Background
The assessment of oocyte quality using an inverted microscope is mainly based on cytological and morphological criteria. However, this evaluation does not fully reflect the level of oocyte maturation competency, fecundability, or its ability to support early embryonic development. It is commonly accepted that a “good quality” oocyte at metaphase II (MII) stage presents a moderately granular vacuole-free cytoplasm, a thin perivitelline space, a non-fragmented round polar body, and a round, homogeneous, non-dense zona pellucida [
1‐
3]. However, it has been reported that more than 50% of collected oocytes after controlled ovarian stimulation (COS) present one or more anomalies [
4,
5], and generally 93% of patients undergoing assisted reproductive technologies (ART) have at least one abnormal oocyte [
6]. Oocyte dysmorphism could be exhibited as the presence of an abnormally granular cytoplasm, vacuoles, refractile bodies, a wide perivitelline space, an abnormal zona pellucida, aggregations in the endoplasmic reticulum, or other anomalies [
7]. Among these abnormalities, cytoplasmic granularity most commonly affects embryo development [
6,
8‐
10].
Centrally located cytoplasmic granulation (CLCG) was first defined by Serhal et al. [
8] as “clearly delimited central granulations that are denser than the adjacent cytoplasm”. Some authors suggest that there is a relationship between CLCG and smooth endoplasmic reticulum clusters (sERC) [
11] or aggregates of tubular smooth endoplasmic reticulum (aSERT) [
12] with mitochondrial disturbance. Furthermore, Otsuki [
13] observed that numerous small refractile (lipofuscin) bodies were located within the CLCG, suggesting that these abnormalities may have a common origin. The biomolecular explanation for this phenomenon is still enigmatic. However, the most plausible hypothesis that could explain CLCG dysmorphism is cytoplasmic immaturity [
14,
15] which would be responsible for embryonic aneuploidy production at 52–57% [
14,
16] and having 50% of risk to occur it in early meiotic maturation [
17,
18]. Moreover, the most affected oocytes by toxic environment exposure are those at the pre-meiotic maturation stage [
14,
16].
Despite the lack of clear explanation involving the real mechanism of CLCG production in oocytes, the impact on ART outcomes cannot be ignored. As already reported by Serhal et al. [
8], the exhibited CLCG by oocytes declined implantation rate from 10% to just 1% without any ongoing pregnancy. Rienzi et al. [
6] could to evaluate therefore the oocyte morphology and conclude that CLCG affects pronuclear morphology (OR: 2,65 [1,45–4,85]) and embryo quality (OR: 2,26 [1,25–4,08]) confirmed by several studies [
9,
19‐
21]. Kahraman et al. [
14] showed that oocyte CLCG results in 28% of clinical pregnancy with high miscarriage rate of 54% confirming Alikani et al. [
22] results. In fact, this negative impact on ART outcomes is evident result of an increased embryonic aneuploidy production risk issued from oocytes with CLCG [
14,
16,
23].
However, there are discrepancies in results as reported by Rienzi et al. [
3] which could be explained by the presence of intra-individual variation in the percentage of oocytes displaying CLCG in participants of the ICSI program. Nevertheless, Kahraman et al. [
14] focused on CLCG phenotype involving its correlation with variables between different couples, either intrinsically or extrinsically linked to environmental conditions. Indeed, oocyte maturation and follicle physiology have been shown to be impaired by persistent environmental pollutants [
24]. This issue calls into question the adverse effects of pesticide exposure on oocyte quality, i.e. CLCG prevalence and its developmental ability impacting eventually ART outcomes.
