
==== Front
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Medicine (Baltimore)
MD
Medicine
0025-7974
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Lippincott Williams & Wilkins Hagerstown, MD

MD-D-24-04777
00076
10.1097/MD.0000000000039645
3
5600
Research Article
Observational Study
Effect of different growth hormone pretreatment times in assisted reproductive therapy for patients with diminished ovarian reserve: A retrospective pilot cohort study
https://orcid.org/0009-0002-3575-7743
Zhang Yongmei MD zhangsongying@zju.edu.cn
ab
Liu Liu PhD liuliu@163.com
ab
Xu Aike BS xuaike1990@126.com
ab
Jin Yuanyang BS 62334058@qq.com
ab
Tong Xiaomei PhD 3406028@zju.edu.cn
ab
Zhou Feng PhD zhoufeng82@zju.edu.cn
ab
Zhang Songying PhD ab*
a Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China
b Zhejiang Provincial Clinical Research Center for Obstetrics and Gynecology.
* Correspondence: Songying Zhang, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China, 310000 (e-mail: zhangsongying@zju.edu.cn)
13 9 2024
13 9 2024
103 37 e3964509 5 2024
19 8 2024
20 8 2024
Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial License 4.0 (CCBY-NC), where it is permissible to download, share, remix, transform, and buildup the work provided it is properly cited. The work cannot be used commercially without permission from the journal.

This study aimed to evaluate the effect of different growth hormone (GH) pretreatment times in assisted reproductive therapy in patients with diminished ovarian reserve (DOR). A retrospective pilot cohort analysis was performed on patients with DOR receiving GH pretreatment in the Assisted Reproduction Unit of Sir Run Run Shaw Hospital. A total of 1459 patients met the criteria and were divided into four groups according to GH pretreatment time as follows: 53 were in the 2-month pretreatment group (GH1), 400 were in the 1-month pretreatment group (GH2), 414 were in the ovulation induction period pretreatment group (GH3), and 592 were in the non-GH pretreatment group (control group). In addition, GH1, GH2, and GH3 were combined in the GH pretreatment group. Baseline characteristics and treatment outcomes were compared between the groups. The number of oocytes retrieved in the GH pretreatment, GH1, GH2, and GH3 groups was significantly higher than that in the control group (all P < .01). The numbers of oocytes retrieved in the GH1 and GH2 groups were similar but were nominally higher than those in the GH3 group. Estradiol concentrations in the GH pretreatment, GH2, and GH3 groups were significantly higher than those in the control group on the day of human chorionic gonadotropin injection (all P < .01). In the GH1 group, 22 patients had >1 assisted reproductive therapy cycle (non-GH pretreatment) before GH pretreatment, and the number of oocytes retrieved in the GH pretreatment cycle was higher than that in the non-GH pretreatment cycle, but this was not significant. These findings suggest that the GH pretreatment time was appropriately prolonged, and the number of oocytes retrieved nominally increased. In patients with DOR, GH pretreatment improved treatment outcomes. More than 1 month of GH pretreatment did not increase the number of oocytes retrieved.

assisted reproductive therapy (ART)
diminished ovarian reserve (DOR)
growth hormone
treatment outcome
the Natural Science Foundation of Zhejiang ProvinceLTGY23H040010 Feng ZhouOPEN-ACCESSTRUE
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pmc1. Introduction

A reduction in the number or quality of follicles in a woman’s ovaries, resulting in reduced fertility, is called a diminished ovarian reserve (DOR). Patients with DOR have a lower quality of ova, fewer ova retrieved after ovulation induction, fewer or even poor-quality embryos, and a lower clinical pregnancy rate.[1–7] DOR seriously affects women’s quality of life and fertility at childbearing ages and even affects reproductive development. Therefore, DOR has increasingly become a research hotspot in the field of reproductive endocrinology.[8]

Assisted reproductive therapy (ART) is an effective method of treating infertility in patients with DOR. In 1988, Homburg et al[9] used growth hormone (GH) for the first time to induce ovulation to enhance the sensitivity of the ovary to gonadotropin and ultimately increase the number of oocytes retrieved.

