Abstract
目的
探讨促性腺激素释放激素拮抗剂（GnRH-ant）方案中添加人绝经期促性腺激素（HMG）的时机对高龄卵巢储备减退（DOR）不孕患者妊娠结局的影响。
方法
行体外受精-胚胎移植（IVF-ET）治疗的≥35岁的DOR患者682例，采用拮抗剂方案，均于月经周期2~3 d启动卵泡刺激素（FSH）促排卵治疗，根据添加HMG的时机分为无添加组371例（不添加HMG）、早期添加组139例（每日加用HMG直至扳机日）、中晚期添加组172例（主导卵泡直径达到10~14 mm时每日加用HMG直至扳机日），比较3组的妊娠结局。
结果
3组扳机日雌二醇、孕酮水平、扳机日子宫内膜厚度、扳机日直径≥14、16、18 mm的卵泡个数、受精率、卵裂率差异均无统计学意义（P>0.05），Gn用量早期添加组和中晚期添加组高于无添加组，中晚期添加组获卵数高于早期添加组，成熟卵子数及可利用胚胎数中晚期添加组高于无添加组及早期添加组（P < 0.05）。新鲜周期临床妊娠率中晚期添加组高于无添加组（P < 0.05），种植率、早期流产率、异位妊娠率、活产率3组差异无统计学意义（P>0.05）。全胚冷冻周期首次移植种植率、临床妊娠率、早期流产率、异位妊娠率、活产率3组差异无统计学意义（P>0.05）。
结论
高龄DOR患者应用GnRH-ant方案时，在卵泡发育的中晚期添加HMG能改善促排卵结局和新鲜周期临床妊娠率。
Keywords: GnRH拮抗剂方案, 人绝经期促性腺激素, 高龄, 卵巢储备减退
Abstract
Objective
To assess the effect and timing of human menopausal gonadotropin (HMG) supplementation in advancedage patients with diminished ovarian reserve (DOR) receiving gonadotropin-releasing hormone antagonist protocol.
Methods
A total of 682 patients with DOR aged over 35 years undergoing IVF-ET treatment were included in this study. All the patients underwent a GnRH antagonist protocol, and controlled ovarian stimulation was initiated on day 2-3 of the menstrual cycle with follicle stimulation hormone (FSH). According to the timing of HMG supplementation, the patients were divided into no supplementation group (n=371) without HMG supplementation; early supplementation group (n=139), in which daily HMG supplementation started on the first day till the trigger day; and late supplementation group (n=172), in which HMG supplementation started when the leading follicle reached 10-14 mm in diameter and lasted until the trigger day. The pregnancy outcomes of the patients were compared among the 3 groups.
Results
The 3 groups showed no significant difference in hCG trigger day E2 and P levels, endometrial thickness, or the number of follicles with comparable fertilization rate and cleavage rate (P>0.05). Gn dose used was the lowest in no supplementation group, and the average number of oocytes retrieved was significantly smaller in early supplementation group than in late supplementation group (P < 0.05). The mean number of mature oocytes and embryos available were significantly higher in late supplementation group than in early supplementation group (P < 0.05). The clinical pregnancy rate of fresh embryo transfer cycle was significantly higher in late supplementation group than in no supplementation group (27.7% vs 45.1%, P < 0.05), but the implantation rate, early miscarriage rate, heterotopic pregnancy rate and live birth rate were comparable among the 3 groups (P>0.05). No significant differences were found among the 3 groups in the implantation rate, clinical pregnancy rate, early miscarriage rate, heterotopic pregnancy rate or live birth rate of the first frozen-thawed embryo transfer cycle with a freeze-all strategy (P>0.05).
Conclusions
HMG supplementation in the middle and late follicular phase can improve the outcomes of controlled ovarian hyperstimulation and increase the clinical pregnancy rate of fresh embryo transfer cycle in advanced-age patients with DOR undergoing GnRH antagonist protocol.
