Introduction
Ovarian stimulation (OS) during in vitro fertilization (IVF) treatment cycle aims to generate multiple oocytes per single aspiration to maximize its success (Trounson et al., Reference Trounson, Leeton, Wood, Webb and Wood1981). During the IVF treatment cycle, resumption of meiosis and final oocyte maturation of the prophase-arrested oocytes is induced by the administration of hCG or GnRH agonist prior to follicle aspiration (Zhang et al., Reference Zhang, Ouyang and Xia2009). Following aspiration, embryologists examined oocyte morphological appearance and classified their maturity as follows: germinal vesicle (GV), metaphase 1 (MI), or metaphase 2 (MII) (Ebner et al., Reference Ebner, Moser, Sommergruber and Tews2003). Within 38 h after triggering final follicular maturation 85% of the retrieved oocytes are expected to be classified as MII, whereas 10% will present as GV, and 5% of the retrieved oocytes appear as MI oocytes (Rienzi et al., Reference Rienzi, Ubaldi, Iacobelli, Minasi, Romano, Ferrero, Sapienza, Baroni, Litwicka and Greco2008).
Oocytes eligible for intracytoplasmic sperm injection (ICSI) are those that have progressed through meiosis to MII, while the remaining immature oocytes are usually discarded (Coetzee and Windt, Reference Coetzee and Windt1996). Although the injection of immature oocytes has been reported to result in a low fertilization rate (De Vos et al., Reference De Vos, Van de Velde, Joris and Van Steirteghem1999; Shu et al., Reference Shu, Gebhardt, Watt, Lyon, Dasig and Behr2007; Li et al., Reference Li, Li, Ma, Feng, Yang, Wu, Zhong, Che and Chen2011; Álvarez et al., Reference Álvarez, García-Garrido, Taronger and González de Merlo2013; De Vincentiis et al., Reference De Vincentiis, De Martino, Buffone and Brugo-Olmedo2013; Ko et al., Reference Ko, Lee, Park, Yang and Lim2015; Margalit et al., Reference Margalit, Ben-Haroush, Garor, Kotler, Shefer, Krasilnikov, Tzabari, Oron, Shufaro and Sapir2019), with the potential for abnormal embryonic development due to chromosomal abnormalities and defective cytoplasmic maturation (Racowsky and Kaufman, Reference Racowsky and Kaufman1992; Smith, Reference Smith2001), it is usually adopted in an attempt to increase the number of embryos achieved to enhance the chance of pregnancy (Vanhoutte et al., Reference Vanhoutte, De Sutter, Van der Elst and Dhont2005).
Sachdev et al. (Reference Sachdev, Grifo and Licciardi2016) demonstrated that embryos derived from immature oocytes that were injected (ICSI) following a delay in vitro maturation within 1 day after aspiration, showed equivalent fertilization and blastocyst formation rates. However, these showed increased aneuploidy rates, as demonstrated by preimplantation genetic testing for aneuploidy (PGT-A) prior to embryo transfer (Sachdev et al., Reference Sachdev, Grifo and Licciardi2016). Moreover, embryos derived from delayed mature oocytes may also show a different morphokinetic profile from their sibling oocytes aspirated at the MII stage (Margalit et al., Reference Margalit, Ben-Haroush, Garor, Kotler, Shefer, Krasilnikov, Tzabari, Oron, Shufaro and Sapir2019).
ICSI for delayed immature oocytes has been described in the published literature as having lower outcomes compared with mature oocytes on the day of ovum pick up (De Vos et al., Reference De Vos, Van de Velde, Joris and Van Steirteghem1999; Shu et al., Reference Shu, Gebhardt, Watt, Lyon, Dasig and Behr2007; De Vincentiis et al., Reference De Vincentiis, De Martino, Buffone and Brugo-Olmedo2013; Ko et al., Reference Ko, Lee, Park, Yang and Lim2015; Margalit et al., Reference Margalit, Ben-Haroush, Garor, Kotler, Shefer, Krasilnikov, Tzabari, Oron, Shufaro and Sapir2019). However, delayed mature oocyte injection increased the number of embryos achieved to enhance the chance of pregnancy (Vanhoutte et al., Reference Vanhoutte, De Sutter, Van der Elst and Dhont2005). Still, when considering patients with very poor ovarian reserve, or those undergoing fertility preservation for different medical backgrounds, any additional embryo might possess a crucial clinical value, including those achieved from the rescue of delayed matured oocytes.
