Smooth endoplasmic reticulum aggregates in oocytes associated with increased risk of neonatal birth defects: A meta-analysis.

Rui Long, Lixia Zhu and Lei Jin contributed to the study design. Qiyu Yang and Yini Zhang contributed to the data acquisition and analysis. Limin Gao was brought in for consensus when there was disagreement. Rui Long, Limin Gao and Meng Wang contributed to the data interpretation. Rui Long contributed to the article writing. All authors made critical revisions and gave final approval of the version to be published.

This study was supported by the National Key Research and Development Program of China (2022YFC2702503), the Key Research of Huazhong University of Science and Technology, Tongji Hospital (2022A20) and Hubei Key Laboratory Open Fund of Environmental and Health Hazards of Persistent Toxic Pollutants (PTS2022‐3).

The search strategy yielded 122 studies. However, 26 were duplicates and 68 did not fulfill the selection criteria. Out of 28 potential studies for the analysis, we further excluded 4 studies that had no available control groups and 10 publications lack of birth defects data. A total of 14 studies were included in the meta‐analysis. 3 , 5 , 7 , 8 , 9 , 10 , 13 , 14 , 19 , 20 , 21 , 22 , 23 , 24 All the included studies were retrospective cohort studies ranged from 2005 to 2021, involving 4633 SERa‐ cycle infants and 711 SERa+ cycle infants, summarized in Table  1 . Seven of the included studies provided detailed data about malformations in children conceived by SERa+/− oocyte in SERa+ cycle, 7 , 8 , 9 , 19 , 20 , 23 , 24 shown in Table  2 . Characteristics of included studies of birth defects in babies from SERa+ cycles and SERa‐ cycles. Abbreviations: ICSI, intracytoplasmic sperm injection; IVF, in vitro fertilization; MAR, medically assisted reproduction; SERa, smooth endoplasmic reticulum aggregates; TOP, terminal of pregnancy. Number of birth defects in children derived from SERa+ oocytes and SERa‐ oocytes. Abbreviation: SERa, smooth endoplasmic reticulum aggregates. Two of the included studies 5 , 20 enrolled children derived from conventional IVF and ICSI cycles, and others only enrolled ICSI cycles. The specific organ system data of birth defects in SERa+ cycles were mentioned in six studies, 3 , 9 , 19 , 23 which are summarized in Table  3 . Notably, neonatal birth defects in the circulatory system, genitourinary system and abnormal chromosome had a high proportion (30.8%, 23.1% and 23.1%, respectively). Most of these studies only observed the birth defects of live birth and two studies 9 , 14 mentioned live birth and termination of pregnancy (TOP) for congenital malformations. In order to evaluate the real risk of SERa+ cycles/oocytes, both birth defects of live birth and TOP for congenital malformations were both included in the calculation of the birth defects rate. Number of birth defects in SERa+ cycles in specific organ system from included studies. Abbreviation: SERa, smooth endoplasmic reticulum aggregates. Three of the studies mentioned stillbirths and neonatal deaths. 3 , 5 , 19 Massarotti et al. 5 mentioned one stillbirth in a twin pregnancy among the newborns of SERa‐ cycle. One preterm death with no visible external malformation in the SERa+ group and two with reasonable explanations (maternal infection and placenta abruption) in SERa‐ group were recorded in the study of Shaw‐Jackson et al. 19 Ebner et al. 3 did not mention the cause of two neonatal deaths in the mixed transfer cycle (double embryo transfer including one SERa+ conceptus). Overall, 2.1% (15/711) of babies conceived by SERa+ cycles and 1.3% (62/4633) of babies conceived by SERa‐ cycles had birth defects. Malformation rates and separate data are shown in Figure  2 . The rate of birth defects was significantly higher in babies derived from SERa+ cycles (risk ratio (RR) = 2.17, 95%CI 1.24–3.81, p  = 0.007). The individual risk estimates for these studies ranged from 0.42 to 5.55. No heterogeneity of risk ratio was among these studies ( I 2  = 0%, p  = 0.97). Sensitivity analysis was performed to observe stability through removing each study and reanalyzing the remaining studies. No significant difference was found when one study 8 was excluded (RR = 1.83, 95%CI 0.88–3.84, p  = 0.11). In the funnel plot, the studies were symmetrically distributed, indicating no publication bias (Figure  S1 ). Comparison of birth defects rate between children conceived by SERa+ cycles and SERa‐ cycles (fix effected model). SERa, smooth endoplasmic reticulum aggregates. Seven included studies mentioned the neonatal outcomes of SERa+ oocytes and SERa‐ oocytes from SERa+ cycles (cycles with at least one SERa+ oocyte). A subgroup analysis was performed to explore the differences in birth defects between SERa+ oocytes and sibling SERa‐ oocytes from the SERa+ cycles. Since birth defects cannot be confirmed in mix embryo transfer, we only included SERa+ or sibling SERa‐ embryo transfer in SERa+ cycles. Figure  3 depicted the results of the fix‐effects model. A higher risk of birth defects was exhibited in children derived from SERa+ oocytes than from sibling SERa‐ oocytes in SERa+ cycles (RR = 3.53, 95%CI 1.21–10.24, p  = 0.02). No substantial heterogeneity was found in this subgroup analysis ( I 2  = 0%, p  = 0.62). Studies in the subgroup were symmetrically distributed (Figure  S2 ). Comparison of birth defects rate between children conceived by SERa+ oocytes and SERa‐ oocytes in SERa+ cycles (fix effected model). SERa, smooth endoplasmic reticulum aggregates.