Keywords: gonadotropin-releasing hormone antagonist protocol, human menopausal gonadotropin, advanced age, diminished ovarian reserve
在全球范围内，促性腺激素释放激素拮抗剂（GnRH-ant）方案已经成为辅助生殖治疗最常用的控制性促排卵方案之一，GnRH-ant方案疗程简化，治疗时间短，明显降低卵巢过度刺激综合征的风险[1]，对垂体抑制程度减轻，停药后卵巢功能恢复迅速，因此被广泛地应用于高龄人群与卵巢储备减退（DOR）患者。
高龄DOR群体周期取消率高，妊娠率低[2]，DOR是一个渐进性发展的过程，促卵泡刺激素（FSH）的升高早于黄体生成素（LH）的变化[3]，导致基础FSH/LH的比值升高。已有研究表明，≥35岁的DOR患者，LH/FSH与卵母细胞数量正相关，加之使用GnRH-ant后内源性LH被抑制，预测卵巢反应性较差，提示可以考虑通过给予HMG，提高LH与FSH的比例，以降低刺激不良风险[4]。有证据显示GnRH-ant方案中添加HMG可以显著提高胚胎种植率和临床妊娠率[5]，亦有相反的观点认为患者应用GnRH-ant后的内源性LH仍足以维持对卵泡发育的刺激作用，需要重新评估添加的必要性[6-7]，目前对GnRH-ant方案中添加HMG的研究较少且结论各异，国内对于特定高龄DOR群体的应用缺乏文献报道，需要进一步探索。
本研究旨在分析应用GnRH-ant方案过程中，高龄DOR患者添加HMG是否受益以及HMG添加时机对妊娠结局的影响，以探求更好的用药方案，为提高高龄DOR群体临床促排卵治疗的有效性提供参考依据。
1. 资料和方法
1.1. 研究对象
回顾性分析2014年1月~2019年12月于本中心采用GnRH-ant方案行常规体外受精/卵胞浆单精子显微注射受精（IVF/ICSI）助孕的高龄DOR患者，纳入标准：年龄≥35岁，且满足以下2项标准中的任意一项：（1）双侧卵巢基础窦状卵泡数（AFC）≤5；（2）基础FSH≥10 U/L，和（或）基础FSH/ LH＞3.6。
1.2. 促排卵方案及分组
所有患者均采用GnRH-ant方案，于月经周期第2- 4天评估卵巢基础状态，给予FSH制剂（注射用重组人促卵泡素，默克雪兰诺；重组促卵泡素β注射液，默沙东；注射用尿促卵泡素，丽珠）150~300 U/d启动促排卵治疗，根据HMG（注射用尿促性素，丽珠）的添加时机，共分为3组。无添加组：不添加HMG，仅使用FSH至扳机日；早期添加组：Gn第1日开始每日添加HMG 75~150 U至扳机日；中晚期添加组：主导卵泡直径约10~14 mm时每日添加HMG 75~150 U至扳机日，同时减少相应FSH剂量。Gn使用第5或6日时皮下注射GnRH-ant 0.25 mg/d直至扳机日，当2~3个优势卵泡直径≥18 mm时给予HCG 5000~10 000 U或联合促性腺激素释放激素激动剂（GnRH-a）0.2 mg扳机，诱发排卵36~38 h后行经阴道超声引导下取卵术，取卵日开始黄体支持。
1.3. 胚胎培养及孕期监测
根据男方精液质量及患者病情决定行IVF或ICSI，取卵后第3天或第5天行新鲜胚胎移植，若患者不适合鲜胚移植（因内膜因素、孕酮升高等）则行全胚冷冻。移植12 d后检测血β-绒毛膜促性腺激素水平，移植后4周通过超声观察到一个或多个孕囊及胎儿心管搏动确定为临床妊娠。优质胚胎标准为2PN来源、卵裂球数目7~8个且评分Ⅰ、Ⅱ级（Edwards标准）的D3胚胎[8]或评分≥3BB的囊胚（Gardner评分法）。
1.4. 统计学分析
数据采用SPSS 22.0软件进行统计分析。计量资料用均数±标准差表示，采用单因素方差分析或KruskalWallis检验；计数资料用率表示，采用χ2检验。P < 0.05（双侧）为差异有统计学意义。
2. 结果
2.1. 患者基础情况比较
本研究共纳入682例≥35岁的DOR患者，其中无添加组371例，早期添加HMG组139例，中晚期添加HMG组172例。3组患者女方年龄、不孕年限、体质量指数（BMI）、原发不孕比例、继发不孕比例、基础FSH、LH、E2、P、AFC、AMH差异均无统计学意义（P>0.05，表 1）。
1.
