In the present study, we found that transfer of MG day 3 cleavage embryos in patients with low RMD3 had similar clinical outcomes as transfer of MG day 3 embryos in patients with high RMD3. However, if seeking blastocysts-stage transfer, the usable blastocyst formation rate from MG day 3 embryos, implantation rate and live birth rate from transfer of MG blastocyts were significantly reduced in patients with low RMD3 as compared to patients with high RMD3.
Previous studies showed that patients with number of good quality embryos (> 3) on day 3 indicated good prognosis, and blastocyst transfer was suggested for those patients (20, 21). Different from those studies, in the present study, we grouped the patients/cycles by RMD3.This definition focused on the formative capacity of overall MG embryos on day 3 rather than the absolute counts. It is known that the follicles within ovary promoted by controlled ovarian stimulation share a common growth environment and genetic foundation. High RMD3 may reflect good maturation of oocytes and ovarian condition, while low RMD3 may reflect weak maturation of oocytes and ovarian function. However, whether the remaining MG embryos from low RMD3 show similar quality with MG embryos from high RMD3 remains elusive Due to fresh cycles with 2PN zygotes = > 5 in the present study, the patients included in the present study were at least normal responders.
In the present, patients with ETC and ETB represented distinct population. This was due to the laboratory strategy. In our reproductive center, around 2 best quality of MG day 3 embryos will be frozen or transferred, and the remaining will be further cultured. For embryo transfer, MG blastocysts have the highest priority, followed by MG day 3 embryos. Therefore, patients having frozen ETC may indicate no available MG blastocysts,and about half of patients having fresh ETC resulted in live birth in the present study. We noticed that patients having ETB showed higher ovarian reserve and response, less Gn use and more oocytes retrieved, and higher usable blastocysts formation rate (from MG or MNG day 3 embryos) in the L or H group, compared to patients having ETC. This was reasonable. Because, with similar RMD3, patients with more number of eggs and high rate of blastocyst formation mean more chance of having MG blastocysts. It has been reported that more dose of Gn use adversely affect the quality of eggs (22, 23). By contrast, a recent study showed that poor responders with less Gn use may result in better oocyte quality(24). Therefore, it was possible that less Gn dose may be associated with better performance of patients in ETB group.
The baseline characteristics of patients and consequent ovulation protocols were grossly comparable between the L and H groups in patients with ETB or ETC .For ETC, patients in the H group showed relatively low response to Gn, evidenced by similar Gn use and reduced oocytes retrieved compared to the L group. However, the reduced oocytes retrieved were compensated by high RMD3, resulting in more available MG embryos on day 3 and subsequent more MG day 3 embryos for expended culture in the H group. For ETB, patients in the H group showed similar Gn use and oocytes retrieved, compared to patients in the L group. Furthermore, we observed that normal fertilization rate was significantly higher in the H group than the L group in either ETC or ETB group. Therefore, grouping by RMD3 didn’t introduce obvious confounding factors regarding baseline characteristics of patients and cycles.
Blastulation rate is also an important indicator reflecting embryo quality. Useable blastocyst formation rate (from MG day 3 embryos) was significantly reduced in the L group in either ETC or ETB group. However, useable blastocyst formation rate from MNG day 3 embryos were similar in the L and H groups in either ETC or ETB group. Due to limited cases of MNG day 3 embryos for further culture in the H group, this observation need large data to confirm. In addition, the difference in useable blastocyst formation rate between the H and L groups is bigger in ETC group than that in ETB group. We proposed that over all good performance of patients in ETB group may partially shorten the difference in usable blastocysts formation rate between the H and L groups. In conclusion, MG day 3 embryos from patients with low RMD3 showed decreased in vitro developmental potential (DP).
The golden maker reflecting the quality of embryo is whether the embryo can result in a live birth (25). For ETB, we found that not only clinical pregnancy rate and live birth rate, but also implantation rate and livebirth rate per embryo were significantly higher in the H group than the L group. It is know that female age and BMI, number of embryos transferred and semen DFI and days of blastocysts may have profound influences on the clinical outcomes of in vitro fertilization (25–31). Conflicting results were reported regarding the effect of male age on the clinical outcomes of in vitro fertilization (32, 33).However, in the present study, we found that there was a trend towards decreased male age in the H group, compared to the L group. Therefore, the male age was also taken as a potential confounding factor. As expected, we showed that number of embryos transferred positively while female age adversely affected clinical pregnancy and live birth, indicating the confidence of our data. In line with a recent study (34), transfer of day 6 blastocysts increase the risk of abortion than transfer of day 5 blastocysts. After controlling factors including female or male age, number of embryos transferred, female BMI, semen DFI and days of blastocyst, we found MG blastocysts from the L group showed decreased chance of implantation or live birth, compared to MG blastocysts from the H group. Therefore, the in vivo DP of MG blastocysts was reduced significantly in patients with low RMD3, compared to patients with high RMD3.
To our surprise, we found that although more ET cycles with grade I or grade I + grade II embryos in the H group, transfer of MG day 3 embryos resulted in similar clinical pregnancy rate, live birth rate, implantation rate per embryo and live birth rate per embryo between the H and L groups. Further analysis showed that semen DFI positively and minimally affected the clinical pregnancy and live birth. This result was inconsistent with previous findings(31, 35). However, DFI of males in the present study was low, it was possible that a higher of DFI (under certain threshold) may minimally benefit patients with transfer of MG day 3 embryos. Consistent with previous studies (36, 37), we observed that female age adversely affected clinical pregnancy and live birth. In addition, grade I embryos did not result in better clinical outcomes as compared to grade II embryos. After controlling factors including female or male age, number of embryos transferred, female BMI, semen DFI and grade of embryos, we found MG embryos from the L group showed similar chance of implantation or live birth, compared to the H group. Therefore, the in vivo DP of MG day 3 embryos was similar in patients with high and low RTD3.
A low in vitro DP and similar in vivo DP of MG day 3 embryos were observed in the L group, compared to the H group. It has been proposed that day 3 embryos which fails to develop into blastocysts in vitro may lead to live births in vivo (38). One reasonable explanation was that MG day 3 embryos from patients with low RMD3 is sensitive to in vitro culture but not to in vivo environment, compared to MG day 3 embryos from patients with high RMD3.
Patients with low RMD3 may experience satisfying 2PN zygotes and limited number of MG day 3 embryos. Our study showed that a single transfer cycle of MG day 3 embryos for patients with low RMD3 had similar clinical outcomes as compared to a single transfer cycle of MG day 3 embryos for patients with high RMD3. However, if seeking blastocysts-stage transfer, the usable blastocysts formation rate from MG day 3 embryos, implantation rate and live birth rate from transfer of MG blastocyts were significantly reduced in patients with low RMD3 as compared to patients with high RMD3. According to our study, we suggested that for patients with low RMD3, it seems that transfer of MG day 3 embryos may benefit patients. This strategy my potentially avoid the possible wastage of MG day 3 embryos during in vitro culture and the possible wastage of MG blastocysts during implantation.
The main limitation of this study is its nature of the retrospective study. Patients with ETB and ETC represented relatively separate population in either the L group or the H group. A concern is whether it was appropriate that results from separate populations were integrated for analysis. This study was a comparison study and did not provide the direct evidence. Furthermore, the data included for analysis were from single center and the cases included in the present study is limited, therefore, the indications from this study still need to be confirmed by large randomized control trial.