While most of the aforementioned studies have focused on oocytes that reached maturity a few hours after oocyte retrieval or specifically MI delayed oocyte (De Vincentiis et al., Reference De Vincentiis, De Martino, Buffone and Brugo-Olmedo2013; De Vos et al., Reference De Vos, Van de Velde, Joris and Van Steirteghem1999; Ko et al., Reference Ko, Lee, Park, Yang and Lim2015; Margalit et al., Reference Margalit, Ben-Haroush, Garor, Kotler, Shefer, Krasilnikov, Tzabari, Oron, Shufaro and Sapir2019; Shu et al., Reference Shu, Gebhardt, Watt, Lyon, Dasig and Behr2007), in the present study, we aimed to assess the outcome of all in vitro matured oocytes, either derived from GV or MI, that reached maturity (MII) and injected (ICSI) 24 h following retrieval. Although it is reasonable to perform ICSI for delayed immature oocytes on the day after ovum pick up in cases in which the fertilization rate is low, in our centre the delayed ICSI was performed in all cases in which it was possible in terms of the workload in the laboratory.
Of notice, Ming et al. (Reference Ming, Liu, Qiao, Lian, Zheng, Ren, Huang and Wu2012) analyzed the outcome of fresh versus frozen–thawed embryo transfer (ET) following rescue ICSI cycles performed in cases of total fertilization failure in conventional IVF cycles. Significantly higher clinical pregnancy and implantation rates were achieved following frozen–thawed compared with fresh ET. They therefore recommended that embryos derived from rescue ICSI cycles should be cryopreserved and subsequently used in frozen–thawed cycles. Prompted by this information, and due to the asynchrony between the endometrium and embryonic development stage of those derived from delayed mature oocytes, we aimed to further evaluate the ET timing, either in the index fresh cycle or during a subsequent frozen–thawed ET.
Materials and methods
Study population
We reviewed the computerized files of all consecutive patients admitted to our IVF programme at Sheba medical centre from September 2016 to December 2020. For the purpose of this study, we included only patients undergoing IVF who had at least one injected day 1 delayed mature oocyte. The decision whether to inject a delayed mature oocyte was according to the workflow in the IVF laboratory and the fertilization oocyte status on day 1.
The study was approved by the Institutional Review Board of the Sheba Medical Center ethical committee.
Ovarian stimulation
The decision as to which type of OS protocol to use was made by the treating physician. Most of the patients underwent OS using the GnRH antagonist protocol and the remaining used the long GnRH agonist protocol. Gonadotropins were administered in variable doses, depending on patient age and/or ovarian responsiveness in previous cycles. These doses were further adjusted according to serum oestradiol (E2) levels and vaginal ultrasound measurements of follicular diameter obtained every 2 or 3 days. Oocyte retrieval (OPU) was undertaken ∼35–38 h post hCG trigger. Endometrial preparation, ET, and other procedures, were performed as previously described (Mohr-Sasson et al., Reference Mohr-Sasson, Orvieto, Blumenfeld, Axelrod, Mor-Hadar, Grin, Aizer and Haas2020).
Embryo culture, assessment and vitrification
Cumulus–oocyte complexes were isolated during follicle aspiration/ovum pick up (OPU) and then held in equilibrated SAGE fertilization medium + protein (Quinn’s advantage protein plus fertilization, SAGE, Cooper Surgical) under SAGE mineral oil (oil for tissue culture, SAGE, Cooper Surgical) in a 37°C 5% O2 and 5.5% CO2 atmosphere incubator (benchtop G210 K-system) for 2–4 h. Oocyte denuding was performed ∼38 h post final follicular maturation triggering and re-examined 2–3 h later for ICSI. Only MII oocytes were eligible for ICSI and the remaining oocytes were cultured for re-evaluation during fertilization evaluation. Spermatozoa collection for ICSI was performed using ICSI dishes prepared by adding multipurpose handling medium (MHM; FUJIFILM Irvine Scientific, USA) drops covered with paraffin oil (SAGE, Cooper Surgical, Inc., USA). Semen were kept at room temperature for an additional 24 h.