Due to the unknown safety problem, embryos derived from SERa+ cycles/oocytes were generally avoided to transfer. As a result, the number of children conceived from SERa+ cycles/oocytes in the previous published studies were limited. It has been difficult to draw the precise conclusions about the safety of SERa+ cycles/oocytes. Therefore, there was no strong evidence for a relationship between SERa+ cycle/oocyte and birth defects based on the basis of the currently published studies. Thus, we included all relevant studies that met the inclusion criteria to enlarge the sample size and performed a standard meta‐analysis. The results indicated that children conceived by SERa+ cycles had a significantly higher risk of birth defects compared with SERa‐ cycles. According to the oocyte type of SERa in the SERa+ cycles, we divided into SERa+ oocytes and sibling SERa‐ oocytes, and found that SERa+ oocytes could increase the risk of birth defects in the offspring. What's more, substantial heterogeneity was not observed in all outcomes. Previous studies have reported malformations or genetic abnormalities in the newborns derived from SERa+ cycles/oocytes, such as Beckwith–Wiedmann syndrome, diaphragmatic hemia, multiple malformations and cardiovascular defects. 3 , 4 , 25 In addition, Ebner et al. 3 mentioned the relationship between SERa+ and an increased tendency for obstetric complications, which is an additional factor leading to the birth defects. Based on these data, the Alpha Scientists and ESHRE guidelines advised against the use of SERa+ oocytes. 11 However, some healthy infants conceived from SERa+ oocyte have been shown in several publications. 5 , 13 , 14 A recent meta‐analysis also found that there was no significant difference in live birth and birth weight between SERa+ and SERa‐ cycles. 12 As a consequence, the polices of IVF centers towards SERa+ cycles/oocytes are not homogeneous, which needs to be further confirmed by long‐term follow‐up and observation of neonates. In this meta‐analysis, the risk of birth defects was significantly increased in children conceived by SERa+ cycles than SERa‐ cycles. In the subgroup analysis, a higher risk of birth defects was found in children derived from SERa+ oocytes than from SERa‐ oocytes. Overall, all the results suggest that SERa may raise the risk of birth defects in newborns. The main function of the smooth endoplasmic reticulum is calcium storage and release after sperm entry, and it plays a key role in energy accumulation throughout oocyte activation and early embryo development. 23 , 26 , 27 Indeed, the presence of aggregates has been shown to disrupt calcium release and oscillation, with one large SERa having a more deleterious effect than several smaller ones. 28 Modification of the amplitude of calcium oscillations has been hypothesized to affect fetal development through epigenetic modifications in rabbits. 29 Increasing evidences suggested that aberrant epigenetic modifications were related to the occurrence of birth defects, such as congenital heart disease, 30 congenital diaphragmatic hernia 31 and cleft palate. 32 We speculate that SERa+ oocytes may exhibit epigenetic disorders which are transmitted to subsequent embryos and fetus. Further studies are needed to verify this hypothesis and to elucidate the molecular mechanisms. In addition, Stigliani et al. 33 compared gene expression profiles between SERa+ and SERa‐ oocytes, and found downregulation of several genes involved in cytokinesis and mitotic/meiotic regulation, spindle assembly and chromosome segregation or genes involved in mitochondrial structure and respiratory activity which may lead to aberrant embryo cleavage. Dal Canto et al. demonstrated that SERa+ oocytes reflect intrinsic damage to the human oocyte cytoskeleton, namely alterations in spindle size, chromosome misalignment and cortical actin disorganization via immunofluorescence staining. 34 The incidence of mitotic cleavage failure after fertilization and meiotic cleavage failure during the second polar body extrusion was significantly higher in SERa+ oocytes than in SERa‐ oocytes. 35 Abnormal chromosome segregation during early embryo development can lead to chromosome aneuploidy, resulting in embryonic death or congenital malformation. 36 Previous studies have compared the blastocyst ploidy outcomes among the SERa+ oocytes and SERa‐ oocytes. Although no significant differences were observed in the aneuploidy rate, an apparently increased trend was shown in the SERa+ oocytes group. 37 , 38 On the contrary, Wang et al. found that SERa+ oocytes had a higher proportion of euploid embryos compared to the sibling SERa‐ oocytes. 