functionality of mitochondria, enhanced oocyte quality, and an elevated number of high-quality embryos following GH supplementation in the population of AMA patients [ 9 ]. Moreover, GH has demonstrated the ability to alleviate oxidative stress-induced mitochondrial damage in oocytes, counteract apoptosis by reversing the effects of FOS and JUN family proteins, and increase oocyte quantity [ 10 , 11 ]. GH also upregulates follicle-stimulating hormone (FSH) and luteinizing hormone (LH) receptor expression in granulosa cells, thereby enhancing ovarian response and bolstering clinical pregnancy and live birth rates [ 12 , 13 ]. A recent high-quality meta-analysis comprising 19 studies found that GH supplementation notably increased the number of retrieved oocytes, reduced gonadotropin doses, and improved the number of embryos available for transfer and the clinical pregnancy outcomes [ 14 ]. Nonetheless, conflicting evidence exists, as a randomized placebo-controlled trial by Norman et al. did not demonstrate increased live birth rates in patients with poor ovarian response, although the study failed to achieve the planned sample size for inclusion [ 15 ]. Additionally, although a meta-analysis by Akanksha et al. indicated a modest enhancement in live birth outcomes associated with GH supplementation (OR 1.77, 95% CI 1.17 to 2.70), the heterogeneity among studies and varied treatment regimens rendered these findings inconclusive [ 15 ]. In summary, existing studies indicated that GH supplementation may enhance the outcomes of women with AMA in ovarian stimulation, while their endpoints primarily focused on metrics such as the number of oocytes retrieved and embryos obtained, as well as the clinical pregnancy rate following transfer. It remains unclear whether GH supplementation has positively affected the rate of live births. Since the euploid status of an embryo is critical for achieving a healthy live birth, the primary aim of ovarian stimulation in this demographic is to maximize the production of euploid embryos. Up to now, only three published cohort studies have evaluated the effect of GH on embryo euploidy, all indicating that GH supplementation positively influences euploidy rates of embryos in women with AMA [ 13 , 16 , 17 ]. One of these studies came from our team, and we found that GH supplementation was associated with an improved probability of acquiring euploid embryos in women with AMA in a retrospective cohort study [ 17 ]. However, it is worth noting that there are no randomized clinical trials (RCT) proving the role of GH, making it challenging to definitively determine the impact of GH on PGT-A outcomes. In addition, it is noticed that even for euploid embryos, around 40% still fail to implant successfully [ 18 ], which raises concerns on how to further improve the developmental potential of euploid embryos with better implantation rate and live birth rate. Our team has developed an artificial intelligence prediction model utilizing time-lapse monitoring (TLM) in conjunction with clinical variables, demonstrating strong predictive capabilities for the developmental potential and implantation rates of aneuploid embryos [ 19 ]. This underscores the influence of morphodynamic indicators on embryo development success. However, existing studies on GH supplementation’s effects on embryo quality have predominantly relied on traditional morphological assessment criteria, and there remains a gap in research examining its impact on the dynamic processes of embryo development. This study protocol describes a prospective RCT examining the impact of GH supplementation on the euploid status of patients with AMA, as well as the morphokinetic characteristics and transfer outcomes of euploid embryos derived from this population. By integrating PGT-A and TLM during in vitro culture, this study will significantly contribute to our understanding of the mechanisms by which GH may enhance the reproductive outcome in women with AMA.

This study protocol outlines a well-designed RCT to evaluate the effect of GH supplementation on the euploidy status and morphokinetic characteristics of blastocysts in women with AMA, a population with significant clinical challenges in ART. By integrating advanced technologies of PGT-A and TLM, this study allows for a comprehensive understanding of the potential benefits of GH in improving embryo quality and reproductive outcomes in this population. If the findings reveal a positive effect of GH on euploidy rates, this could facilitate the incorporation of GH supplementation into ART protocols for patients with AMA, thus enhancing their prospects for successful pregnancy and live birth. Conversely, if no significant effects are observed, this will inform against the unnecessary application of GH in this demographic. The results of the trial will be disseminated in peer-reviewed journals and international conferences, potentially shaping clinical practice through evidence-based guidance on GH utilization in ART for AMA patients. While this study is designed to provide high-quality evidence, it is not without limitations. The single-center design may limit the generalizability of the findings to other settings or populations. Additionally, the open-label nature of the trial could introduce bias, although blinding of embryologists and statisticians will help mitigate this risk. Besides, the sample size is calculated to detect a 15% increase in euploidy rates, it may not be sufficient to identify smaller but clinically significant effects or subgroup differences. Future multi-center studies with larger sample sizes and longer follow-up periods are needed to confirm these findings and explore the long-term effects of GH on offspring health. To conclude, this RCT will compare the euploid rate of the blastocysts acquired with or without GH supplementation in AMA patients. This will answer whether AMA patients will benefit from GH supplementation based on high-quality evidence.

Supplementary Material 1. Supplementary Material 1.