Effect of the time interval between oocyte retrieval and ICSI on embryo development and reproductive outcomes: a systematic review

Intra-cytoplasmic sperm injection (ICSI) is a technique to achieve fertilisation for couples with severe male infertility, in which the sperm is inserted into the cytoplasm of the oocyte [1]. After oocyte collection, the cumulus cells need to be denudated before ICSI, a process called oocyte denudation (DN). However, different assisted reproductive technology (ART) laboratories implement different protocols depending on their daily workload and different time intervals, including the time of oocyte pick up (OPU), DN, and ICSI [2‐5].

During follicular development, cumulus cells that surround the oocytes can promote maturation of cytoplasm and nucleus of the oocytes through an autocrine/paracrine mechanism and communicate through gap junction [6, 7]. Before ovulation, cumulus cells proliferate widely and provide basic nutrition for oocytes to support their development. Additionally, cumulus cells also participate in meiotic arrest, transcription regulation, and induction of cytoplasmic maturation [8, 9]. After ovulation, oocytes resume meiotic separation under Leutinizing Hormone (LH) stimulation; they complete the first meiosis, stop at the second meiosis metaphase (MII), and reach meiotic maturation [10]. Simultaneously, cumulus cells produce hyaluronic acid and expand in vitro, and further induce oocyte cytoplasmic maturation by stimulating gene expression and reducing oxidative stress [11, 12]. Research shows that after ovulation induction, gap junctions between cumulus cells around oocytes continue to exist, whereas distant junctions disappear. Using this strategy, intact cumulus cells can use gap junctions between oocytes to promote oocyte maturation [13]. However, DN must be performed prior to ICSI for two reasons: a) the cumulus cells affect the entrance of the injection needle, and that a cumulus-oocyte-complex (COC) cannot be held properly by the holding pipette; b) oocyte maturity must be assessed since only mature oocytes that have reached the MII stage should be injected. Moreover, the polar body has to be placed at 12 or 6 o’clock positions to avoid the oocyte spindle from being damaged [14].

Therefore, an important question arises: When should the cumulus cells around the oocytes be removed to obtain the optimal ICSI outcome? Though many researchers have tried to confirm the optimal timing intervals in ICSI procedures, these time intervals and their impact on the outcomes of ICSI cycles remain controversial. Some studies have found that prolonging the incubation periods between oocyte pickup (OPU) and DN can improve the rates of fertilisation and blastocyst formation, which is conducive to embryo development. It is suggested that oocytes should be incubated for a few hours before DN [15‐17]. However, recent studies in mice have shown that prolonged incubation time before DN can induce oocyte apoptosis. In contrast, some studies suggest DN immediately after oocyte retrieval [18]. Other studies found that prolonging the time of OPU-DN could not improve the rates of fertilisation or clinical pregnancy [5, 19]. A negative correlation between the time of DN and ICSI has also been reported, i.e., longer DN-ICSI time lowers the fertilisation rate, and may lead to adverse pregnancy outcomes [17, 20]. Therefore, the aim of this study was to systematically analyse and review the published literature on the impact of time intervals on embryo development and clinical outcomes and determine the optimal time intervals during ICSI cycles.

PubMed and the Embase databases were searched for relevant studies and reviews using the following keyword combinations: 1) ‘Oocyte Denudation/Denudation/Oocyte’ and ‘Intra-cytoplasmic Sperm Injection/ICSI’ and 2) ‘Oocyte/Oocyte maturation/ cumulus’ and ‘Cumulus removal/ removal.’ The search strategy was limited to articles published in English involving human subjects, with the last search performed on 25 July 2020. Articles that described the effect of DN time on embryo development and clinical outcomes were included in this study, with the additional requirement that they should include at least one of the following indicators: mature egg rate, fertilisation rate, high-quality embryo rate, cleavage rate, blastocyst formation rate, implantation rate, clinical pregnancy rate, and live birth rate. The specific selection process is shown in Fig. 1.