GH pretreatment is often used during the ovulation induction period in patients with DOR, and some patients with DOR start receiving GH pretreatment before ovulation induction. At present, there are no unified criteria for GH pretreatment. Therefore, this study aimed to analyze the clinical efficacy of GH pretreatment at different times during ART cycles in patients with DOR.

2. Patients and methods

2.1. Patients

A retrospective pilot cohort analysis was performed on 1900 patients with DOR who received GH pretreatment during ART in the Assisted Reproduction Unit of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China, from January 1, 2021 to December 31, 2022. DOR patients according to the 2017 DOR diagnostic criteria[10] and 2011 Bologna criteria.[11] The following inclusion criteria were used: age 40 years or younger or any other risk factors for a poor ovarian response; a previous history of a poor ovarian response (i.e., ≤3 oocytes were retrieved with the conventional stimulation protocol); abnormal ovarian reserve function (i.e., antral follicle count < seven or anti-Mullerian hormone [AMH] concentrations < 1.1 ng/mL); the husband and wife had normal karyotypes; a preimplantation genetic testing cycle; complete clinical data; and patients with ART. The exclusion criteria were as follows: acute and chronic medical and surgical diseases such as uterine malformation, endometrial polyps, endometrial inflammation, and hydrosalpinx; use of other pretreatment drugs; preimplantation genetic testing cycle; and severe oligoasthenospermia or teratospermia. There are 41 patients refused GH injection or canceled the cycle, and 1459 patients met the criteria. The selection criteria are illustrated in Figure 1.

Figure 1. Flowchart for selection criteria. ART = assisted reproductive therapy, DOR = diminished ovarian reserve.

The 1459 patients were divided into four groups according to the GH pretreatment time as follows: 53 were in the two-month pretreatment group (GH1, GH had been used continuously for two months before inducing ovulation), 400 were in the 1-month pretreatment group (GH2, GH had been used continuously for one month before inducing ovulation), 414 were in the ovulation induction period pretreatment group (GH3), and 592 were in the non-GH pretreatment group (control group). Additionally, the combination of the GH1, GH2, and GH3 groups was called the GH pretreatment group. This retrospective pilot cohort study was approved by the ethics committee of Sir Run Run Shaw Hospital (ethics batch number: Run Run Shaw Hospital-Lun-Shen [2023] Research No. 0276).

2.2. Research Methods

2.2.1. Treatment

Each DOR patient received GH pretreatment when the patients entered the ovulation induction cycle. Because the size of basal antral follicles of some patients are not uniform, pretreatment with GH continued for 1 to 2 months until eligibility for ART was established. A subcutaneous injection of GH (15 IUs/5 mg/1.5 mL/injection, Guo-Yao Zhun-Zi No. S20050024; Changchun GeneScience Pharmaceutical Co., Ltd., Changchun City, Jilin Province, China) was started with a dosage of 0.67 mg (2 IU/day). During ovulation induction, a dosage of 2 IU/day GH was maintained until the day of human chorionic gonadotropin (HCG) administration. Follicular growth and sex hormone concentration were closely monitored during ovulation induction. When there were at least one to two follicles with a diameter > 18 mm during monitoring, HCG was injected intramuscularly, and venous blood was drawn on the same day to detect estradiol (E2) concentrations. Thirty-six hours after HCG administration, oocytes were retrieved via vaginal puncture under ultrasound guidance. In vitro fertilization (IVF) was performed after oocyte retrieval; fertilization was observed at 18 hours, and the embryos were scored at 72 hours.

2.2.2. Embryo quality assessment criteria

Blastomeres were assessed according to their grade. In grade I, blastomeres were uniform and fragmented. In grade II, blastomeres were slightly nonuniform in size, with a fragmentation rate of < 20%. In grade III, blastomeres were nonuniform in size, with a fragmentation rate of 20% to 50%. In grade IV embryos, the number and grade of embryonic blastomeres showed a downward trend, and the fragmentation rate was > 50%. Grades I and II embryos were defined as high-quality embryos, and blastocysts were scored according to the Gardner[12] scoring system.

