Embryological Characteristics and Clinical Outcomes of Oocytes with Heterogeneous Zona Pellucida During Assisted Reproduction Treatment: A Retrospective Study

Background

The use of assisted reproduction technologies accounts for over 1 million births each year worldwide [1]. The morphological assessment of oocytes and subsequent embryos is a crucial part of assisted reproduction techniques such as in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) [2] because it helps ensure the selection of metaphase II (MII) oocytes with an optimal potential to successfully develop into viable embryos for transfer [3].

The human zona pellucida (ZP) is an extracellular glycoprotein matrix that is synthesized and secreted by oocytes during follicular development. The ZP is made of 4 glycosylated proteins (ZP1, ZP2, ZP3, and ZP4) [4,5]. The human ZP is vital for specific sperm-oocyte binding, acrosome reaction induction, and polyspermy prevention [6]. In addition, in vivo, the ZP protects the embryo during its passage from the oviduct to the uterus or, in vitro, the ZP protects the embryos from mechanical and chemical stress [7,8].

The ZP shows different morphologies among oocytes of women undergoing assisted reproductive technology treatment. Abnormal ZP morphology can be observed in 2–5% of all oocytes [9]. It is likely that any abnormality in composition, shape, color, and thickness of the ZP may affect the outcomes of IVF. Oocytes with an oval-shaped ZP have a high risk of abnormal embryo cleavage and are associated with lower rates of implantation and pregnancy after IVF [10,11]. The fertilization rate and clinical outcomes may depend upon variations in ZP thickness [12,13]. In contrast, the fertilization or clinical outcomes seems unaffected by a dark appearance of the ZP [14–16], but contradictory results have been reported [17]. Oocytes with heterogeneous zona pellucida (HZP) have a bright vitreous appearance with an irregular outer edge. A study with a limited sample size found that HZP is associated with reduced oocyte maturity and reduced rates of fertilization and high-quality embryos [18]. Further investigation into the outcome of oocytes with HZP is needed.

Therefore, this study investigated the embryological characteristics and clinical outcomes of oocytes with HZP during IVF and ICSI treatments, as compared to oocytes with normal zona pellucida (non-HZP).

Material and Methods

PATIENTS:

This study retrospectively examined the IVF, and ICSI treatment cycles carried out at The Reproductive Medicine Center of The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, China, between June 2006 and March 2016. Only the cycles completed under controlled ovarian hyperstimulation with the protocol using gonadotropin-releasing hormone (GnRH) agonist were included. Twenty-nine IVF and 46 ICSI cycles with HZP oocytes were included (HZP group). Another 521 IVF and 206 ICSI cycles with oocytes retrieved on the same day were included as controls (non-HZP group). The detailed baseline information from the women is shown in Table 1.

The exclusion criteria were: 1) known previous poor ovarian response to ovarian stimulation; and 2) endometriosis, polycystic ovary syndrome (PCOS), hydrosalpinx, or uterine pathology.

This study was reviewed and approved by the Ethics Committee of The First Affiliated Hospital of Wenzhou Medical University (No. 2019-099), and the requirement for informed consent was waived due to the retrospective nature of the study.

STUDY DESIGN:

The patients were grouped according to the presence or absence of HZP in the oocytes used during IVF/ICSI. When all retrieved oocytes showed HZP and no normal oocytes could be found, the HZP oocytes were used for the procedure and assigned to the HZP group. The control (non-HZP group) group consisted of cycles with oocytes retrieved on the same day and considered to be morphologically normal.

STIMULATION REGIMENS:

During the study period, the luteal long downregulation protocol (LP) or the short flare-up protocol (SP) were routinely used, as previously described [19]. Specifically, in the LP, a 0.5–0.9 mg depot of a GnRH agonist (diphereline, 3.75 mg; IpsenPharma Biotech) was administered in the mid-luteal phase. Stimulation using gonadotropin (Gonal-F; EMD Serono) was initiated at 14 days, the injection of diphereline (usually on cycle day 3–9) or when pituitary downregulation was achieved, as previously described [19]. Downregulation was confirmed by an endometrial lining of up to 5 mm, serum estradiol <50 pg/L, and serum luteinizing hormone (LH) <5 U/L. For SP, the GnRH agonist (decapetyl, 0.1 mg; Ferring GmbH) was given as a daily dose of 0.1 mg starting on cycle day 2, followed by gonadotropin (Gonal-F; EMD Serono) starting on day 3, as previously described [19]. For both protocols, Gonal-F (150 or 225 U/day) was given for 5–6 days and adjusted based on follicle growth and serum levels of estradiol. Human chorionic gonadotropin (HCG; Livzon, China) was administered at 5000–10 000 IU when at least 3 follicles were observed to have reached 17 mm in diameter as assessed by B-mode ultrasound; oocyte retrieval was carried out 34–36 h later, as previously described [20].

