Oocytes morphology is commonly used to predict embryo competence and pregnancy outcomes in ART treatment, especially in ICSI cycles, as cytoplasmic structures are distinctively displayed in denuded oocytes. However, dysmorphisms always puzzle embryologists because of its repetitive occurrence [1, 15] and the controversies surrounding both the causes of formation and effects on clinical outcomes in previous studies [3, 7, 10, 13–15]. CLCG is one of these abnormal phenotypes and previous studies proposed that CLCG might be a sign of oocytes cytoplasmic immaturity [7, 8, 9] and its formation could be related to patients’ age and Gn type [10]. A very recent case study showed that an antagonist protocol could improve the oocytes morphology with regards to CLCG, as well as the embryo quality, which indicates that the stimulation protocol is also related to the occurrence of CLCG for specific patients [15], Moreover, it was reported that long term exposure to a pesticide contaminated environment could cause CLCG in human oocytes, and severely affect the fertility of local people [11, 12]. Although there is no agreement regarding the causes of formation, we believe that exogenous stress and some patient innate features might be related to the manifestation of CLCG in human oocytes, because of repetitive occurrence in some patients and the effects of pesticide or Gn stimulation.
Most studies on the factors relevant to CLCG formation analyzed patients where CLCG was found in all oocytes in a sibling cohort [7, 14, 18] or patients where at least one CLCG oocytes was found in one cohort [10]. In our preliminary observation, we found that there were always some patients repetitively showing either pCLCG or aCLCG. Whether these signatures of CLCG occurrence represent different populations of patients is unknown. Hence, in this study, we compared patients’ features among three groups of patients (Control, pCLCG and aCLCG), and surprisingly, pCLCG patients had a significantly lower BMI and higher AMH level. Corresponding to this distinctive feature, the pCLCG group showed lower fertilization and blastocyst formation rates compared to the control group. In contrast, aCLCG patients did not show any difference from the control group in any of the analyzed clinical and embryo competence features, which is consistent to other studies [10, 13, 14, 16, 19]. Consistent with previous studies, aCLCG did not show any effects on all three clinical outcomes in fresh cycles [14, 20, 21] but the clinical outcomes in pCLCG were dramatically worse. According to our results, we infer that the detrimental effects on the fertilization and blastocyst formation in the pCLCG group is probably not caused by the CLCG structure, and that the pCLCG group may represent a specific patient population distinctive from both control and aCLCG patients.
Organelles and cytoplasmic structures are sensitive to the freeze/thaw process [22, 26]. As a type of cytoplasmic structure and probably aggregations of organelles [1], CLCG structures could affect the clinical result after cryopreservation. A recent study showed different tolerance of vitrification and thawing among granulation patterns and thawed oocytes with appearance of smooth center and course edge of CLCG significantly affect the clinical outcomes [17]. Our tracing observation showed that the CLCG structure can be inherited by cleavage embryos and possibly blastocysts, which suggest that the embryos derived from CLCG oocytes may also have a higher sensitivity to the freeze/thaw process. An early study showed that freeze/thaw survival rate of embryos derived from CLCG oocytes was dramatically reduced [22]. Consistently, this study found a higher degradation of embryos after the freeze/thaw process in CLCG patients (Fig. 4d). However, much better survival rates compared to the early study [22] can be noticed, which reflects a general improvement of cryopreservation technique in current days.
Surprisingly, few studies pay attention to the effect of CLCG structure on the clinical outcomes in FET cycles, even though FET has been extensively adopted in ART treatment. For the first time, this study clearly showed a significant reduction of all three clinical outcomes in aCLCG patients in FET cycles compared to the control, and this compromising effect was not found in pCLCG patients. These results strongly suggest that CLCG structures may have long term detrimental effects on the embryos subjected to cryopreservation, even though the survival rate after freeze/thaw has been remarkably improved nowadays (Fig. 4d). The comparison of clinical outcomes between fresh transfer cycles and FET cycles among three groups of patients, revealed similar results in the control group, improved results in FET cycles in pCLCG patients, and compromised results in FET cycles in aCLCG patients. These distinctively different outcomes between the pCLCG and the aCLCG groups regarding fresh transfer and FET cycles further indicate that pCLCG patients probably represent an unique population with distinctive clinical features i.e. lower BMI and higher AMH, as well as being more suitable for FET rather than fresh transfer. It has been proven that clinical outcomes can be improved by performing FET for polycystic ovary syndrome (PCOS) patients [27]. Although we didn’t find a significant relevance between this group of patients with the diagnosis of PCOS (Table 1, p > 0.05), a particular type of PCOS—lean PCOS with lower BMI has been proposed and well analyzed [28]. Hence, pCLCG patients with lower BMI and higher AMH might be related to the diagnosis of lean PCOS, which may explain the improvement of clinical outcomes in FET cycles for PCOS patients. However, further investigation need to be done to clarify the relationship between pCLCG and lean PCOS.