Study inclusion and exclusion criteria
Table of Contents
From November 2018 to December 2019, 375 patients who underwent IVF in the Reproductive Medicine Center of Shiyan People’s Hospital Affiliated to Hubei University of Medicine were analyzed. All patients were treated with the follicular-phase GnRH agonist protocol. Serum vitamin B12 levels were measured by chemiluminescence in all patients before the initiation therapy. After applying the inclusion and exclusion criteria, 356 patients were included in the final analysis.
Inclusion criteria
Aged 20–45 years; Diagnosed with infertility; Undergoing their first IVF cycle; AMH 1.0–4.0 ng/mL (In the literature, the AMH level in individuals with normal ovarian reserve (NOR) is consistently reported to exceed 1 ng/ml, whereas the threshold values established for patients with PCOS range from 3.5 to 5 ng/ml [9, 10]. To eliminate confounding effects of DOR and PCOS, we included only participants with an AMH concentration of 1–4 ng/ml).
Exclusion criteria
Severe medical conditions (e.g., hypertension, diabetes, gastrointestinal diseases); Indication for preimplantation genetic testing (PGT); Other endocrine disorders affecting metabolism (e.g., thyroid disease, hyperprolactinemia); Intake of vitamin B supplements within 3 months prior to inclusion.
The study was approved by the Ethics Committee of Renmin Hospital, Hubei University of Medicine, and informed consent forms were obtained from all couples. We confirm that all methods were performed in accordance with the relevant guidelines and regulations.
We determined the serum vitamin B12 level of the study population, as tertile division allows for exploring non-linear relationships [11]the population was divided into three equal groups according to the distribution of vitamin B12: Low (96.89pg/ml-292.70pg/ml, n = 119), Average (293.31pg/ml −422.44pg/ml, n = 118), High (422.88pg/ml −1215.28pg/ml, n = 119).
Ovarian stimulation in IVF
In the follicular-phase GnRH agonist protocol, the patients received a single intramuscular injection of 3.75 mg long-acting triptorelin acetate (Decapeptyl; Ferring, SaintPrex, Switzerland) on Day 2 or 3 of the cycle. After 30–42 days of downregulation, gonadotrophin (Gn, r-hFSH, Serono, Switzerland, 75 IU/day) and/or hMG (Lipo Biochem, 75 IU/day) were added for ovulation induction. When there were more than 2 follicles with a diameter of 18 mm, r-hFSH and hMG were stopped, recombinant human chorionic gonadotrophin (r-HCG, Serono, Switzerland) 250 µg was injected, and ultrasound-guided aspiration was performed 36 h later. On the 3rd to 5th day after oocyte retrieval, embryo morphology was scored according to morphological parameters [12]and high-quality embryos were selected for embryo transfer. Intrauterine transfer was performed under the guidance of ultrasound. Clinical pregnancy was defined as a positive serum hCG level with ultrasound evidence of a gestational sac at approximately 6 weeks of gestation.
Serum FSH, LH, E2and P levels on the 2nd-3rd day of menstruation and the trigger day were measured. Hormone assays were performed using commercial chemiluminescence kits. Detailed information on assay performance (including precision, reproducibility, and calibration curve characteristics) is provided in the Supplementary Materials.
Outcome measures
The primary outcome was the clinical pregnancy rate (CPR). Secondary outcome was the cumulative clinical pregnancy rate (CCPR). Clinical pregnancy was defined as ultrasound-confirmed intrauterine gestational sac at approximately 6 weeks of gestation, in accordance with the International Glossary on Infertility and Fertility Care [13].
The CCPR per oocyte retrieval cycle was calculated as follows:
$$mathrm{CCPR};=;frac{mathrm{Number};mathrm{of};mathrm{first}; mathrm{clinical};mathrm{pregnancies};mathrm{in};mathrm{fresh};ma thrm{and};mathrm{frozen};mathrm{cycles}}{mathrm{Number};mathrm{of} ;mathrm{oocytes};mathrm{retrieved};mathrm{cycles}};times;100%$$
(1)
The observation standard is to obtain at least one clinical pregnancy or use up all the embryos in this ovulation induction, and the observation time is 2 years [14, 15].
Statistical analysis
All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) version 22.0 and EmpowerStats ( X&Y Solutions, Inc., Boston, MA, USA). Normally distributed continuous variables are expressed as the mean ± standard deviation (x¯±s), and categorical variables are expressed as percentages (%).
We conducted univariate analysis to identify potential confounding factors affecting the clinical pregnancy rate. Subsequently, multivariate logistic regression analysis was employed to examine the relationship between vitamin B12 levels and clinical pregnancy rate after adjusting for identified confounders. Results are reported as odds ratios with corresponding 95% confidence intervals.
To further explore the relationship, we applied smooth curve fitting to assess the linear association between vitamin B12 level and clinical pregnancy rate. A piecewise regression model was used to evaluate potential threshold effects and determine the threshold value, while adjusting for confounding variables. A two-sided P value < 0.05 was considered statistically significant.
