探討在人體外授精過程中胚胎早期分裂用不同卵巢刺激策略之關聯與濾泡液中基質金屬蛋白酶的活性變化

Abstract

本論文分成兩個部分，第一部分主題為評估是否使用促性腺協同劑方案或促性腺拮抗劑方案，胚胎早期分裂是否為一個好的懷孕率的預測值；第二部分主題為評估人體濾泡液中基質金屬蛋白酶 (matrix metalloproteinases, MMPs)活性及其抑制因子(tissue inhibitor of MMPs, TIMPs)濃度是否與卵子成熟度有任何相關。

第一部分：此部份的研究為回溯性資料分析，534個接受人工生殖技術的病人，其中331個病人接受促性腺協同劑方案 做為卵巢刺激，而203個病人接受促性腺拮抗劑方案，在每組中，病人如果有至少一顆早期分裂的胚胎植入就歸為早期分裂的組別；而病人沒有早期胚胎可植入就歸為晚期分裂的組別。我們比較早期分裂組及晚期分裂組的懷孕率在使用不同的方案是否有差異。研究結果顯示在使用促性腺協同劑方案的這一組中，植入早期分裂的胚胎，懷孕率明顯比植入晚期分裂的胚胎高(50% vs. 37.8%, P < 0.0001)，而在使用促性腺拮抗劑方案的這一組中，植入早期分裂或是晚期分裂的胚胎，懷孕率並沒有顯著差別。而使用邏輯回歸統計方式，我們發現在使用促性腺協同劑方案這組，年齡越大，早期分裂的胚胎數目越少( P < 0.001)；使用促性腺拮抗劑方案這組， 年齡越大，早期分裂的胚胎數目並不隨年齡改變( P = 0.61)。本部分研究結論為使用促性腺協同劑方案，胚胎早期分裂是一個好的懷孕率的預測值；而使用促性腺拮抗劑方案，胚胎早期分裂並不是一個好的懷孕率的預測值。使用促性腺協同劑方案，年齡越大 早期分裂的胚胎數目越少；但使用促性腺拮抗劑方案，早期分裂的胚胎數目並不隨年齡改變。

第二部分：本研究為前瞻性研究，共有150個接受人工生殖技術的病人進行這個研究。我們收集取卵過程中濾泡液去分析MMP-2、MMP-9、TIMP-1及TIMP-2對卵子成熟度或是胚胎發育是否有任何關連。共有1504個卵子進行分析。研究結果顯示人體濾泡液中MMP-2活性與卵子成熟度有顯著性正相關(P < 0.001) 。而人體濾泡液中MMP-2活性 越高，則胚胎正常受精率越高(P < 0.01)。人體濾泡液中MMP-9、TIMP-1、TIMP-2與卵子成熟度無關。研究結論為人體濾泡液中MMP-2活性越高與卵子成熟度越好。且人體濾泡液中MMP-2活性越高，則胚胎正常受精率越高。人體濾泡液中MMP-2活性可作為一個好的卵子成熟度的預測值。

關鍵詞：胚胎早期分裂、促性腺協同劑、促性腺拮抗劑、濾泡液、體外授精、卵子成熟度、基質金屬蛋白酶、基質金屬蛋白酶組織抑制因子

There are two parts of topics performed in this study. In the first part, we wonder to test if early-cleavage was a strong predictor of pregnancy in patients receiving either a GnRH agonist long protocol or a GnRH antagonist protocol for in-vitro fertilization treatment (IVF) and intracytoplasmic sperm injection (ICSI). In the second part, we wonder to determine whether matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMP-1 and TIMP-2) in human follicular fluid, have any relationships with oocyte maturation in vitro and subsequent fertilization during IVF or ICSI.

Part I of the study: This retrospective study included 534 patients undergoing a fresh cycle of oocyte retrieval and the day-3 embryo transfer (from 22 to 46 years old). Of the 534 patients treated, 331 received a GnRH agonist long stimulation protocol (GnRH agonist group) for ovarian stimulation and 203 patients received a GnRH antagonist protocol (GnRH antagonist group). In each group, patients who had at least one early-cleavage embryo transferred were designated as the ‘early-cleavage’ subgroup. Patients who had no early-cleavage embryos transferred were designated as the ‘late-cleavage’ subgroup. The early cleavage rate was significantly lower in the GnRH antagonist group compared with that in theGnRH agonist group (IVF cycles: 34% vs. 20%; ICSI cycles: 50% vs. 37.8%, respectively, P < 0.0001). In the GnRH agonist group, the pregnancy rates were significantly higher in the early-cleavage subgroup than those in the late-cleavage subgroup (53.7% vs. 33.9%, P < 0.0001). In the GnRH antagonist group, the pregnancy rates were not significantly different between the early-cleavage and late-cleavage subgroups (45.9% vs. 43.8%, P > 0.05). By logistic regression analysis, the rate of embryonic early-cleavage was significantly decreased with increasing age of women in the agonist (P < 0.001) but not in antagonist groups (P = 0.61). Based on the results of this study, maternal age is a critical factor for embryonic early-cleavage in agonist protocol but not in antagonist protocol. The results also showed that early-cleavage embryos were of better quality and resulted in a higher pregnancy rate than late-cleavage embryos in the GnRH agonist group. However, embryo quality and pregnancy rate was not significantly different between early and late cleavage embryos in the GnRH antagonist group. Early cleavage of zygote is not a reliable predictor for embryo implantation potential in using the GnRH antagonist protocol. The implantation rates between the GnRH agonist and GnRH antagonist groups were comparable. We also conclude that embryonic early-cleavage status is negatively correlated with aging in women receiving GnRH agonist long down-regulation but not in GnRH antagonist protocols. Early cleavage of the zygote is not a reliable predictor for pregnancy potential using the GnRH antagonist protocol.

Part II of the study: This was a prospective study. The follicular fluids were obtained from 150 female patients undergoing IVF/ICSI cycles and a total of 1,504 oocytes were retrieved for analysis. MMP-2 and MMP-9 activities were measured using zymography assay. TIMP-1 and TIMP-2 concentrations were quantitatively assessed using enzyme-linked immunosorbent assay (ELISA). Human follicular fluid MMP-2 level was significantly associated with the rate of maturity of oocytes (P < 0.001). Furthermore, the MMP-2 was significantly associated with the higher fertilization rate (P < 0.01). There was no significant correlation between follicular MMP-9 and the maturation rate of oocytes. The TIMP-1 and TIMP-2 also showed no correlation with the oocyte maturation rate. We found that the level of gelatinase MMP-2 in human follicular fluid might be a reliable marker of mature oocytes during IVF/ICSI cycles. Therefore, the MMP-2 expression has a strong association with higher fertilization rate. Further studies are needed to support this theory.

Key Words: embryonic early cleavage, GnRH agonist, GnRH antagonist, follicular fluid, in vitro fertilization, oocytes maturation, matrix metalloproteinase, tissue inhibitor of matrix metalloproteinase