患者基础情况比较
Baseline characteristics of the patients
| Index | No addition | Early addition | Middle and late addition | P |
| No. of cycles | 371 | 139 | 172 | - |
| Age (year) | 39.66±2.96 | 39.58±3.25 | 39.22±2.70 | 0.263 |
| Years of infertility | 5.09±4.58 | 5.55±4.74 | 4.50±3.83 | 0.110 |
| BMI (kg/m2) | 22.23±2.71 | 22.06±2.46 | 21.87±2.72 | 0.344 |
| Type of infertility | ||||
| Primary infertility | 22.1% (82/371) | 23.7% (33/139) | 18.6% (32/172) | 0.689 |
| Secondary infertility | 77.9% (289/371) | 76.3% (106/139) | 81.4% (140/172) | 0.689 |
| BasalFSH (U/L) | 10.30±4.54 | 10.75±3.81 | 10.10±4.03 | 0.402 |
| BasalLH (U/L) | 5.05±3.99 | 4.86±2.98 | 4.75±2.36 | 0.623 |
| BasalE2 (pg/mL) | 42.03±20.83 | 40.27±25.69 | 41.18±25.76 | 0.746 |
| BasalP (ng/mL) | 0.42±0.34 | 0.49±0.45 | 0.47±0.58 | 0.358 |
| Antral follicle count | 4.93±2.34 | 5.10±2.49 | 5.15±2.31 | 0.588 |
| AMH (ng/mL) | 0.76±0.67 | 0.94±0.74 | 0.70±0.41 | 0.549 |
2.2. 卵巢刺激情况和促排卵结局
3组扳机日LH、E2、P水平、扳机日子宫内膜厚度、扳机日直径≥14 mm、≥16 mm、≥18 mm的卵泡个数、受精率、卵裂率、优质胚胎数差异均无统计学意义（P>0.05）；早期添加组、中晚期添加组Gn用量高于无添加组，中晚期添加组获卵数高于早期添加组，中晚期添加组成熟卵子数、可利用胚胎数均高于无添加组及早期添加组（P < 0.05，表 2）。
2.
