Integrating Yangjing Zhongyu Decoction into IVF/ICSI-ET Cycles in Patients with Diminished Ovarian Reserve

Summary

This study demonstrates that Yangjing Zhongyu decoction enhances assisted reproduction outcomes in women with diminished ovarian reserve by improving oocyte and embryo quality. In a rat model of premature ovarian failure, the decoction restored hormone balance, promoted follicle development, and modulated autophagy and lipid metabolism.

Abstract

Yangjing Zhongyu Decoction (YZD), a traditional Chinese herbal formula, has been suggested to improve ovarian function and fertility outcomes, but standardized clinical evidence remains limited. This study evaluated the effects of YZD in women with diminished ovarian reserve (DOR) undergoing IVF/ICSI-ET, alongside mechanistic validation in a rat model of premature ovarian failure (POF). In the clinical trial, DOR patients received either the standard GnRH antagonist protocol alone or in combination with YZD. Baseline characteristics between groups were comparable, and the YZD group showed higher numbers of retrieved oocytes and high-quality embryos, as well as improved traditional Chinese medicine syndrome scores. In parallel, POF was induced in rats, and animals treated with YZD exhibited improved estrous cyclicity, restoration of serum estradiol levels, reduction of follicle-stimulating hormone, and healthier ovarian histology. Electron microscopy revealed that YZD reduced excessive autophagosome formation and lipid accumulation in ovarian cells, suggesting regulation of the autophagy-lipid metabolism axis. Together, these findings indicate that YZD enhances ovarian reserve function, improves embryo development, and may increase pregnancy potential in women with DOR. This standardized protocol provides a reproducible framework for integrating YZD into assisted reproductive technology cycles, supporting its translational potential in clinical reproductive medicine.

Introduction

Diminished ovarian reserve (DOR) is a clinical condition that compromises female reproductive health and is characterized by a reduction in the number or quality of ovarian follicles¹. Clinically, it is reflected by decreased antral follicle count, reduced anti-Müllerian hormone (AMH) levels, or elevated follicle-stimulating hormone (FSH) concentrations². DOR accounts for approximately 10% of infertility cases and represents a major barrier to successful conception³. In the context of assisted reproductive technology (ART), the prevalence of DOR is estimated at 19%-26% in the United States⁴, and it is associated with reduced pregnancy rates, higher incidence of preeclampsia, and an increased risk of recurrent miscarriage⁵. Despite the availability of multiple therapeutic approaches, improving ART outcomes in DOR patients remains a considerable challenge, underscoring the need for novel and effective interventions⁶.

Yangjing Zhongyu Decoction (YZD), a classical multi-herb traditional Chinese medicine (TCM) formulation, has long been applied in reproductive practice⁷. It is believed to enhance endocrine balance and support ovarian function, thereby improving outcomes in in vitro fertilization and intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET) cycles⁸. Preliminary clinical data suggest that YZD can improve ovarian response, but rigorous mechanistic and translational studies are still limited. The precise biological pathways through which YZD acts in DOR patients remain unclear^9,10. Current literature indicates that Chinese herbal compounds may influence the ovarian microenvironment through multiple mechanisms, such as modulating AMH expression, inhibiting follicular atresia-related signaling pathways including PI3K/AKT/mTOR, and reducing oxidative stress injury. However, these mechanistic hypotheses are largely indirect and lack experimental validation.

This study addresses these gaps by integrating clinical evaluation with experimental verification. Specifically, we examined the effects of YZD in women with DOR undergoing ART and investigated its mechanistic underpinnings using a tripterygium glycoside-induced DOR rat model. In the animal experiments, we monitored endocrine markers (estradiol and FSH), antral follicle counts, autophagosome formation, and lipid accumulation in ovarian tissue. By combining clinical outcomes with experimental mechanistic data, this work provides a standardized protocol for incorporating YZD into IVF/ICSI-ET cycles and offers novel evidence supporting its potential as an adjunctive therapy for improving ovarian reserve and reproductive outcomes in DOR patients. The novelty of this study lies in its dual approach-linking standardized clinical application with mechanistic insights-which advances both the clinical translation and theoretical understanding of YZD in reproductive medicine.

Protocol

All clinical procedures were conducted in accordance with the Declaration of Helsinki and were approved by the Ethics Committee of Shanghai Changhai Hospital (Approval No. CHEC2022-156). Written informed consent was obtained from all participants. Animal experiments were approved by the Ethics Committee of Changhai Medical College (Approval No. CHEC2022-156) and complied with national laboratory animal care standards.