OOCYTE INSEMINATION AND EMBRYO CULTURE:

IVF and ICSI were carried out according to standard protocols, as previously described [17,19]. Specifically, ICSI was used when sperm concentration was below 5×106/mL or when sperm motility was <10%. Insemination was performed by IVF or ICSI 3–6 h after oocyte retrieval. Sixteen to eighteen h later, fertilization was confirmed based on the observation of 2 pronuclei and 2 polar bodies. Fertilized oocytes were cultured in fresh 20 μl of G1 plus medium (Vitrolife AB, Sweden) in individual microdroplets covered with mineral oil at 37ºC in an atmosphere of 6% CO2. In the morning of day 3, embryos were graded according to morphology, blastomere number, blastomere size, and fragmentation, as described by the Istanbul Consensus [21]. The embryos with 7, 8, or 9 blastomeres, and those with less than 20 fragments were considered to be of high quality.

EMBRYO TRANSFER AND PREGNANCY DETECTION:

On day 3, embryo transfer was performed under abdominal ultrasound guidance, as previously described [22]. Specifically, 2 or 3 transferred embryos were selected according to embryo quality. If there were only poor-quality embryos or no embryos from the IVF/ICSI cycle, embryo transfer was canceled.

Pregnancy was confirmed as previously described [19]. Specifically, 2 weeks later, an HCG test was performed. In the case of positive signals, clinical pregnancy was defined as the presence of fetal heartbeat 4 to 5 weeks after embryo transfer, confirmed by ultrasound. Early abortion was defined as spontaneous abortion before 20 weeks of gestation.

OBSERVATION OF THE ZP: The ZP was evaluated under an OLYMPUS IX73 microscope (OLYMPUS, Japan) equipped with a digital camera at 200× magnification. We defined HZP as a ZP with the bright and vitreous appearance and an irregular outer edge, as previously described [18]; an oocyte with HZP had overtly absent or extremely narrow perivitelline space (Figure 1).

DEFINITIONS AND OUTCOMES:

The numbers of oocytes retrieved, MII oocytes, fertilized oocytes, cleaved embryos, high-quality embryos, and available embryos for transfer were recorded. The corresponding rates were calculated as previously described [19], including MII oocyte rate (MII oocyte number by that of all retrieved oocytes×100), normal fertilization rate (number of fertilized oocytes by that of all retrieved oocytes [for IVF] or MII oocytes [for ICSI]×100), cleavage rate (number of cleaved embryos by that of all fertilized oocytes×100), high-quality embryo rate (the number of high-quality embryos [7, 8, or 9 blastomeres with less than 20 fragments] divided by that of cleaved embryos×100) and abandoned transfer rate (number of cycles of abandoned transfer divided by that of all cycles×100). For successfully transferred embryos, the number of transferred embryos, positive or negative HCG, implantation, pregnancy, gestational period, and miscarriage status were recorded. The positive HCG rate was defined as the total cycles of clinical, biochemical, intrauterine, and ectopic pregnancies divided by all transfer cycles×100. The implantation rate was defined as the number of fetal heartbeats divided by that of transferred embryos×100. The pregnancy rate was defined as the number of patients with 1 or more gestational sac(s) visualized by ultrasound at 6–7 weeks’ gestational age by that of transferred cycles×100. The miscarriage rate was defined as the number of miscarriage cycles divided by that of cycles with clinical pregnancy×100. The live birth rate was defined as the number of cycles with premature or mature delivery divided by that of transferred cycles×100.

STATISTICAL ANALYSIS:

Statistical analyses were carried out with SPSS version 16.0 (SPSS, Inc., USA). Continuous variables are presented as means±standard deviations (SD); comparisons between groups were performed using the t test. Categorical variables were reported as frequencies (percentages) and compared by the chi-square test. Multivariable logistic regression models were used to assess the associations of risk factors with the main outcome (pregnancy or not) in both ICSI and IVF cycles. P<0.05 was considered statistically significant.