2.2.3. Observational indicators

Early embryonic cleavage was evaluated every 24 hours after fertilization. The development and number of embryos were recorded 72 hours after oocyte retrieval to evaluate the embryo quality. Grades I to III embryos were transferred, frozen, or continuously cultured for two or three days. Only embryos that formed blastocysts better than grade III were transferred or frozen. Embryos with fewer than four cells were discarded. Transferable embryos were limited to ultra-long ovarian stimulation, but patients with ovarian hyperstimulation syndrome or uneven endometrium did not undergo embryo transfer.

2.3. Statistical analysis

The normality of the data was tested using the K-S method. All descriptive parameters are expressed as mean ± standard deviation, and the K-sample independent test is a non-parametric test used for the comparison of variables between the groups. Enumeration data were expressed as the constituent ratio or rate (%), and the chi-square test was used for comparison between the groups. Logistic regression was performed for all patient data. Statistical significance was set at P < .05. The observed difference was statistically significant with P < .01. Statistical analysis was performed using the IBM SPSS software (version 26.0).

3. Results

None of the patients pretreated with GH experienced side effects or ovarian hyperovulation induction syndrome. We compared the baseline characteristics and clinical outcomes between the GH pretreatment and control groups (Table 1). There were no significant differences in age, years of infertility, body mass index, basic endocrine follicle-stimulating hormone concentrations, basic endocrine E2 concentrations, AMH concentrations, insemination methods, or treatment options between the GH pretreatment and control groups.

Table 1 Baseline characteristics of all patients.

	GH pretreatment group (n = 867)	GH 1 group (n = 53)	GH 2 group (n = 400)	GH 3 group (n = 414)	Non-GH pretreatment group (n = 592)	
Age (yrs)*,†	35.54 ± 5.14	35.38 ± 5.12	35.38 ± 5.13	35.71 ± 5.16	35.99 ± 5.58	
BMI (kg/mL)*,†	22.61 ± 4.16	23.28 ± 3.86	22.28 ± 3.12	22.83 ± 4.98	22.73 ± 4.25	
Years of infertility (yr)*,†	3.11 ± 3.16	3.23 ± 3.08	3.13 ± 3.15	3.07 ± 3.19	3.10 ± 2.94	
Baseline sex hormone	
FSH (IU/L)	9.85 ± 3.15	9.46 ± 3.30	9.93 ± 3.32	9.99 ± 2.95	10.10 ± 3.47	
 Estradiol (ng/mL)*,†	33.87 ± 18.31	39.35 ± 26.02	33.18 ± 15.81	33.84 ± 19.28	35.10 ± 19.14	
 AMH (ng/mL)*,†	0.69 ± 0.30	0.72 ± 0.29	0.71 ± 0.29	0.68 ± 0.31	0.68 ± 0.36	
Ratio of insemination methods (%)	
 IVF*,†	59.3 (514/867)	64.2 (34/53)	57.8 (231/400)	60.1 (249/414)	63.9 (378/592)	
 ICSI*,†	40.7 (353/867)	35.8 (19/53)	46.2 (160/400)	39.9 (165/414)	36.1 (214/592)	
Treatment protocols	
 Ultro-long protocols, n (%)*,†	4.5 (39/867)	7.5 (4/53)	5.0 (17/400)	4.3 (18/414)	2.9 (17/592)	
 Antagonist protocol, n (%)*,†	8.1 (70/867)	5.7 (3/53)	9.0 (36/400)	7.5 (31/414)	6.6 (39/592)	
 Mini-stimulation protocol, n (%)*,†	57.1 (495/867)	58.5 (31/53)	55.5 (222/400)	58.5 (242/414)	55.4 (328/592)	
 Luteal phase stimulation protocol, n (%)*,†	30.3 (263/867)	34.0 (18/53)	30.1 (122/400)	29.7 (123/414)	35.1 (208/592)	
AMH = anti-Mullerian hormone, BMI = body mass index, FSH = follicle-stimulating hormone, GH = growth hormone, ICSI = intracytoplasmic sperm injection, IVF = in vitro fertilization, No. = number.

* All values are presented as the mean ± standard deviation or n (%).

† The GH pretreatment, GH1, GH2, and GH3 groups were compared with the non-GH pretreatment group (controls).