卵巢刺激情况及促排卵结局
Outcomes of controlled ovarian hyperstimulation in the patients
| Index | No addition | Early addition | Middle and late addition | P |
| *P < 0.05 vs early addition group; #P < 0.05 vs Middle and late addition group. | ||||
| No. of cycles | 371 | 139 | 172 | - |
| Dosage of Gn used (U) | 2528.10±754.38*# | 2911.15±920.21 | 2921.73±727.68 | < 0.001 |
| Value of LH on hCG trigger day (U/L) | 4.52±5.18 | 5.09±8.80 | 3.70±3.08 | 0.169 |
| Value of E2 on hCG trigger day (pg/mL) | 1295.89±800.18 | 1410.06±1301.22 | 1400.07±1238.19 | 0.507 |
| Value of P on hCG trigger day (ng/mL) | 0.70±0.78 | 0.80±1.46 | 0.75±0.56 | 0.670 |
| Endometrial thickness on hCG trigger day (mm) | 9.13±2.89 | 9.41±3.09 | 9.30±2.54 | 0.574 |
| No. of follicles on hCG trigger day No. of follicles with diameter≥14 mm | 3.97±2.45 | 4.17±2.60 | 4.30±2.37 | 0.360 |
| No. of follicles with diameter≥16 mm | 2.94±1.78 | 3.30±2.12 | 3.18±1.83 | 0.178 |
| No. of follicles with diameter≥18 mm | 1.63±1.21 | 1.81±1.36 | 1.63±1.32 | 0.418 |
| No. of oocytes retrieved | 5.12±3.53 | 4.57±3.16# | 5.65±3.54 | 0.020 |
| No. of mature oocytes | 4.52±3.16# | 4.11±2.83# | 5.06±3.20 | 0.020 |
| Fertilization rate | 75.89%(1442/1900) | 80.63%(512/635) | 77.75%(755/971) | 0.689 |
| Cleavage rate | 98.02%(1140/1163) | 97.22%(420/432) | 95.93%(590/615) | 0.709 |
| No. of embryos available | 1.95±1.50# | 1.96±1.28# | 2.27±1.59 | 0.029 |
| No. of high quality embryos | 1.30±1.48 | 1.35±1.34 | 1.44±1.45 | 0.371 |
2.3. 新鲜移植周期妊娠结局
3组间新鲜周期种植率、早期流产率、异位妊娠率、活产率比较均无统计学差异（P>0.05）。中晚期添加组临床妊娠率高于无添加组（P < 0.05），但早期添加组与无添加组及中晚期添加组比较均无统计学差异（P>0.05，表 3）。
3.
新鲜移植周期妊娠结局
Clinical outcomes of fresh embryo transfer cycle
| Index | No addition | Early addition | Middle and late addition | P |
| *P < 0.05 vs early addition group; #P < 0.05 vs Middle and late addition group. | ||||
| No. of cycles | 130 | 24 | 71 | - |
| No. of embryos transferred | 1.93±0.59 | 1.96±0.55 | 2.06±0.53 | 0.315 |
| Implantation rate | 15.94% (40/251) | 21.28% (10/47) | 24.66% (36/146) | 0.289 |
| Clinical pregnancy rate | 27.7% (36/130)# | 33.3% (8/24) | 45.1% (32/71) | 0.045 |
| Early miscarriage rate | 8.3% (3/36) | 25.0% (2/8) | 21.9% (7/32) | 0.234 |
| Heterotopic pregnancy rate | 2.8% (1/36) | 0.0% (0/8) | 0.0% (0/32) | 0.570 |
| Live birth rate | 21.5% (28/130) | 25.0% (6/24) | 31.0% (22/71) | 0.334 |
2.4. 全胚冷冻周期首次冻融胚胎移植（FET）妊娠结局
中晚期添加组临床妊娠率较早期与无添加组妊娠率稍高，但未达到统计学差异，3组间种植率、早期流产率、异位妊娠率、活产率比较均无显著性差异（P>0.05，表 4）。
4.