For ART to achieve a positive outcome, it is important to obtain high-quality mature oocytes. Oocytes maturation involves both nuclear and cytoplasmic maturation [36]. Nuclear maturation involves recovery from the first meiosis, germinal vesicle breakdown, and the first polar body formation. Cytoplasmic maturation can help prepare the oocyte for fertilisation and subsequent embryonic development, and it can provide enough energy, enzymes, and protein synthesis reserve to meet the needs of new functional protein synthesis during embryonic development [8, 9]. Unlike the nucleus, there is no clear standard for defining and detecting cytoplasmic maturation, which is a highly complex process. In the natural process, the cytoplasm and nucleus may mature synchronously in some way, but they may not be completely synchronised in the ovulation induction cycle [21]. If the cytoplasm of oocytes is not mature during ICSI, this may directly affect fertilisation and embryo development. It may also impair the supply of material to the embryo, resulting in early embryo death and pregnancy failure. Whether intact or incomplete cumulus cells should be incubated before ICSI remains controversial. Therefore, this issue is discussed in the following sections.

After the introduction of ICSI, based on experience with in vitro fertilisation, some researchers hypothesised that incubation of the obtained oocytes before ICSI for a certain duration before DN and/or ICSI might help to achieve a better outcome. However, to date, no consensus has been established. We found that most studies (11/16) concluded that OPU-DN time (0–12 h) did not affect oocyte maturation rate. However, some studies (5/16) showed the opposite trend, which found the oocyte maturation could be improved through a period of incubation [2, 26]. Considering the differences in methodologies used, there may have been certain confounding factors, one of which was the HCG-OPU time. Studies have confirmed that appropriate prolongation of in vivo maturation time, i.e. HCG-OPU > 36 h, can improve oocyte maturation, thereby improving embryo and pregnancy outcomes [37, 38]. In this review, we found that the HCG-OPU time in five studies was less than 36 h [5, 26, 31, 34, 35]. However, in other studies, HCG-OPU was approximately 36 h, with a better homogeneity. Among these five studies, two suggested that prolonging the HCG-OPU and OPU-DN time was beneficial to oocyte cytoplasmic maturation [26, 31]. Ho et al. [26] found that when HCG-OPU was 34 h, the oocyte maturity in the group that were degranulated within 2.5 h was significantly lower than that of other groups, with no differences in fertilization rate and pregnancy outcome. And they suggested that prolonging HCG-OPU time could shorten in vitro incubation [26]. Maggiulli et al. [35] found no correlation between HCG-OPU and mature egg rate and embryo development. Two studies had divided groups into either HCG-OPU < 36 h or > 36 h and found the time of OPU-DN had no effect on fertilization and clinical pregnancy rates regardless of whether HCG-OPU was more or less than 36 h [5, 34]. Therefore, for good homogeneity, we only collected data in groups with HCG-OPU > 36 h in the two studies.

A study has found evidence that meiosis of oocytes might be blocked by the corona-cumulus-complex, suggesting that oocytes could complete meiosis after the removal of cumulus cells [23]. However, almost all the included studies have concluded that DN-ICSI time does not affect oocyte maturation. Hassan [2] compared the effects of OPU-DN and DN-ICSI on oocyte maturation rate, and found that incubation with intact cumulus cells before DN could improve the maturation of oocytes, whereas incubation after DN could not improve the maturation of oocytes. Some studies have suggested that oocyte regulation and the consequent gene expression of cumulus cells and bidirectional control require expression gap junction-associated genes and structural integrity [39]. The expression of connexin and gap junctions was related to oocyte maturation after a short time but decreased with oocyte maturation [40]. Therefore, the role of cumulus cells in controlling oocyte maturation is significant before germinal vesicle breakdown. After ovulation induction, gap junctions between cumulus cells around oocytes continue to exist, whereas distant junctions disappear. Using this strategy, intact cumulus cells can use gap junctions between oocytes to promote oocyte maturation [13]. The specific mechanism underlying this process needs further research.

There is no consensus on the effect of OPU-DN on fertilisation rate; most studies have concluded that there is no significant effect. It has been suggested that this may be due to the synchronous development of the nucleus and cytoplasmic maturation of oocytes during ovulation induction rather than the beneficial effect of peripheral cumulus cells, or that the ICSI may have avoided some mechanisms associated with cytoplasmic maturation [21, 35]. Bárcena et al. [19] have suggested that oocytes from young and fertile people are more tolerant to long-term incubation in vitro without affecting their subsequent development. Patrat et al. [17] suggested that even if the number of mature oocytes could not be increased, incubation would further promote the cytoplasmic maturation of oocytes, improving their fertilisation potential. Peripheral cumulus cells may secrete some paracrine substances and growth factors or express some adhesion molecules to promote the nuclear or cytoplasmic maturation of oocytes [7, 39]. For example, brain-derived neurotrophic factor secreted by cumulus cells is important for the development of the nucleus and cytoplasm of oocytes [41].