Clinical outcomes are shown in Table 2. On the day of HCG injection, mean E2 concentrations in the GH pretreatment, GH2, and GH3 groups were significantly higher than those in the control group (all P < .01), but there was no difference in the mean endometrial thickness on the same day among these groups. There was no significant difference in E2 concentrations between the GH1 and control groups on the day of the HCG injection. The mean endometrial thickness on the day of HCG injection was significantly higher in the GH1 group than in the control group (P < .01). The mean number of oocytes retrieved in the GH pretreatment group was significantly higher than that in the control group (P < .01).

Table 2 Comparison of clinical outcomes of all patients.

	GH pretreatment group (n = 867)	GH 1 group (n = 53)	GH 2 group (n = 400)	GH 3 group (n = 414)	Non-GH pretreatment group (n = 592)	
E2 levels on hCG day (ng/mL)*,†	1056 ± 746.70‡	1020.69 ± 716.22	1068.33 ± 786.73‡	1049.42 ± 709.94‡	842.35 ± 734.74	
Endometrial thickness on hCG day (mm)*,†	7.97 ± 2.73	8.89 ± 2.85‡	8.05 ± 3.17	7.80 ± 2.22	7.69 ± 2.48	
No. of oocytes retrieved*,†	2.91 ± 2.14‡	3.02 ± 2.23‡	3.06 ± 2.17‡	2.76 ± 2.08‡	2.39 ± 2.31	
Normal fertilization rate (%)*,†	
 IVF	64.3 (%)	60.9 (%)	64.6 (%)	64.1 (%)	67.8 (%)	
 ICSI	76.0 (%)	81.0 (%)	74.1 (%)	77.9 (%)§	71.4 (%)	
Cleavage rate*,†	74.1 (%)	72.3 (%)	73.5 (%)	74.2 (%)	71.7 (%)	
Available embryo ovum utilization rate (%)*,†	50.7 (%)	48.1 (%)	49.9 (%)	51.8 (%)	50.0 (%)	
Good-quality embryo rate*,†	64.5 (%)	53.2 (%)	63.8 (%)	66.6 (%)	63.6 (%)	
GH = growth hormone, hCG = human chorionic gonadotropin, ICSI = intracytoplasmic sperm injection, IVF = in vitro fertilization.

* All values are presented as the mean ± standard deviation or n (%).

† The GH pretreatment, GH1, GH2, and GH3 groups were compared with the non-GH pretreatment group (controls).

‡ P < .01 versus non-GH pretreatment group.

§ P < .05 versus non-GH pretreatment group.

The rates of normal IVF, intracytoplasmic sperm injection (ICSI) normal fertilization, available embryo/ovum use, cleavage, and high-quality embryos in the GH pretreatment group were not significantly different from those in the control group. The mean number of oocytes retrieved in the GH1, GH2, and GH3 groups was significantly higher than that in the control group (all P < .01). There were no significant differences in the rates of normal IVF, ICSI normal fertilization, available embryo/ovum use, cleavage, and high-quality embryos between the GH1 and control groups. There were also no significant differences in these variables between the GH2 and control groups. However, the rates of ICSI normal fertilization, cleavage, and high-quality embryos in the GH2 group were higher than those in the control group (P > .05). The embryo/ovum use rates were similar between the GH2 and control groups. In the GH3 group, the ICSI normal fertilization rate was significantly higher than that in the control group (P < .05). The rates of available embryo use, cleavage, and high-quality embryos in the GH3 group were higher than those in the control group, but the differences were not significant.

Table 3 shows that there was no significant difference in the number of oocytes retrieved or endometrial thickness on the HCG injection day between the GH pretreatment groups (GH1, GH2, and GH3). However, the mean numbers of oocytes retrieved in patients in the GH1 and GH2 groups appeared to be higher than that in the GH3 group, but this was not significant (P > .05). The mean endometrial thickness on the HCG injection day appeared to be higher in patients in the GH1 group than in those in the GH2 and GH3 groups, but this was not significant (P > .05). E2 concentrations on the HCG injection day, and the rates of normal IVF, ICSI normal fertilization, available embryo/ovum use, cleavage, and high-quality embryos were not significantly different among the three groups.