8 They attributed the reason to the increased blastocyst self‐repair and renewal capacity after self‐selection and the uneven sample size of cases. In our analysis, the birth defects of children conceived by SERa+ cycle had a high proportion of abnormal chromosomes (3/13, 23.1%), although the numbers were too small for statistical comparisons. The aberrant epigenetic modifications and abnormal chromosome segregation may partly explain our conclusions. In the future, mechanism explorations and clinical studies with a larger samples size are needed to explore the underlying mechanism. Furthermore, previous study demonstrated that the large (18 μm) and the medium (10–17 μm) size of SERa can be observed by light microscopy. However, the small sized SERa (2–9 μm) were not visible under the conditions used. 3 , 4 Therefore, even transfer the SERa‐ oocytes derived from the SERa+ cycle may also lead to a detrimental effect on the obstetric and neonatal outcome. Although embryos originating from SERa+ cycles/oocytes were not recommended to be discarded easily due to the comparable implantation and developmental potential as SERa‐ cycles/oocytes, 2 clinicians should fully assess the quality of whole embryos and make prudent decisions about embryo transferring. Comprehensive counseling on the pregnancy chance and birth defects risk should be offered to patients confronted with SERa+ on a case‐by‐case basis. Significantly, pregnancy and neonatal outcomes should be closely monitored. Besides, the mechanism of SERa formation is still unclear. In the current studies, SERa+ has been reported to be associated with prolonged duration and dose of gonadotropin, which leads to increasing estradiol concentrations during ovarian stimulation. Alternatively, Saito et al. suggested that an elevation of serum estradiol may not be the cause of SERa but a consequence of their common trigger. 39 Interestingly, SERa were not observed in oocytes from unstimulated patients, contrasting with a frequency of 2%–7% in cycles with ovarian stimulation, which further supports the idea that exposure to extracorporeal gonadotrophins during the final period of oocyte maturation might provoke the appearance of SERa. 40 Therefore, a modulated controlled stimulation protocol, like dose reduction, may be a good strategy to reduce the occurrence of SERa in the subsequent cycle. In previous recommendation, it was preferable to inject/inseminate oocytes and transfer embryos without SERa rather than SERa+, in order to avoid potential adverse perinatal outcomes. 2 Our findings demonstrated that the risk of birth defects in children conceived by SERa+ oocytes was higher than by sibling SERa‐ oocytes which also supported this proposal. Although previous studies indicated that the presence of SERa in oocytes was not associated with adverse effects on embryological, clinical or neonatal outcomes, 19 , 20 the sample size was too small to draw reliable conclusions. Our results from the enlarged sample size may offer some instructions to embryologists in selecting embryos for transfer in consideration of priority factors of embryo morphological grading or oocyte SERa. So far, the safety of SERa+ cycle/oocyte is still unclear. No meta‐analyses have investigated the association between SERa+ cycles/oocytes and congenital malformations. Evidence‐based proof is urgently needed to instruct its application. To the best of our knowledge, this is the first and largest meta‐analysis to estimate the risk of birth defects in SERa+ cycles/oocytes which raised the level of evidence and had directed significance of clinical treatment. An improved understanding may help clinicians better managing IVF patients with SER+ oocytes and providing relevant counseling. This study also has inherent limitations. Firstly, due to the unknown safety problem, transferring the embryos derived from SERa+ cycles/oocytes were generally avoided. The number of children conceived from SERa+ cycles/oocytes in this meta‐analysis was limited. Furthermore, the clinical outcomes of SERa+ cycles are largely attributable to the transfer of embryos derived from unaffected oocytes from SERa+ cycles, leading to a small number of newborns from affected SERa+ oocytes, which may cause a selection bias. Secondly, as mentioned above, SERa+ oocytes may affect the function of spindle assembly, spindle size defects and chromosome segregation, embryonic cells that experience cleavage failure during mitosis could become tetraploid and cause chromosomal abnormalities in the embryo, leading to an increased rate of TOP due to fetal defect. 34 However, only two included studies reported the data about TOP. 9 , 14 The real birth defects rate of SERa+ cycles/oocytes may be higher than what we calculated. The sensitivity analysis found that one included study 9 influenced the stability of this result. This study was considered as a high‐quality study (NOS = 7). We reread the study and found that the reason might be related to the data on TOP. The inclusion of TOP would increase the birth defect rate. In order to evaluate the true birth defect rate, this study was still included. Additionally, all the included studies only collected the congenital malformation data at birth. Some chromosomal abnormalities or mental dysplasia may not be detected or may only be detected later in life. Thus, long‐term follow‐up studies are necessary to comprehensively analyze the safety of SERa. Thirdly, two of the included studies 5 , 20 enrolled the SERa+ cycles/oocytes derived from the conventional IVF. Given that SERa were always observed 16–18 h after IVF, it may affect the accuracy of the judgment in SERa+ oocytes. Therefore, a time‐lapse system may be obligatory to solve this tricky problem in the future.