全胚冷冻周期首次FET妊娠结局
Clinical outcomes of the first frozen-thawed embryo transfer cycle with a "freeze-all" strategy
| Index | No addition | Early addition | Middle and late addition | P |
| No. of cycles | 139 | 73 | 70 | - |
| No. of embryos transferred | 1.72±0.53 | 1.74±0.50 | 1.81±0.43 | 0.354 |
| Implantation rate | 17.15% (41/239) | 19.69% (25/127) | 20.47% (26/127) | 0.823 |
| Clinical pregnancy rate | 24.5% (34/139) | 28.8% (21/73) | 31.4% (22/70) | 0.537 |
| Early miscarriage rate | 20.6% (7/34) | 33.3% (7/21) | 9.1% (2/22) | 0.147 |
| Heterotopic pregnancy rate | 0.0% (0/34) | 4.8% (1/21) | 0.0% (0/22) | 0.259 |
| Live birth rate | 15.1% (21/139) | 16.4% (12/73) | 27.1% (19/70) | 0.204 |
3. 讨论
本研究资料显示，中晚期添加组的成熟卵子数、可用胚胎数、新鲜周期临床妊娠率明显升高，提示GnRHant方案中中晚期添加HMG能改善高龄DOR人群的促排卵结局和新鲜周期临床妊娠率。此外，无添加组扳机日的LH水平为4.52±5.18 IU/L，与早期和中晚期添加组相比无显著差异，表明添加HMG后不影响血清LH水平，不能够通过血清LH水平反应添加HMG后的LH水平。
HMG是外源性LH制剂的一种，是从绝经期妇女尿中提取的促性腺激素（Gn），含FSH∶LH比例1∶1，HMG中LH的活性主要由hCG驱动[9]，与LH相比，hCG半衰期更长[10]，与黄体生成素/人绒毛膜促性腺激素受体（LHCGR）亲和性更高[11]，其分泌不受性激素反馈调节机制的影响，能够为卵泡生长提供更稳定的刺激环境。
女性35岁以后LH的生物活性下降[12]，使用GnRHant会导致卵泡液中LH、AMH水平显着降低，而AMH与卵母细胞的着床潜力正相关，补充HMG能诱导卵泡分泌雄激素，刺激AMH分泌，改善卵母细胞质量[13]，对于高龄DOR患者，推测添加HMG能增加IVF成功率[14-15]。有学者观察到，高龄患者添加rLH与单用rFSH相比，获卵数、种植率、流产率等均无差异，并无额外益处[16-18]。Alviggi[19]则提出，补充外源性LH制剂可能对两类患者有利，包括预期对卵巢刺激反应较迟钝者以及36~39岁者，与本文研究结果一致。既往研究多集中于长方案以及rLH添加与否对妊娠结局的影响，本文根据添加时机进行分组，首次明确阐述了GnRH-ant方案中高龄DOR患者中晚期添加HMG可获益。
本研究中，与中晚期添加组相比，早期添加组的患者扳机后获卵数偏少，差异有统计学意义。根据LH窗[20]和LH ceiling学说[21]，甾体激素的合成及优势卵泡的继续生长需要在合适的LH作用水平下，即达到LH阈值，但不能超过一定的LH范围，过低或过高均不利于卵泡发育。不同发育阶段的卵泡LH上限不同，与未成熟卵泡相比，成熟卵泡的LH上限更高。考虑早期添加组获卵数下降可能是由于GnRH-ant方案允许内源性LH的早期作用，再添加HMG会使得LH活性超过上限值，早卵泡期，未成熟卵泡耐受性较低，LH活性过高，将会抑制颗粒细胞增殖，造成过早黄素化或卵泡闭锁[22]。一方面，颗粒细胞功能丧失和过早黄素化，会导致许多与卵母细胞发育有关的细胞因子如抑制素、胰岛素样生长因子等合成受阻，干扰卵丘-卵母细胞复合体的成熟过程[23]；另一方面，获卵数的减少造成最终可利用的胚胎数少，这可能是早期添加组成熟卵子数、可利用胚胎数均低于晚期添加组的原因。
