探讨血清25-羟维生素D[25(OH)D]水平，对体外受精-胚胎移植(IVF-ET)助孕患者妊娠结局的影响及其与外周血、卵泡液淋巴细胞亚群的相关性。

选取2023年1月至2024年6月在粤北人民医院生殖医学中心进行IVF-ET助孕的19例患者为研究对象。根据血清25(OH)D水平是否正常，将其分为异常组[n＝10，血清25(OH)D水平<30 ng/mL(偏低)]与正常组[n＝9，血清25(OH)D水平≥30 ng/mL(正常)]。对2组患者均采用黄体期长方案促排卵。对2组患者人绒毛膜促性腺激素(hCG)扳机次日血清及取卵日卵泡液25(OH)D水平均采用化学发光法测定，而患者hCG扳机次日外周血及取卵日卵泡液淋巴细胞亚群水平，则均采用流式细胞仪测定。采用成组t检验、Mann-Whitney U检验或χ²检验，对2组患者的IVF-ET妊娠结局指标、血清与卵泡液25(OH)D水平、外周血与卵泡液淋巴细胞各亚群水平等进行比较。采用Pearson相关性分析或Spearman秩相关性分析方法，分析患者血清与卵泡液25(OH)D水平、外周血与卵泡液淋巴细胞亚群4者之间的相关性。本研究遵循的程序符合粤北人民医院医学伦理委员会规定，并通过该伦理委员会审批(审批文号：KY-2021-146)。2组患者年龄、人体质量指数(BMI)、促性腺激素(Gn)总量等临床资料分别比较，差异均无统计学意义(P>0.05)。

①2组患者hCG日雌二醇及孕酮水平、获卵数、正常受精率、受精后第3天(D3)优质胚胎率、临床妊娠率、活产率分别比较，差异均无统计学意义(P>0.05)。正常组患者hCG日子宫内膜厚度较异常组显著增厚，并且差异有统计学意义(t＝2.47，P＝0.025)。②2组患者卵泡液25(OH)D水平、外周血及卵泡液淋巴细胞各亚群水平分别比较，差异均无统计学意义(P>0.05)。异常组血清25(OH)D水平显著低于正常组，并且差异有统计学意义(t＝－6.50，P<0.001)。③血清25(OH)D水平与卵泡液25(OH)D、外周血淋巴细胞亚群、卵泡液淋巴细胞亚群水平，以及卵泡液25(OH)D与卵泡液淋巴细胞亚群水平的相关性，差异均无统计学意义(P>0.05)。外周血B细胞百分比与卵泡液CD3^＋CD4^＋ T细胞百分比呈正相关(r＝0.480，P＝0.037)，外周血CD3^＋CD4^＋ T细胞百分比与卵泡液CD3^＋CD8^＋T细胞百分比、CD3^＋CD4^＋ T细胞与CD3^＋CD8^＋ T细胞比值(CD4^＋/CD8^＋)，则分别呈负相关关系(r＝－0.509，P＝0.026)与正相关关系(r_s＝0.655，P＝0.002)，外周血与卵泡液CD4^＋/CD8^＋呈正相关关系(r_s＝0.609，P＝0.006)，外周血CD3^＋CD4^＋ T细胞百分比与外周血CD3^＋CD8^＋ T细胞百分比呈负相关关系(r＝－0.658，P＝0.002)。

本研究初步发现血清25(OH)D水平，对IVF-ET助孕患者超排卵过程中子宫内膜可能产生有利影响，而有助于改善妊娠结局；未发现患者25(OH)D水平与淋巴细胞亚群之间存在相关性。IVF-ET助孕患者外周血淋巴细胞亚群与卵泡液淋巴细胞亚群有一定相关性，检测其外周血淋巴细胞亚群，可为预测卵泡液中淋巴细胞亚群提供一定参考。

To investigate the effect of serum 25-hydroxyvitamin D [25(OH)D] levels on pregnancy outcomes of patients with in vitro fertilization-embryo transfer (IVF-ET) and its correlation with peripheral blood and follicular fluid lymphocyte subsets.