The meta‐analysis indicated that the risk of birth defects in SERa+ cycles and SERa+ oocytes were significantly increased compared with SERa‐ cycles and sibling SERa‐ oocytes, respectively. Our study provides evidence‐based proofs for clinicians and embryologists to make prudent decisions about SERa+ oocytes transferring and offer reliable counseling to couples. Rigorous long‐term follow‐up of neonates derived from SERa+ cycles/oocytes is essential, and more well‐conducted observational studies are needed to confirm the safety of SERa.

Since the first report of smooth endoplasmic reticulum aggregates (SERa) in 1997, 1 several studies have indicated the association between SERa and poorer medically assisted reproduction outcomes, such as increased miscarriage rates, lower oocyte maturation and fertilization rates, poorer embryo quality and decreased pregnancy rates. 2 Moreover, SERa+ women tended to deliver earlier than healthy women which may relate to a higher percentage of obstetric complications (placenta previa, premature rupture of the membranes, hemorrhage, gestosis) during pregnancy. 3 The formation of SERa has been reported to be associated with the dose and duration of gonadotropin administration, not related to patient age, number of retrieved oocytes, presence of endometriosis in ovaries or thickness of the endometrium. 3 , 4 , 5 Brage et al. have reported that SERa affected the quality of the inner cell mass of blastocysts in intracytoplasmic sperm injection (ICSI) cycle. 6 The risk of birth defects in children conceived by SERa+ cycles (one or more SERa were visible by light microscopy after denudation)/oocytes is still unknown. Several previous studies have reported an increased risk of birth defect in infants born from SERa+ cycles/oocytes compared with control group, with or without significant differences. 7 , 8 , 9 , 10 This may be related to the limited sample size of SERa+ cycles/oocytes. Given that the published reports suggested an association between SERa and early fetal demise with certain imprinting disorders, 4 the Istanbul Consensus Workshop in 2011 strongly recommended that oocytes with SERa should not be inseminated. Normally fertilized oocytes in which SERa disks are observed should not be transferred. 11 Whereas a recent meta‐analysis demonstrated that SER+ did not affect the live birth rate or infant body weight. 12 Healthy babies derived from SERa+ cycles, even from SERa+ oocytes have also been reported in several studies. 5 , 13 , 14 Two system reviews have summarized the perinatal outcomes of babies originating from SERa+ cycles, and both recommended that SER+ embryos can be considered when embryos of sufficient quality are not available. 2 , 15 Thus, a revision was conducted in the Vienna consensus in 2017 that the expert panel recommended the decision to inject SERa+ oocytes should be reviewed by the clinical team on a case‐by‐case basis. 16 Overall, a long‐term follow‐up is still needed to verify the effects of SERa on newborns. Due to the unclear neonatal outcomes, SERa+ embryos are only suitable for patients without sufficient quality embryos. Thus, the number of babies born from SERa+ embryos was limited, and the sample size of current studies was too small to draw any firm conclusions. What's more, it is still uncertain whether the SERa+ oocytes and the sibling SERa‐ oocytes in the same cycle have a comparable risk in birth defects. The association between SERa and neonatal outcomes is still controversial and open to debate. Therefore, in order to have a clear proof of the safety in SERa+ cycles/oocytes, we conducted this meta‐analysis to enlarge the sample size and explore the safety of children conceived by SERa+ cycles/oocytes.