Conflicting results concerning the effect of DN-ICSI time on fertilisation were found in the literature. Most authors have concluded that there is no effect, but Patrat et al. [17] have found that DN-ICSI time is negatively related to fertilisation rate. Another study has suggested that a longer incubation time results in spindle instability and chromosomal material loss in oocytes [42]. Therefore, the authors suggested that ICSI should be carried out immediately after degranulation. Some studies found that it was positively correlated with fertilisation rate but significantly decreased from 5 h after degranulation [33]. Some studies suggest that the reason for the increase in fertilisation rate is not only related to the further maturation of cytoplasm, but is also related to oocyte ageing [20, 33]. Oocyte ageing is related to the activities of the M-phase promoter (MPF) and mitogen-activated protein kinase (MAPK), which are important regulators of the second meiosis [43]. With the ageing of oocytes, the activities of MPF and MAPK decrease, which leads to the spontaneous activation of oocytes [44]. Therefore, ageing oocytes with less MPF levels are more likely to be activated by ICSI to form the pronucleus, thus explaining the increase in the fertilisation rate with increased OPU-ICSI time [20].

There is a special situation that cannot be ignored, oocyte spontaneous activation, which is a rapid and uncontrollable process. Normally, oocytes are obtained naturally or COH remains arrested at MII until fertilization, at which the oocyte resumes meiosis [45, 46]. Meiotic arrest is achieved through a series of cytostatic factor activities, such as c-Mos/ MAPK and EMI2 [46‐48]. However, in certain cases, advanced maternal age, high FSH exposure for a long time in COH, or high vacuum pressure during oocyte retrieval, may induce oocyte spontaneous activation [49‐51]. Oocyte spontaneous activation can lead to premature separation of sister chromatids which are then scattered in the cytoplasm. Once re-activated by a sperm, these separated or scattered chromatids will form 3PN or multiple pronuclei (MPN) [45, 52]. Repeated abnormal fertilization has been previously reported, which may be caused by genetic defect resulting in spontaneous activation [45, 50, 51, 53, 54]. Additionally, the unexplained infertility or repeated pregnancy loss may also be associated with the parthenogenesis caused by spontaneous activation of oocytes prior to ovulation [45, 54]. Therefore, for those who failed to conceive after multiple cycles, especially those who repeat 3PN or MPN, the intervals in ICSI should be avoided since oocyte spontaneous activation could be a contributing factor. To minimize the effect, oocytes should be denuded immediately after collection, followed by a careful and rapid ICSI to mitigate oocyte spontaneous activation-induced abnormal fertilization and possible aneuploidy.

More than half of the studies examined concluded that prolonging OPU-DN time could not improve the rate of good-quality embryos. Some studies found that incubation for 2–4 h before DN could increase the rate of high-quality embryos [15, 16], but that longer times led to a decrease in the rate of high-quality embryos [22]. Underlying reasons include the possibility that cumulus cells may promote the further maturation of oocyte cytoplasm, conducive to subsequent embryonic development. Conversely, COC is sensitive to oxidative stress in vitro, and reactive oxygen species (ROS) can accelerate oocyte ageing, which decreases MAPK activity, meiosis acceleration, the non-separation of sister chromatids, and the increase of aneuploid chromosome number in oocytes [55, 56]. Studies have shown that cumulus cells can secrete glutathione, which may delay the oocyte ageing process and improve embryo quality in a short-term incubation [57, 58]. However, if incubation is prolonged, the antioxidants produced by cumulus cells are not enough to counteract ROS. Therefore, long-term accumulated oxidative stress damages mitochondrial DNA and reduces the ratio of ATP and glutathione/glutathione disulfide in cells, resulting in abnormal cytoskeleton fibres and calcium signalling in the endoplasmic reticulum. It can also induce abnormal calcium oscillation after fertilisation, which manifests as abnormal fertilisation and embryo development [59‐61]. One study found that the rate of high-quality embryos in the group performing ICSI immediately after DN was higher than that in the group incubated for 3 h [27]. If the oocytes were incubated for more than 5–6 h, the frequency of high-quality embryos decreased, but the frequency of inferior embryos in the latter three groups was considerably higher than that in the former three groups [3]. The authors suggested that the results indicated that the quality of embryo segmentation depended on the period of oocyte preincubation before injection. In this review, 10 articles had mentioned culture environment, of which only 3 had oocyte incubation at 5% O₂ [15, 32, 35], while most incubated at 20% O₂. It has been confirmed that 20% O₂ can accelerate the formation of ROS in culture medium, induce histone modification, inactivate enzymes, and cause membrane lipid peroxidation, thereby damaging the surface of embryo membrane and affecting its development [56, 62]. Therefore, the obtained oocytes should be incubated in hypoxia environment, which can effectively reduce the production of exogenous ROS and improve the embryonic development potential and outcome of assisted reproduction.