生理状态下，卵泡早期，FSH刺激卵泡募集，使小窦状卵泡免于凋亡。卵泡中期，血液中升高的E2通过负反馈机制抑制FSH，但被选择的优势卵泡依然可以进一步发育生长，很大程度上是由于卵泡逐渐依赖于LH[24]，该阶段颗粒细胞开始表达自身LHCG受体并能直接对LH产生应答，同时卵母细胞、颗粒细胞和卵泡膜细胞产生的卵泡内调节因子使得颗粒细胞对FSH敏感性降低，LH的作用日益增大。高龄DOR患者处于生殖功能的老化阶段，颗粒细胞有丝分裂速度减慢，凋亡比例增高[25]，LHCG受体敏感性下降。在卵泡中后期，当生长的卵泡处于对类固醇生物合成刺激和最终成熟的最大需求时，内源性LH水平快速下降，可能会对卵子正常发育造成不利影响[26]。此时添加HMG符合生理需求，中晚期添加HMG能上调FSHR和LHR的表达，增加芳香化酶的活性，增加颗粒细胞对FSH的敏感性[27-28]，刺激雄激素- AMH轴，从而改善卵子发育能力，提高胚胎质量[29]。因此无添加组及中晚期添加组在获卵数上虽无差异，但在成熟卵子数及可利用胚胎数上中晚期添加组明显更高，这与国内外学者研究一致，卵泡中后期添加HMG可以弥补内源性LH活性不足带来的负面影响，促进卵母细胞发育成熟[30-31]。
研究中观察到，中晚期添加组鲜胚移植周期临床妊娠率明显高于无添加组，除了由于中晚期添加HMG促进卵子发育成熟，子宫内膜上存在LHCG受体[32]，研究证实，HMG中的hCG成分对蜕膜化、着床、血管生成等过程具有重要的旁分泌作用[33-34]，hCG可以直接作用于内膜调节蜕膜代谢，诱导基底层成纤维细胞中与蜕膜化相关的α-平滑肌细胞肌动蛋白合成，调节血管内皮生长因子、巨噬细胞集落刺激因子、胰岛素样生长因子结合蛋白1等的表达，同时保护内膜基质细胞免于氧化应激相关的凋亡[35]。此外，当子宫内膜过早或过久暴露于hCG作用时，会触发LHCG受体下调和下游信号减敏，细胞外信号调节激酶1/2磷酸化消除，内膜细胞的粘附能力和紧密连接调节受损，继而影响妊娠率，与Evans等的研究一致[36]，中晚期适量添加HMG能改善内膜容受性，但过早添加反而削弱hCG的生物学作用，这可能是早期添加组临床妊娠率高于无添加组，而低于中晚期添加组的原因。3组中，中晚期添加组活产率仍最高，比无添加组高9.5%，但差异无统计学意义，考虑与中晚期添加组样本量较少有关，有待扩大样本量进一步研究。
新鲜移植周期中晚期添加组临床妊娠率高于无添加组，而在全胚冷冻周期两组差异在统计学上不显著，推测可能原因是卵巢刺激周期中，中晚期添加HMG能改善子宫内膜的接受能力，促进内膜血管化，而冻胚移植可能消除了HMG对内膜的积极作用。Andersen等[37]的研究也表明，促排卵过程使用HMG会使血清和卵泡产生不同的激素水平波动，继而影响内分泌反应和子宫内膜接受性。
本研究中，早期添加组与中晚期添加组Gn使用量均高于无添加组，可能是由于HMG含有较多的尿蛋白杂质，制剂批次间差异较大，其生物活性存在内变异，不如通过基因工程单克隆技术合成的FSH制剂纯度高，生物活性和均一性好。但国产的HMG价格仅为进口rFSH的1/9，仍然不失为是一种经济有效的促排卵药物选择。
综上所述，应用GnRH-ant方案时，中晚期添加HMG促进卵泡发育，可能可以提高子宫内膜的容受性，有利于改善高龄DOR患者的新鲜周期临床妊娠率。本研究为回顾性研究，有待于严谨的前瞻性随机对照试验进一步分析，在药代动力学和分子水平上对其调控机制进行验证。
Biography
吴晓敏，硕士研究生，E-mail: 1549947824@qq.com
Funding Statement
国家重点研发计划(2017YFC1001100);南方医科大学临床研究启动计划(LC2016ZD010);南方医科大学南方医院临床研究计划(2018CR016)
Supported by National Key Research & Developmental Program of China (2017YFC1001100)
Contributor Information
吴 晓敏 (Xiaomin WU), Email: 1549947824@qq.com.
陈 士岭 (Shiling CHEN), Email: chensl_92@vip.163.com.
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