Table 3 Comparison of clinical outcomes in the GH pretreatment groups.

	GH 1 group (n = 53)	GH 2 group (n = 400)	GH 3 group (n = 414)	P	
E2 levels on hCG day (pg/mL)*	1020.69 ± 716.22	1068.33 ± 786.73	1049.42 ± 709.94	0.904	
Endometrial thickness on hCG day (mm)*	8.89 ± 2.85	8.05 ± 3.17	7.80 ± 2.22	0.061	
No. of oocytes retrieved*	3.02 ± 2.23	3.06 ± 2.17	2.76 ± 2.08	0.119	
Normal fertilization rate (%)	
 IVF*	60.9 (%)	64.6 (%)	64.1 (%)	0.760	
 ICSI*	81.0 (%)	74.1 (%)	77.9 (%)	0.283	
Cleavage rate*	72.3 (%)	73.5 (%)	74.2 (%)	0.706	
Available embryo ovum utilization rate (%)*	48.1 (%)	49.9 (%)	51.8 (%)	0.380	
Good-quality embryo rate (%)*	53.2 (%)	63.8 (%)	66.6 (%)	0.069	
* All values are presented as the mean ± standard deviation or n (%).

GH = growth hormone, hCG = human chorionic gonadotropin, ICSI = intracytoplasmic sperm injection, IVF = in vitro fertilization.

In the GH 1 group, 22 patients had more than one ART cycle (non-GH pretreatment) before GH pretreatment. The clinical outcomes of the two ART cycles are shown in Table 4. On the day of HCG injection, the mean E2 concentration and the mean endometrial thickness did not differ between the GH pretreatment and non-GH pretreatment cycles. The number of oocytes retrieved in the GH pretreatment cycle was higher than that in the non-GH pretreatment cycle; however, the difference was not significant. There was no significant difference in the rates of normal IVF fertilization, ICSI normal fertilization, available embryo/ovum use, cleavage, and high-quality embryos between the GH and non-GH pretreatment cycles.

Table 4 Comparison of the clinical outcomes of patients in the GH1 group who had more than one ART cycle.

	2-mo pretreatment group (n = 22)	Non-GH pretreatment cycle (n = 25)	P	
E2 levels on hCG day (pg/mL)*	881.61 ± 894.97	729.42 ± 609.63	0.277	
Endometrial thickness on hCG day (mm)*	6.55 ± 1.27	6.77 ± 2.66	0.652	
No. of oocytes retrieved*	1.95 ± 1.91	1.56 ± 1.64	0.482	
Normal fertilization rate (%)	
 IVF*	66.7 (%)	59.1 (%)	0.584	
 ICSI*	66.7 (%)	84.6 (%)	0.274	
Cleavage rate (%)*	78.6 (%)	79.2 (%)	0.958	
Available embryo ovum utilization rate (%)*	51.2 (%)	48.8 (%)	0.808	
Good-quality embryo rate (%)*	45.5 (%)	47 .6 (%)	0.887	
* All values are presented as the mean ± standard deviation or n (%).

3.1. Logistic regression analysis

To further evaluate the influencing factors, logistic regression analysis was performed using the number of oocytes retrieved and good-quality embryo rate as dependent variables, and GH pretreatment or non-GH pretreatment, GH pretreatment time, age, body mass index, years of infertility, baseline sex hormone (follicle-stimulating hormone, LH), AMH, insemination methods, treatment protocols (ultro-long protocols, antagonist protocol, mini-stimulation protocol, and luteal phase stimulation protocol) as independent variables. The logistic regression analyses were set at P < .05. The observed difference was statistically significant with P < .01. The logistic regression analyses were performed using the IBM SPSS software (version 26.0).

In Table 5, univariate logistic regression analyses reveal that GH pretreatment exerts significant impacts on the number of oocyte and the rate of high-quality embryos. Specifically, two months of GH pretreatment was associated with alterations in both oocyte yield and high-quality embryo rate, whereas one month of GH pretreatment influenced the high-quality embryo rate. Furthermore, serum levels of follicle-stimulating hormone and AMH were significant predictors of oocyte yield and high-quality embryo production. Variations in the oocyte retrieval protocols also significantly influenced the rate of high-quality embryos. In Table 6, multivariate logistic regression analysis of the effects of four treatment groups on oocyte yield and the rate of high-quality embryos revealed findings consistent with those presented in Table 5.