Clinical features

Patient recruitment and grouping
From August 2022 to March 2023, 78 infertility patients diagnosed with kidney-yin deficiency¹⁰ syndrome and seeking ART were recruited from the Reproductive Medicine Center of the First Affiliated Hospital of Naval Medical University and the outpatient department of Yueyang Hospital, Shanghai University of Traditional Chinese Medicine. Participants were randomly assigned to either a control group or a treatment group (39 cases each) using SPSS 23.0 random number generation.

Diagnostic criteria
Western medical diagnosis followed the American Society for Reproductive Medicine (ASRM) 2020 Committee Opinion on ovarian reserve indicators¹¹. Criteria included FSH ≥ 10 IU/L, estradiol ≥ 80 pg/mL, Antral Follicle Count (AFC) < 7, and AMH < 0.5-1.1 ng/mL. Kidney-yin deficiency syndrome was diagnosed according to Gynecology of Traditional Chinese Medicine¹² and Diagnostic Efficacy Criteria of TCM Syndromes¹³.

Inclusion and exclusion criteria
Inclusion criteria were planned IVF/ICSI-ET using an antagonist protocol; history of poor ovarian response (< 5 retrieved oocytes); age 20-40 years; meeting both DOR and TCM syndrome criteria; and signed informed consent. Exclusion criteria included endometrial pathology (polyps, tuberculosis, dysplasia, cancer), uterine malformation, comorbidities (diabetes, lupus), recent hormone therapy (< 3 months), history of pelvic surgery, chromosomal abnormalities, or drug allergy. Elimination criteria were applied for protocol deviation, adverse events, withdrawal, or spontaneous conception within 2 weeks of enrollment.

Medication method
Both groups underwent baseline evaluation on menstrual day 2-3 by transvaginal ultrasound and hormone testing. The control group received the antagonist regimen: recombinant FSH or human menopausal gonadotropin, initial dose 225 IU/day intramuscularly for 5 days, followed by dose adjustment based on follicle growth. When the leading follicle diameter exceeded 12 mm, GnRH antagonist (0.125 mg/day subcutaneous) was started and continued until trigger. When ≥ 1 follicle reached 18 mm, ≥ 2 reached 17 mm, or ≥ 3 reached 16 mm, ovulation was triggered with 250 µg recombinant hCG, followed by oocyte retrieval after 34-36 h.

The treatment group followed the same protocol with the addition of Yangjing Zhongyu Decoction (YZD), administered orally 2x daily (morning and evening), one decoction per day, starting from menstrual cycle days 2-3 until the trigger day. The clinical formula contained Rehmannia 12 g, Paeonia lactiflora 12 g, Cornus officinalis 10 g, and Angelica sinensis 12 g.

Observation indicators
Clinical outcomes included baseline parameters of age, BMI (calculated as weight (kg)/height² (m²)), infertility duration, FSH, gonadotropin dose/duration, kidney-yin deficiency syndrome scores, number of antral follicles (assessed by transvaginal ultrasound, 2-9 mm in diameter), retrieved oocytes, Metaphase II (MII) oocytes (identified at oocyte retrieval), fertilized embryos, cleavages, high-quality embryos(defined by standard embryology laboratory criteria), and clinical pregnancy rate, which was defined as the presence of a gestational sac on ultrasound at 6-7 weeks of gestation.

Experimental animals
Here, 30 SPF-grade female Sprague-Dawley rats (8 weeks old, 160-200 g) were acclimatized under controlled conditions (23 ± 2 °C, 40%-45% humidity, 12 h light/dark cycle) with ad libitum food and water.

Drug preparation
YZD was prepared with Rehmannia 30 g, Angelica 15 g, Paeonia lactiflora 15 g, and Cornus officinalis 15 g. The herbs were decocted in water, filtered through a 200-mesh filter, and concentrated under reduced pressure at 70 °C. The final solutions for low, medium, and high doses were 0.34 g/mL, 0.68 g/mL, and 1.35 g/mL, corresponding to 0.5x, 1.0x, and 1.2x of the clinical dose of YZD. Decoctions were stored at 4 °C.

Modeling and grouping
A DOR model was induced by intraperitoneal injection of tripterygium glycosides (40 mg/kg daily for 15 days). Rats were randomly assigned to blank (n = 6), model (n = 6), and YZD treatment groups (low, medium, and high doses; n = 6 each). Gavage administration (10 mL/kg body weight) was done 1x daily, starting from the first day of tripterygium glycosides injection continued for 15 days.