Following ICSI, oocytes were divided into two groups, in the same dish and culture medium: half were incubated in equilibrated Global Total medium (Global Total, Life Global, Cooper Surgical), and the remaining were incubated in equilibrated continuous single culture medium (FUJIFILM Irvine Scientific). The medium drops were covered with SAGE mineral oil. At ∼16–20 h later, oocytes were examined for the presence of pronuclei.
Embryos were assessed on day 3 (68 h ± 1 h post-ICSI) based on the individual embryo scoring parameters according to the Istanbul consensus (Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology, 2011). A top-quality embryo (TQE) was defined as having seven or eight blastomeres on day 3, having equally sized blastomeres, and up to 10% fragmentation with no vacuoles or multinucleation. For cycles in which embryos were vitrified or transferred on day 2, four blastomere embryos with up to 10% fragmentation, with equally sized blastomeres and with no vacuoles or multinucleation were also considered to be top-quality embryos. Embryo transfer had been performed mostly on cleavage-stage embryos. According to our laboratory policy, embryos not eligible for transfer/cryopreservation at the cleavage stage are cultured up to days 5–6. Only those that developed to good quality blastocysts were transferred/cryopreserved. Grading of blastocysts was according to the Gardner method (Gardner et al., Reference Gardner, Lane, Stevens, Schlenker and Schoolcraft2000, Reference Gardner, Lane and Schoolcraft2002), based on the assessment of the inner cell mass and trophectoderm appearance. Only type A and type B blastocysts were transferred or vitrified.
The vitrification–warming methods were carried as previously described (Aizer et al., Reference Aizer, Noach-Hirsh, Dratviman-Storobinsky, Haas and Orvieto2021).
Fertilization, clinical pregnancy, and live-birth rate
For fertilization analysis, sibling oocytes were divided into two groups: Mature oocytes were those documented as MII on day 0 post follicle aspiration and delayed mature oocytes were those documented as MII on day 1 post follicle aspiration (and were defined as MI or GV on day 0). Fertilization rate was defined as the presence of two pronuclear (2PN) divided to the total MII oocyte.
Clinical pregnancy was defined as visualization of a gestational sac and fetal cardiac activity on transvaginal ultrasound. Implantation rate was defined as the number of gestational sacs observed, divided by the number of embryos transferred. The live-birth rate was defined as the number of live offspring delivered, divided by the number of ETs.
Clinical pregnancy and live-birth rate were calculated for embryos obtained from delayed mature oocytes that were transferred in the fresh or adjacent frozen–thawed cycles.
Statistical analysis
Statistical analysis was performed using Student’s t-test and chi-squared test, as appropriate. Continuous variables are presented as means ± standard deviations (SD). The significance threshold was set as P < 0.05.
Results
The study population consisted of 362 women who underwent 476 IVF cycles, with at least one injected day 1 delayed mature oocyte. The clinical characteristics of the study group are presented in Table 1.
Patients’ baseline clinical characteristics
Table 2 presents the laboratory and embryological variables of the corresponding ICSI cycles. In total, 4023 oocytes were aspirated with a mean of 8.5 ± 5.8 oocytes per OPU; 2150 oocytes were mature (MII) on day 0 and 851 of the remaining immature oocytes were injected on day 1. The mean time interval between OPU and injection was 4.5 ± 0.9 and 23.6 ± 3.7 h for mature and delayed mature oocytes, respectively. Figure 1 describes the pronuclear (PN) status on day 2 post OPU during fertilization check. In total, 68/851 (8%) were degenerative, 33/851 (3.9%) contained multi-PN, 24/851 (2.8%) were mono-PN (1PN), 497/851 (58.4%) were 2PN, and 67 had undocumented fertilization. Fertilization rates per injected mature oocytes were comparable between the delayed mature oocyte group compared with injection in the day 0 group (58.4% vs 62%, respectively; P = 0.07) (Table 2).
IVF cycles’ characteristics
Allocation of delayed mature oocytes according to fertilization outcomes.
In contrast, the TQE rates per injected MII day 0 oocyte were significantly higher compared with day 1 injected oocytes (57.5% vs 44.4%, respectively; P < 0.001) (Table 2).
Clinical outcomes are presented in Table 3. Following fresh embryo transfer, implantation rate and the clinical pregnancy and live-birth rates (LBR) per transfer were 3.9%, 3.3% and 1.6% respectively (Table 3). When considering the following thawed ET cycles, implantation, pregnancy and LBRs were non-significantly higher (10%, 8.3% and 8.3%, respectively) (Table 3).