Table 5 Univariate logistic regression analysis of the risk factors for no. of oocytes retrieved and good-quality embryo rate.

	No. of oocytes retrieved	Good-quality embryo rate	
OR (95% CI)	P	OR (95% CI)	P	
GH pretreatment	0.430 (0.281–0.658)	0.000*	0.675 (0.574–0.834)	0.000*	
 2-mo GH pretreatment	0.552 (0.334–0.912)	0.020*	0.643 (0.498–0.830)	0.001*	
 1-mo GH pretreatment	0.721 (0.239–2.170)	0.056	0.551 (0.310–0.979)	0.042*	
 In the ovulation induction period pretreatment	2.080 (1.000–4.326)	0.050	0.976 (0.735–1.294)	0.864	
Age	0.996 (0.958–1.036)	0.996	1.001 (0.981–1.021)	0.932	
BMI	0.995 (0.949–1.044)	0.850	1.014 (0.988–1.040)	0.307	
Years of infertility	0.966 (0.907–1.029)	0.288	0.984 (0.951–1.018)	0.359	
Baseline sex hormone	
 FSH	0.923 (0.863–0.988)	0.021*	0.955 (0.922–0.989)	0.011*	
 LH	1.036 (0.922–1.164)	0.553	0.960 (0.907–1.018)	0.172	
 AMH	7.496 (3.641–15.433)	0.000*	1.937 (1.397–2.684)	0.000*	
Insemination methods	1.407 (0.901–2.198)	0.134	1.225 (0.990–1.517)	0.062	
Treatment protocols	0.889 (0.812–0.973)	0.011*	0.925 (0.886–0.965)	0.000*	
AMH = anti-Mullerian hormone, BMI = body mass index, FSH = follicle-stimulating hormone, GH = growth hormone, LH = luteinizing hormone, OR = odds ratio.

* P < 0.05.

Table 6 Multivariate logistic regression analysis of the risk factors for no. of oocytes retrieved and Good-quality embryo rate.

	No. of oocytes retrieved	Good-quality embryo rate	
OR (95% CI)	P	OR (95% CI)	P	
GH pretreatment	0.455 (0.295–0.703)	0.000*	0.690 (0.556–0.857)	0.001*	
 2-mo GH pretreatment	0.571 (0.342–0.954)	0.032*	0.641 (0.494–0.832)	0.001*	
 1-mo GH pretreatment	0.647 (0.209–2.003)	0.450	0.501 (0.278–0.903)	0.021*	
 In the ovulation induction period pretreatment	2.014 (0.959–4.231)	0.064	0.936 (0.701–1.249)	0.652	
Age	1.016 (0.975–1.059)	0.441	1.013 (0.992–1.035)	0.215	
BMI	0.997 (0.954–1.043)	0.908	1.012 (0.986–1.040)	0.369	
Years of infertility	0.952 (0.890–1.019)	0.158	0.974 (0.940–1.009)	0.148	
Baseline sex hormone	
 FSH	0.917 (0.857–0.982)	0.014*	0.952 (0.918–0.986)	0.007*	
 LH	1.031 (0.914–1.162)	0.620	0.955 (0.900–1.013)	0.123	
 AMH	6.908 (3.358–14.212)	0.000*	1.781 (1.273–2.491)	0.001*	
Insemination methods	1.397 (0.875–2.231)	0.158	1.261 (1.009–1.576)	0.041*	
Treatment protocols	0.910 (0.827–1.002)	0.054	0.925 (0.884–0.967)	0.001*	
AMH = anti-Mullerian hormone, BMI = body mass index, FSH = follicle-stimulating hormone, GH = growth hormone, LH = luteinizing hormone, OR = odds ratio.

* P < 0.05.

4. Discussion

Currently, there are no standard protocols for extending the duration of GH treatment. A strength of this study is that we analyzed the effects of different GH pretreatment durations on the outcome of patients with DOR in the ART cycle. Additionally, the sample size was large, providing strong evidence for the standardized use of GH in the future.