Vaginal cytology
Estrous cycle stages were assessed by vaginal exfoliative cytology. Vaginal smears were collected once daily (08:00-10:00) with sterile cotton swabs, diluted in 30 µL of saline, and examined under a light microscope at 100x magnification. The observation period lasted 15 consecutive days. The normal estrous cycle index was defined as the proportion of rats exhibiting regular 4-5 day estrous cycles over this period.

Hormone assays
Whole blood was collected under anesthesia by cardiac puncture at the end of the 15-day treatment period, allowed to clot at room temperature for 30 min, and centrifuged at 1000 x g for 15 min at 4 °C to obtain serum. E2 and FSH levels were measured by ELISA according to the manufacturer's instructions.

Ovarian histology
Ovarian tissue was collected immediately after euthanasia by surgical removal of both ovaries, rinsed in cold saline to remove blood, and then fixed in 4% paraformaldehyde at 4 °C for 24-48 h. Tissues were dehydrated in graded ethanol, cleared in xylene, embedded in paraffin, and sectioned at 4-5 µm. Sections were stained with hematoxylin and eosin, dehydrated, cleared, and mounted.

Statistical analysis
Data were analyzed using SPSS 26.0. Normal variables were expressed as mean ± SD and compared by t-test; non-normal variables as median (IQR) and compared by Mann-Whitney U test. Categorical variables were expressed as % and compared by the chi-square test. p < 0.05 was considered statistically significant. For consistency, the same statistical methods were applied to figure analyses (no separate Student's t-test description).

Safety and waste disposal
Paraformaldehyde, xylene, and tripterygium glycosides were handled in certified fume hoods with gloves, goggles, and lab coats; spills and contaminated disposables were collected in labeled hazardous-waste containers and disposed of per institutional biosafety SOP. All procedures involving paraformaldehyde, xylene, and tripterygium glycosides were performed in fume hoods with appropriate personal protective equipment (gloves, lab coats, goggles). Chemical waste and animal carcasses were disposed of according to institutional biosafety guidelines.

Results

Clinical features

During the study, 3 patients in the control group withdrew due to COVID-19 infection, and 1 was excluded because of abnormal liver function, leaving 35 patients. In the treatment group, 4 withdrew due to COVID-19, and 35 patients completed the study. Thus, a total of 70 subjects were included in the final analysis. There were no significant differences between groups in age, body mass index (BM...

Discussion

In this study, we investigated the potential role of Yangjing Zhongyu Decoction (YZD) as an adjunct therapy for diminished ovarian reserve (DOR) in assisted reproductive technology (ART). Clinical findings demonstrated that patients receiving YZD in addition to the standard antagonist protocol achieved significantly higher numbers of retrieved oocytes and high-quality embryos. Although pregnancy rates did not differ significantly between groups, a favorable trend was observed. These results suggest that YZD may enhance f...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Centrifuge	Serum separation (1000 x g, 15 min, 4 °C)	Eppendorf 5810R	5811 000.012
Cotton swabs (sterile)	Collection of vaginal cytology samples	Jiangsu Kangjian Medical Supplies	KJ-MW-01
ELISA Kit for E2	Serum estradiol quantification (rat)	Elabscience Biotechnology Inc.	E-EL-0152
ELISA Kit for FSH	Serum FSH quantification (rat)	Elabscience Biotechnology Inc.	E-EL-R0391
Ethanol (70–100%)	Graded dehydration for histology	Sinopharm Chemical Reagent Co., China	100092683
Glass slides & coverslips	Sample mounting	Citotest Scientific, China	7101/1812
Hematoxylin & Eosin	Staining solution for histology	Beyotime Biotechnology, China	C0105S
Optical Microscope	Histology and cytology observation	Olympus (generic, no trademark)	CX23
Paraffin	Embedding medium	Leica Biosystems	39601095
Paraformaldehyde (4%)	Fixative for tissue preparation	Sigma-Aldrich	P6148
Saline solution (0.9%)	Vaginal smear preparation	Sinopharm Chemical Reagent Co., China	10019318
Sprague–Dawley rats	Experimental animals (female, 8 weeks, 160–200 g)	Beijing Weitong Lihua Experimental Animal Tech Co., Ltd.	SCXK(Beijing)2024-0011
SPSS Software	Statistical analysis (ver. 26.0)	IBM Corp.	RRID:SCR_002865
Transmission Electron Microscope	Ultrastructural observation	JEOL Ltd.	JEM-1400Plus
Tripterygium glycosides	Induction of DOR rat model	Generic supplier (no trademark)	TG-001
Xylene	Clearing reagent for histology	Sinopharm Chemical Reagent Co., China	10023418