Reproductive outcomes stratified according to fresh vs frozen cycles of delayed mature oocyte-derived embryos
Discussion
Ovarian hyperstimulation is a fundamental step in the success of IVF, enabling the recruitment of multiple oocytes. However, due to the extreme heterogeneity in ovarian response to OS, some patients may yield only a few mature follicles, if any (Ben-Rafael et al., Reference Ben-Rafael, Orvieto and Feldberg1994). The follicular cohort might consist of small-sized follicles, yielding immature oocytes that did not complete cytoplasmic maturation (Ectors et al., Reference Ectors, Vanderzwalmen, Van Hoeck, Nijs, Verhaegen, Delvigne, Schoysman and Leroy1997; Triwitayakorn et al., Reference Triwitayakorn, Suwajanakorn, Pruksananonda, Sereepapong and Ahnonkitpanit2003). The presence of immature oocytes among the cohort of the retrieved oocytes is a common feature that results from heterogeneous, asynchronous follicles at different developmental stages (Smitz and Cortvrindt, Reference Smitz and Cortvrindt1999) following OS.
Our strategy to perform rescue of delayed mature oocyte was intended for women with zero fertilized oocytes on the next day of OPU, but not only this. It was suitable for patients with low ovarian reserve, patients undergoing preimplantation genetic diagnosis (PGT), in which there were few embryos suitable for ET, and patients undergoing fertility preservation for medical reasons, etc. (Table 1).
In the present study, the fertilization rate of delayed mature oocytes was comparable with mature oocytes after injection on day 0, in which TQE rate was significantly lower. Moreover, following fresh ET, implantation rate and the clinical pregnancy and LBRs per transfer were non-significantly lower compared with the following thawed ET cycles. These observations were in accordance with Ming et al. (Reference Ming, Liu, Qiao, Lian, Zheng, Ren, Huang and Wu2012), who emphasized the importance of synchronization between embryo development and the endometrium. Therefore, for good quality embryos derived from delayed mature oocytes, we recommend that the ongoing embryo should be frozen and transferred in a subsequent frozen–thawed cycle. According to our findings, although the clinical outcomes of pregnancy and live-birth rate are relatively low, when comparing fresh versus frozen–thawed ET of delayed mature oocyte, the frozen–thawed cycle revealed a better outcome as reflected by non-significantly higher implantation and clinical pregnancy and LBRs.
Whereas delayed mature oocyte may resume meiosis and reach the MII stage, it has lower developmental competence. This could be observed by comparing high-quality embryos derived from sibling oocytes of matured versus delayed mature oocytes. We found that delayed mature oocytes produced significantly lower numbers of high-quality embryos. This observation was in agreement with other studies, demonstrating more cleavage-stage arrest (Chen et al., Reference Chen, Chen, Lien, Ho, Chang and Yang2000; Wang and Keefe, Reference Wang and Keefe2002), higher numbers of multinucleated blastomeres (Chen et al., Reference Chen, Chen, Lien, Ho, Chang and Yang2000) and a reduced development to the blastocyst stage (Li et al., Reference Li, Feng, Cao, Zheng, Yang, Mullen, Critser and Chen2006). Generally, the aforementioned outcomes were in accordance with evidence indicating that delayed matured oocytes had an increased incidence of spindle abnormalities and disrupted chromosomal alignments compared with mature oocytes on day of OPU (Balakier et al., Reference Balakier, Sojecki, Motamedi and Librach2004; Strassburger et al., Reference Strassburger, Friedler, Raziel, Kasterstein, Schachter and Ron-El2004).
The limitations of the present study are its retrospective nature, which limits the ability to control for confounding factors, and its limited sample size. Although CPR and LBR are significantly higher when using embryos derived from mature oocyte compared with delayed mature oocyte, additional embryos derived from delayed mature oocytes might contribute to the embryo cohort and increase the cumulative live-birth rate per retrieval. Moreover, the embryos derived from delayed mature oocyte favoured a transfer in a frozen–thawed cycle rather than in a fresh cycle.
Acknowledgements
The authors would like to thank Moran Madari for her contributions to data collection and secretarial assistance.
Financial support
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Conflict of interest
The authors report no financial or commercial conflicts of interest.
Ethical standards
The authors assert that all procedures contributing to this work complied with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008.
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