In this study, we performed a retrospective pilot cohort analysis of GH pretreatment during the ART ovulation induction cycle in patients from a single institute in China over the past two years. We found that >1 month of GH pretreatment increased the number of oocytes retrieved. However, patients with two months of GH pretreatment had a similar number of oocytes retrieved to that in patients with one month of GH pretreatment. The number of oocytes retrieved significantly increased after GH pretreatment. The rates of cleavage, high-quality embryos, and available embryos in the GH pretreatment groups were significantly higher than those in the control group. In the GH1 group, 31 patients were in the first cycle owing to poor ovarian function, and ART therapy was initiated two months after GH pretreatment. Twenty-two patients had >1 ART cycle (non-GH pretreatment) before GH pretreatment. When we compared the clinical outcomes of different cycles in the same patient, the number of oocytes retrieved increased in the GH pretreatment cycle. However, because of the small number of patients, the sample size should be increased to further validate this finding.

In 2017, studies showed that GH promotes the recruitment of follicles, improves the speed and quality of follicular development, and inhibits follicular atresia, thereby increasing the quantity and quality of available oocytes, as well as the pregnancy rate.[13] In addition, a meta-analysis showed that in IVF embryo transfer, adding GH considerably improved the clinical pregnancy rate during the IVF cycle.[14] Another meta-analysis showed that more oocytes were retrieved in the GH group than in the non-GH group in women with poor ovarian response during the IVF/ICSI cycle.[15] Some animal studies have suggested that GH treatment has a beneficial role in the function of animal ovaries, which supports the development of follicles and oocytes and improves the efficiency of embryo production.[16,17]

GH is a peptide hormone secreted by eosinophilic cells in the anterior pituitary gland. GH receptors are present in granulosa cells, theca cells, oocytes, cumulus cells, mammary glands, placenta, and the uterus.[18] Silva et al[19] reported that GH plays an important role in the growth, recruitment, and differentiation of preantral follicles by binding to its receptors. GH can also directly act on the ovary to improve its responsiveness to gonadotropin, cooperate with gonadotropin to stimulate the growth of preantral follicles, and enhance the responsiveness of antral follicles to gonadotropins. GH also increases the activity of mitochondria, inhibits follicular atresia, and increases the number of follicles, thereby directly improving oocyte quality.[20] This process promotes oocyte maturation and regulates estrogen and progesterone concentrations. In addition, GH promotes the maturation of immature oocytes.[21] Fan et al[22] suggested that co-treatment with GH during controlled ovarian stimulation alters follicular fluid metabolite profiles and increases the number of oocytes retrieved in patients with DOR, which is consistent with our results. Many studies showed that the effectiveness of GH on pregnancy outcomes of women who underwent IVF is significant, especially in those who were poor responders under ovarian stimulation.[23–25]

Insulin-like growth factor I is an important factor involved in early folliculogenesis[26] and is dependent on GH both in vivo and in vitro.[27] GH pretreatment promotes the generation of IGF and insulin-like growth factor I to enhance the response of preantral follicle granulosa cells to gonadotropin.

GH binds to granulosa cell receptors to improve the proliferation and differentiation of granulosa cells[28,29] and enhances mitochondrial function in oocytes during follicular development and oocyte maturation.[30] IGF-binding protein-3 secreted by granulosa cells can inhibit apoptosis of follicles[31] and is a necessary cytokine for the development of follicles and maturation of oocytes.[17,32,33]

Appropriately prolonging the use of GH can enhance the effect of GH on follicular development, and this study found that more than one month of GH pretreatment increased the number of oocytes retrieved. However, no redundant literature supports this conclusion.