There are a few studies on blastocyst formation rates. Although two studies found that the blastocyst formation rate in the group incubated for 2 or 4 h after DN was higher than that in the immediate degranulation group with no incubation, significant differences were not found [2, 16]. One recent study reported the effect of ICSI immediately after degranulation and after four HCG-DN periods, namely < 37 h, 37–37.5 h, 37.5–38 h, and > 38 h, and found that the blastocyst formation rate decreased with time (44.6% ± 27.5, 39.8% ± 27.2, 36.9% ± 28.4, and 33.0% ± 27.8%, respectively) [35]. They suggested that this was unrelated to the in vitro maturation of immature oocytes, and that the specific mechanism should be studied further [35].

Most of the studies examined for this review have concluded that OPU-DN does not affect the implantation and pregnancy rates. Some found that the highest implantation rate was achieved with 1.5–2 h incubation before DN, a duration that also optimised the pregnancy and live birth rates [17]. The clinical pregnancy rate following incubation for 1, 2, or 4 h before DN was higher than that in the immediate degranulation group [2, 4]. It was suggested that the reason for this could be the appearance of spindles, which resulted from further maturation of the oocyte cytoplasm through the incubation of cumulus cells [4]. It has been confirmed that if the spindles in oocytes are visible upon observation, most of which appears 39–40.5 h after hCG administration, the fertilisation and implantation rates are higher, but after this duration, the oocyte begins to deteriorate. Therefore, it is recommended that ICSI should be performed 39–40.5 h after hCG administration [63, 64]. Considering the effects of the time of DN-ICSI on the pregnancy outcome, Pujol et al. [20] found that, for each 1 h increase in DN-ICSI time, the biochemical and clinical pregnancy rates decreased by 7.5 and 7.9%, respectively, but the continuous pregnancy and live birth rates were unaffected. Moreover, they suggested in vitro ageing of human oocytes significantly affected the chance of becoming pregnant, and ICSI should not be delayed whenever possible [20]. Two studies found that oocytes incubated for 4 h or 6 h resulted in a similar or increased pregnancy rate, which then decreased remarkably [3, 33]. This may be due to the oocytes reaching their best states for fertilisation after incubation within a certain duration before DN. Over-incubation of the oocyte can lead to changes in ultrastructure and gene expression, and increase the incidence of spindle abnormalities, which will affect subsequent embryonic development and reduce the clinical pregnancy rate [63, 65]. Currently, there is no indication that DN time affects the live birth rate.

According to our literature review on the effects of the time intervals on embryo development and pregnancy outcomes, the results showed that the incubation time before degranulation usually had a small positive effect on ICSI outcome and no negative effect. A short incubation time may be beneficial for embryo development and pregnancy outcome; however, excessive incubation (> 4 h) should be avoided. However, the incubation time after degranulation remains controversial, and negative effects have been observed upon varying this interval, in addition to studies showing no effect or a favourable outcome. Therefore, whether ICSI should be performed after a period of recovery following degranulation should be investigated further. In conclusion, further multicenter, randomised controlled studies with large sample sizes are warranted to optimise the precise timing of the ICSI procedure in the future.