Our ART center is located in Picardy (northern France), a region well known for its high annual pesticide consumption of about 3900 tons used in the agricultural sector. Pesticides and their degradation products can contaminate water, soil, and air; therefore humans can be exposed to these compounds. Moreover, endocrine disruptor chemicals derived from certain pesticides (2,2-Bis [p-hydroxyphenyl]-1,1,1-trichloroethane [HPTE]; antiandrogenic endocrine disruptors; high concentration of total bisphenol A [BPA]; diethylstilbestrol [DES]; methoxychlor [MXC]; polychlorinated biphenyl [PCB] congeners, p,p’-dichlorodiphenyltrichloroethane [DDT], and its persistent metabolite p,p’-dichlorodiphenyldichloroethylene [DDE]) are among the factors that are incriminated for having an adverse impact on several aspects of ovarian biology including oocyte quality [
25,
26].
The aim of the present study was to investigate the ICSI outcomes of couples presenting different CLCG prevalence’s of retrieved oocytes (low prevalence of CLCG; fewer than 25% [LCLCG] and high prevalence of CLCG; over 75% [HCLCG]) and its correlation to pesticide exposure zones in the region of Picardy, France.
Discussion
Since the beginning of implementation of ART to treat human infertility, oocyte quality has always been considered the most important key to IVF success, involving various variables to determine its final ability to produce a normal embryo. One-quarter of oocytes have double anomalies, 6% have triple anomalies, and 3% have abnormal shapes [
6,
8‐
10]. Furthermore, the most damaging oocyte dysmoprhism is that linked to its cytoplasmic immaturity, as in the case of CLCG leading to heterogeneity in the oocyte cohort [
14]. This condition is implicitly reflected in the problem of COS response with unbalanced ovarian microenvironment, resulting in a negative impact on IVF outcomes [
26].
Several studies have shown that CLCG prevalence varies from 32% to 63% for mature oocytes, showing lack of consensus between several authors about the negative impact of CLCG on fertilization [
11,
19], embryo cleavage [
29], its quality [
8,
14,
19] and its ability to reach blastocyst stage [
5]. Nevertheless, almost of studies were more agreed about the negative impact of CLCG on clinical outcomes [
3,
5,
6,
8,
11,
14,
29]. In our study, among 633 initial ICSI cycles, 482 couples (76%) had moderate CLCG with a prevalence of 26–74% and were excluded from our study to avoid the non-interpretable results with such a heterogeneous population. For this reason, we decided to focus on analyzing the ICSI outcomes of couples with LCLCG (in 13% of initial cycles [83/633]) and those with HCLCG (in 11% of initial cycles [68/633]), suggesting that CLCG prevalence could have relative impact on ICSI outcomes.
It has been reported that CLCG presence affects the fertilization rate (OR [95%CI]: 1.22 [1.03–1.45]) [
30] and Ebner et al. [
5] were able to show the association between lower fertilization rate and CLCG. In our study, the negative effect on fertilization rate was not sufficient to be significant between LCLCG and HCLCG (Table
2), while other researchers failed to observe any impact of CLCG on fertilization rates [
3,
4,
14,
20,
22,
27,
31‐
33]. This issue could be explained by the ability of endoplasmic reticulum in the oocyte to composite the intrinsic dysregulation by stimulating calcium flux after injection of spermatozoa by ICSI and assuring the necessary activation for fertilization despite CLCG presence [
34‐
36], whereas oocytes with aggregation of the endoplasmic reticulum were associated with a lower fertilization rate and poor embryo quality [
29,
31].
However, the correlation between CLCG exhibited by oocytes and their impaired developmental competence has been discussed in detail by many authors [
6,
37,
38]. These works confirm our results regarding embryo cleavage rate (99% for LCLCG vs 82% for HCLCG) (Table
2). Nevertheless, discrepancies between studies that show a difference in cleavage rates could be explained by the involved other factors on embryo cleavage decrease. Among them, we can cite in one hand the lack of rigorous oocyte morphology evaluation including other possible less apparent of oocyte dysmorphisms. In the other hand, there is risk of sperm genome decays presence on the selected spermatozoa for ICSI which is morphologically normal [
25].