Our study is consistent with the findings of Liu et al.[34] E2 concentrations on the day of HCG injection were also significantly higher in the GH pretreatment group than in the control group. This finding may be related to increased E2 production from more developed follicles, consistent with the findings of Bassiouny et al.[35]

Recent research[36] indicates that in poor ovarian responders (POR) undergoing assisted reproductive therapies, the daily administration of 4 to 8 IU of GH during the follicular phase of ovarian stimulation cycles yields optimal therapeutic outcomes. These outcomes include increased numbers of oocytes retrieved and matured, enhanced endometrial thickness, and reduced requirements for gonadotropins, compared to the control group. Our study employed lower doses of GH and found that pretreatment with GH—whether for 2 months, 1 month, or during the follicular phase of the stimulation cycle—significantly improved the number of oocytes retrieved. This finding suggests that even reduced GH dosages can be efficacious, potentially leading to more accessible and cost-effective treatment protocols for POR patients.

The intricate relationship between Vitamin D and GH disorders, encompassing both GH deficiency and excess, represents a complex nexus that has attracted significant attention in recent scholarly investigations. Ciresi and Giordano[37] have demonstrated that GH directly modulates the activity of renal 1 alpha-hydroxylase, a critical enzyme in the metabolism of Vitamin D. Concurrently, emerging evidence[38] suggests that Vitamin D may elevate levels of growth factors and GH, potentially impacting the efficacy of varying pretreatment durations with GH in assisted reproductive therapies for patients with DOR. Additionally, previous studies[39] have established a link between both serum and follicular Vitamin D concentrations and the degree of embryo fragmentation. Our investigation did not assess the serum and follicular Vitamin D levels in the study participants, which we recognize as a limitation of this study.

In addition, this is a retrospective pilot cohort study, and there is a possibility of patient selection bias. Furthermore, the number of patients who underwent embryo transfer was small, the return visits were incomplete, and the statistical sample size for the clinical pregnancy rate was insufficient. Therefore, we need to continue to collect data and assess the serum and follicular Vitamin D levels. We plan to address this limitation by conducting a prospective randomized controlled trial (RCT) to further validate our findings.

5. Conclusions

GH pretreatment has a major effect on patients with DOR undergoing ART and can increase the number of oocytes retrieved in the ovulation induction cycle of patients with DOR. However, appropriately prolonging the use of GH cannot further increase the number of oocytes retrieved and the rates of cleavage, high-quality embryos, and available embryo/ovum use.

Acknowledgments

We thank Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, for their support in this research. We thank Ellen Knapp, PhD, from Liwen Bianji (Edanz) (www.liwenbianji.cn/) for editing the English text of the draft of this manuscript. We extend our heartfelt thanks to Professor Liang Shuwen from the Clinical Research Centre at Run Run Shaw Hospital for her invaluable statistical assistance. Her expert guidance significantly enhanced the rigor of our analysis.

Author contributions

Data curation: Yongmei Zhang, Aike Xu, Yuanyang Jin.

Formal analysis: Yongmei Zhang, Songying Zhang, Feng Zhou.

Investigation: Yongmei Zhang, Liu Liu, Xiaomei Tong, Songying Zhang.

Methodology: Yongmei Zhang, Xiaomei Tong.

Project administration: Yongmei Zhang.

Resources: Yongmei Zhang.

Software: Yongmei Zhang.

Writing – original draft: Yongmei Zhang, Liu Liu, Aike Xu.

Writing – review and editing: Yongmei Zhang, Liu Liu, Songying Zhang.

Funding acquisition: Feng Zhou.

Abbreviations:

AMH anti-Mullerian hormone

ART assisted reproductive therapy

DOR diminished ovarian reserve

E2 estradiol

GH growth hormone

HCG human chorionic gonadotropin

ICSI intracytoplasmic sperm injection

IVF in vitro fertilization.

Funding Statement: the Natural Science Foundation of Zhejiang Province (Nos. LTGY23H040010).

This work was supported by grants from The Natural Science Foundation of Zhejiang Province (no. LTGY23H040010).

The authors declare that they have no competing interests regarding the present study. The authors are accountable for all aspects of the work and must certify that their manuscript is a unique submission and is not being considered for publication by any other source in any medium. Furthermore, the manuscript has not been published, in part or in full, in any form.

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

How to cite this article: Zhang Y, Liu L, Xu A, Jin Y, Tong X, Zhou F, Zhang S. Effect of different growth hormone pretreatment times in assisted reproductive therapy for patients with diminished ovarian reserve: A retrospective pilot cohort study. Medicine 2024;103:37(e39645).
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