Results

BASELINE FEATURES OF THE INCLUDED PATIENTS:

When the ICSI cycles of both groups were compared, age and BMI were similar, but the duration of infertility was longer in the HZP group (p=0.002), and the diagnosis of infertility was more likely to be primary infertility (p<0.001) vs. the non-HZP group. When IVF cycles were compared between the 2 groups, the same factors were significant: the duration of infertility was longer in the HZP group (p=0.003), and the diagnosis of infertility was more likely to be primary infertility (p<0.001) compared with the non-HZP group, but age and BMI were similar. Other baseline features are shown in Table 1.

EMBRYO CHARACTERISTICS:

Embryological outcomes are shown in Table 2. For ICSI cycles, no differences were observed between the HZP and non-HZP groups in the number of oocytes retrieved, normal fertilization rate, and cleavage rate. The rates of MII oocytes and high-quality embryos were lower in the HZP group vs. the non-HZP group (p<0.05). In IVF cycles, no differences were detected between the HZP and non-HZP groups in the number of oocytes retrieved or high-quality embryo rate. MII oocyte rate, normal fertilization rate, and cleavage rate were lower in the HZP group vs. the non-HZP group (all p<0.001).

CLINICAL OUTCOMES:

Clinical outcomes are shown in Table 2. In ICSI cycles, 36 embryo transfers were performed in the HZP group and 161 in the non-HZP group. There were no significant differences in the abandoned transfer rate per retrieval cycle, positive HCG, implantation, pregnancy, and miscarriage rates between the 2 groups.

In IVF cycles, 12 embryo transfers were performed in the HZP group and 442 in the non-HZP group. The rate of abandoned transfer per retrieval cycle was higher in the HZP group vs. the non-HZP group (p<0.001). Although the number of transferred embryos per cycle in the HZP group was lower than that of the non-HZP group, there were no differences in positive HCG, implantation, and pregnancy and miscarriage rates between the 2 groups.

UNIVARIATE AND MULTIVARIATE ANALYSES:

Univariate analyses of factors related to pregnancy after ICSI are shown in Table 3. The results revealed that the woman’s age (OR=0.894, 95% CI 0.835–0.959; p=0.002), duration of infertility (OR=0.921, 95% CI 0.851–0.998; p=0.044), number of retrieved oocytes (OR=1.077, 95% CI 1.025–1.131; p=0.003), number of MII oocytes (OR=1.097, 95% CI 1.037–1.160; p=0.001), and number of D3 high-quality embryos (OR=1.286, 95% CI 1.109–1.491; p=0.001) were significant factors. The multivariate analysis is shown in Table 3, and no factors were independently related to pregnancy after ICSI.

Table 4 shows the univariate analysis of factors related to pregnancy after IVF. The results demonstrated that the woman’s age (OR=0.895, 95% CI 0.855–0.936; p<0.001), duration of infertility (OR=0.918, 95% CI 0.855–0.985; p=0.017), number of retrieved oocytes (OR=1.061, 95% CI 1.026–1.097; p=0.001), number of MII oocytes (OR=1.074, 95% CI 1.035–1.114; p<0.001), and number of D3 high-quality embryos (OR=1.205, 95% CI 1.098–1.323; p<0.001) were significant factors. As also shown in Table 4, the multivariate analysis found that the woman’s age (OR=0.916, 95% CI 0.873–0.962; p<0.001) and the number of D3 high-quality embryos (OR=1.120, 95% CI 1.004–1.249; p=0.043) were significantly associated with pregnancy. Whether the oocyte had HZP or not was not found to be related to pregnancy in case embryos were available for transfer in either ICSI or IVF cycle.

Discussion

LIMITATIONS:

Firstly, the definition of HZP has no established guidelines and could vary by study. Secondly, patients had different degrees of HZP, which were not graded or distinguished in this study. Thirdly, the sample size was relatively small. A larger study, including patients from multiple centers, might provide more evidence for these findings. Finally, this was a retrospective study, with possible selection bias.

Conclusions

Overall, women with oocytes showing HZP can become pregnant after either IVF or ICSI treatment. However, these women should avoid low fertilization and high abandoned transfer rates by choosing ICSI.