Moreover, Ebner et al. [
5] proved the negative correlation between CLCG and blastulation rate (44%). Contrary to previous findings on embryo cleavage rate, some authors did not find lowered in vitro developmental ability in oocytes with CLCG compared to those with complete absence of granulation in the cytoplasm [
14,
20,
29,
32]. When other cytoplasmic features were compared to CLCG, the assessment of their predictive value showed less consistent results, whereas CLCG presence showed the strongest association with a value of f 2.7 presenting the major point in the Rienzi’s score (Metaphase II oocyte morphological scoring system MOMS) [
3]. The negative impact of CLCG on embryonic morphology could not be significantly demonstrated [
4]. It could not be proved for the embryo quality rate either, with a risk of 1.15 [0.56–2.36] [
33]. Generally, oocyte morphology could affect embryo cleavage contrarily to embryo quality. There was no real significant difference between abnormal extra-cytoplasmic or cytoplasmic morphological features in oocytes [
39‐
41]. This could explain why we found a significant difference in embryo cleavage rate and not in embryo quality rate (Table
2). With more available embryos, it was easier to obtain more cryopreserved embryos from an oocyte cohort with a CLCG under 25% (two embryos per patient) than those with a CLCG over 75% (one embryo per patient) (Table
2). Nevertheless, Balaban et al. [
37] reported that embryos derived from oocytes with CLCG had decreased survival rate and impaired in vitro development after cryopreservation. Hence, the cryopreservation of embryos issued from dysmorphic oocytes need to be studied, perhaps using a genetic evaluation and aneuploidy testing is still unclear and has sparked a debate among authors [
14,
15,
18,
32,
42,
43].
With a selection based only on morphological features from MII oocytes until the embryo transfer stage, seemingly appropriate and normal but intrinsically the impaired embryos might be transferred, resulting in poor clinical outcomes [
16]. Balaban and Urman [
29] did not find any effect comparing all oocyte anomalies with miscarriage rates (20% for abnormal oocytes vs 14% for normal oocytes) nor with pregnancy rates in CLCG compared to a control group (39% and 42%, respectively). However, most oocytes with an abnormal morphology have been shown to be associated with a poor implantation or pregnancy rate [
6,
8,
9,
14,
19,
32,
34,
39]. Indeed, significantly lower pregnancy rates were reported with oocytes with cytoplasmic anomalies (3% for abnormal oocytes vs 24% for normal oocytes) [
44]. In the present study, we were able to demonstrate a clear negative impact of HCLCG compared to LCLG on clinical outcomes including ongoing pregnancy and live birth rates which were decreased in couples with HCLCG (32% and 30% vs 14% and 13%, respectively) (Table
2), as confirmed by the study of Kahraman et al. [
14]. Nevertheless, though the pregnancy rate was affected by CLCG presence as reported by some authors [
6,
14,
23]; the difference in pregnancy rate between groups in the present work was not significant. Wilding et al. [
20] also reported these findings, demonstrating the need to calculate the study power with more consistent study populations. However, this result could be explained by a possible presence of embryonic aneuploidies [
14,
16,
18,
25,
34,
35] issued from oocytes with CLCG which are responsible for implantation failures before clinical pregnancy. There is probable involvement of other intra-uterine dysregulating factors [
28] which are associated with oocyte dysmorphism [
11,
13,
35].
Early miscarriage rates in our study were increased from 11% for LCLCG to 47% for HCLCG, and this result was confirmed after multivariate analysis, resulting in a risk of 3.1 (2.1–4.1) when CLCG was over 75% (Table
2). This topic was previously elucidated by Otsuki et al. [
11] and Ebner et al. [
5], highlighting the association between CLCG and other cytoplasmic organelle dysregulations involving the endoplasmic reticulum and the mitochondria with high levels of oxidative stress and division spindle anomalies [
11,
14,
35]. This condition could produce aneuploidies [
1,
11,
14,
34] and could very well explain why Otsuki et al. [
11] could report the case of a newborn with Beckwith-Wiedemann syndrome, a genetic imprinting disorder affecting chromosome 11p15.5, associating it with oocyte dysmorphism with sERC. They suggested that it was linked to CLCG production in oocytes whereas the endoplasmic reticulum in the oocyte stores and redistributes calcium, enabling cell activation during fertilization and energy accumulation by mitochondria necessary for appropriate embryo cleavage and an eventual maintained pregnancy [
34‐
36]. Although there is no clear difference between oocyte dysmorphisms based on oocyte morphology evaluation, other investigations are needed to explore further associations between each oocyte morphologic anomaly and aneuploidy affecting clinical outcomes as well as epigenetic modifications with a risk of impairment of offspring [
45].