The authors declare that they have no conflicts of interest.

This meta‐analysis has been registered on PROSPERO (CRD42022313387) and reported according to the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta‐Analyses (PRISMA). 17 We searched PubMed, Embase, Cochrane Libraries, Web of Science, and Chinese databases including China National Knowledge Infrastructure (CNKI) and Wan Fang from inception to July 2023 for studies reporting on SERa+ and fetal outcomes. A search strategy was conducted using the following keywords and/or medical subject heading (MeSH) terminology: smooth endoplasmic reticulum, SER, SERa, oocyte, oocytes. We also hand‐searched the reference list of the included studies, and relevant reviews to identify any additional studies that may have met the inclusion criteria. All available prospective and retrospective comparative studies (cohort or case–control studies) comparing neonatal birth defects from SERa+ cycles/oocytes and SERa‐ cycles/oocytes were included. Moreover, the included studies were human studies published in English or Chinese with original data, and in vitro fertilization (IVF) or ICSI was used for fertilization. Studies were excluded if: (i) no data about the birth defects; (ii) studies had no available comparable control groups (SERa‐ cycles/oocytes); (iii) review articles, case reports and animal experimental studies; (iiii) the samples and data were duplicated. After a primary screening of all titles and abstracts, the full texts of all potentially eligible studies were retrieved for detailed evaluation by two independent researchers. Disagreements were resolved by discussion with a third reviewer. The search strategy and included studies are shown in Figure  1 . A PRISMA statement flow diagram. The quality of the studies was assessed in accordance with the Newcastle–Ottawa Scale. 18 According to the Newcastle–Ottawa Scale, non‐randomized studies were assessed in three domains: selection (representativeness, selection of the non‐exposed cohort, ascertainment of exposure, whether the outcome of interest was present), comparability (controlled for confounders), and ascertainment of the outcome (assessment of outcome; duration and proportion for follow‐up), with each item given one star. Total stars of <7 and ≥7 were considered as low and high quality of studies, respectively. Two investigators made the revolution independently. Disagreements were resolved under discussion. Two authors extracted the relevant data independently as follows: authors, geographic region, sample size of infants derived from the SERa+ cycle/oocyte group and the control group (SERa‐ cycle/oocyte), number and system of birth defects and other related information. The data were rechecked by other researchers. For dichotomous data, the number of events in the SERa+ cycle/oocyte group and control group of each study were keyed into Review Manager 5.1 software and pooled the findings by Mantel–Hansel method assuming a fixed‐effected model. The statistical heterogeneity was evaluated by forest plots and I 2 statistic (significance level at I 2 >50%). Funnel plot was produced to assess publication bias.

Figure S1. Funnel plot for publication bias of birth defects in babies from SERa+ cycles and SERa‐ cycles. SERa, smooth endoplasmic reticulum aggregates. Figure S2. Funnel plot for publication bias of birth defects in babies from SERa+ oocytes and sibling SERa‐ oocytes in the SERa+ cycle. SERa, smooth endoplasmic reticulum aggregates.