A total of 19 infertile patients who underwent IVF-ET at Reproductive Medicine Center of Yuebei People′s Hospital from January 2023 to June 2024 were selected as the research subjects. Based on the serum 25(OH)D level of infertile patients, 19 infertile patients were divided into abnormal group [n=10, serum 25(OH)D level <30 ng/mL] and normal group [n=9, serum 25(OH)D level ≥30 ng/mL]. The patients in two groups underwent ovulation induction using the long luteal phase protocol. Serum 25(OH)D levels on the day after trigger shot of human chorionic gonadotropin (hCG) and follicular fluid 25(OH)D levels on the day of oocyte retrieval were detected using chemiluminescence immunoassay. Flow cytometry was used to detect peripheral blood lymphocyte subsets on the day after trigger shot and follicular fluid lymphocyte subsets on the day of oocyte retrieval. Independent samples t-test, Mann-Whitney U test, or chi-square test were used to compare IVF-ET pregnant outcomes, serum and follicular fluid 25(OH)D levels, and peripheral blood and follicular fluid lymphocyte subset levels between two groups. Pearson correlation analysis or Spearman rank correlation analysis was used to analyze the correlations among serum and follicular fluid 25(OH)D levels, and peripheral blood and follicular fluid lymphocyte subsets. The procedures followed in this study complied with the regulations of the Medicine Ethics Committee of Yuebei People′s Hospital and were approved by the committee (Approval No. KY-2021-146). There were no statistically significant differences in clinical data such as age, body mass index (BMI), and total gonadotropin (Gn) levels between the two groups (P>0.05).

①There were no statistically significant differences between the two groups in estradiol and progesterone levels on hCG day, number of oocytes retrieved, normal fertilization rate, high-quality embryo rate on day 3 post-fertilization (D3), clinical pregnancy rate, and live birth rate (P>0.05). The endometrial thickness on hCG day was significantly higher in the normal group than those in the abnormal group (t=2.47, P=0.025). ②There were no statistically significant differences in follicular fluid 25(OH)D level, peripheral blood and follicular fluid lymphocyte subset levels between two groups (P>0.05). The serum 25(OH)D level in abnormal group was significantly lower than that in normal group, and the difference was statistically significant (t=-6.50, P<0.001). ③There was no significant correlation between serum 25(OH)D and follicular fluid 25(OH)D, peripheral blood lymphocyte subsets, follicular fluid lymphocyte subsets, and follicular fluid 25(OH)D and follicular fluid lymphocyte subsets (P>0.05). There was a positive correlation between percentage of peripheral blood B cells and percentage of follicular fluid CD3⁺ CD4⁺ T cells (r=0.480, P=0.037), and a negative correlation between percentage of peripheral blood CD3⁺ CD4⁺ T cells and percentage of follicular fluid CD3⁺ CD8⁺ T cells (r=-0.509, P=0.026), and a positive correlation between percentage of peripheral blood CD3⁺ CD4⁺ T cells and ratio of CD3⁺ CD4⁺ T cells to CD3⁺ CD8⁺ T cells (CD4⁺ /CD8⁺ ) in follicular fluid (r_s=0.655, P=0.002). Peripheral blood CD4⁺ /CD8⁺ was positively correlated with follicular fluid CD4⁺ /CD8⁺ (r_s=0.609, P=0.006). And percentage of peripheral blood CD3⁺ CD4⁺ T cells was negatively correlated with percentage of peripheral blood CD3⁺ CD8⁺ T cells (r=-0.658, P=0.002).

This study preliminarily found that serum 25(OH)D level may have a beneficial effect on the endometrium during the superovulation process in IVF-ET patients. No significant correlation was found between 25(OH)D and lymphocyte subsets in IVF-ET patients. There is a certain correlation between peripheral blood lymphocyte subsets and follicular fluid lymphocyte subsets in IVF-ET patients, detection of lymphocyte subsets in peripheral blood can provide certain reference for predicting lymphocyte subsets in follicular fluid.