Although several authors are debating the effect of oocyte dysmorphism on clinical outcomes [
6,
8,
9,
19‐
22], we cannot neglect the importance of oocyte morphology evaluation particularly when it is directly correlated to the negative effect of toxic environments.
Indeed, a large proportion of human oocytes resulting from exogenous gonadotropin-stimulated cycles have different morphological attributes or dysmorphisms [
12,
42]. Conversely, other authors did not find negative effects of COS protocol on oocyte quality and IVF outcomes [
3,
7,
39] involving other factors such as female age [
46], AMH, total FSH dose, COS duration [
5], or estradiol level [
47].
Apart from the toxic intra-ovarian environment produced intrinsically by COS with exogenous gonadotropins, the developing ovarian organ is particularly sensitive to environmental harm caused by pesticide exposure affecting oocyte quality [
48].
Some pesticides, (e.g. dichlorodiphenyltrichloroethane [DDT], methoxychlor, DES, and vinclozolin) are endocrine disruptors with estrogen-like effects. In women, exposure to bisphenol A affects folliculogenesis (via the granulosa and theca cells [
49]), induces meiotic aberrations (aneuploidy), and leads to a decreased oocyte quality during IVF.
In contrast, diethylstilbestrol (DES), which represents a well-studied estrogenic chemical, was proved in murine model to cause oocyte dysmorphism with condensed chromatin along the nuclear membrane [
45], ovarian cysts, and ovarian tumors [
50]. DDE, the most stable metabolite of DDT, and vinclozolin are persistent organic pollutants in follicular fluid, targeting androgen receptors. They stimulate aromatase and act in synergy with FSH to induce a premature rise in estradiol levels, affecting oocyte maturation. In addition, they have been found to induce the intra-ovarian inflammatory process involving VEGF and IGF-1; negatively affecting intra-ovarian mechanisms [
51‐
54]. Moreover, methoxychlor (MXC), an organochlorine pesticide, has had anti-estrogen activity in the ovary [
55], inhibiting growth and inducing atresia of antral follicles through an oxidative stress pathway with an effect on AMH production [
54].
It is known that the fetus is particularly vulnerable to pesticide exposure because of its rapid growth, the sensitivity of its developing organs, and the immaturity of its metabolic pathways and enzymatic defenses. A Canadian study described an association between pesticide exposure and increased miscarriage rate [
56], contrary to Willis et al. [
57] in a Californian study. In our study focusing on the region of Picardy, France, pesticide exposure zones were relatively correlated to CLCG prevalence as the risk zone having the highest pesticide exposure over 3000 g/ha was the residence place of 67% of couples with HCLCG compared to 33% of couples with LCLCG (Fig.
2).
The limitations of our study relate to our lack of data on the type of pesticides used (because one can legitimately consider that different types of products have different effects), the impact of meteorological and hydrological conditions which can modify the pesticide profile, and the type of exposure (acute or chronic). Moreover, despite this study was focused on evaluation of the correlation presence between residence couples included in risk zones of pesticides exposure and CLCG, this issue cannot prove the exposure occurrence especially that duration of residence was unknown. Nevertheless, this study could to demonstrate a relative correlation between pesticide exposure and risk of CLCG and the extension of this study will be the evaluation of different pesticide concentrations in serum and follicular fluid to establish the causality of certain compounds on oocyte quality, embryonic development, and clinical outcomes.