Complete zona pellucida removal from blastocysts prior to transfer in a cryo-cycle does not affect the efficiency of in vitro fertilization programs

Blastocyst hatching is a prerequisite for successful adhesion and implantation of a human embryo into the endometrium and the onset of pregnancy. The resistance of zona pellucida (ZP) to the action of lysins is one of the adverse events that occur during the cryopreservation and thawing of oocytes and embryos. The hardening of THE ZP leads to an increase in its resistance to the action of lysines secreted by the embryo trophectoderm, thus failing a blastocyst to hatch from the ZP [1]. However, this phenomenon can be minimized using assisted hatching.

The human blastocyst vitrification can lead to hardening of the embryo ZP. As has been previously reported in the literature, vitrification is associated with a significant increase in the resistance to enzymatic removal of the ZP by pronase, as compared to embryos not subjected to the freezing and thawing procedures [2]. Assisted hatching has also been reported to improve clinical outcomes in frozen-thawed embryo transfer cycles [3]. It seems likely that blastocysts with reduced viability fail to hatch even after partial removal or thinning of ZP. Besides, escaping through the hole in the ZP as a result of assisted hatching requires a considerable energy-consuming effort from blastocysts and can cause a dramatic distortion of their shape [4].

Vajta G. et al. [4] suggested that that complete removal of the ZP at the blastocyst stage has a greater positive effect on the further development of embryos than a partial removal or dissection of ZP. Hiraoka K. reported a preliminary study showing that complete removal of the ZP is associated with higher pregnancy, implantation, and delivery rates compared with a partial opening for vitrified blastocyst transfer [5].

This study aimed to investigate whether a complete mechanical removal of the ZP from vitrified and thawed blastocysts in IVF transfer results in better outcomes than the transfer of embryos with intact ZP.

Materials and methods

The study was conducted at the AltraVita clinic, Moscow. A total of 431 patients undergoing IVF received an embryo transfer with blastocysts that were vitrified and thawed in a cryo-cycle (total number of blastocysts - 476). The patients were divided into two groups to receive either an embryo transfer ofvitrified/thawed blastocysts with completely removed ZP (experimental group, n = 209, 230 blastocysts) or an embryo transfer with vitrified/thawed ZP-intact blastocysts (control group, n = 222, 246 blastocysts).

Cryopreservation of all good quality blastocysts was carried out on day 5-6 of development, and they were thawed on the day of embryo transfer. The embryos of the patients were randomly assigned to the experimental or control group. After embryo transfer, the following outcomes were analyzed: embryo implantation rate, clinical pregnancy rate, ongoing pregnancy rate, and multiple pregnancy rates.

The patients’ age ranged from 23 to 49 years with median (Me) age 33.5 [Q1, Q3 (29.8; 36.6)] and 33.0 [Q1, Q3 (30.0; 38.0)] years in the experimental and control groups (p = 0.32), respectively.

Ovarian stimulation Ovarian superovulation was induced by a gonadotropin-releasing hormone antagonist protocol from 2-3 days of the menstrual cycle using recombinant and/or urinary gonadotropins in a daily dose of 150-300 IU. Ovulation was induced by the administration of human chorionic gonadotropin at a dose of 10,000 IU 34–36 h before a planned follicular puncture.

Oocyte retrieval Transvaginal ultrasound-guided follicle aspiration was performed under intravenous anesthesia 34–36 h after administration of an ovulatory dose of chorionic gonadotropin resulting in retrieval of oocytes surrounded by a layer of cumulus cells.

Fertilization Fertilization of the retrieved oocytes was performed using the ICSI or IMSI Hoffman modulation procedure [6, 7], depending on the sperm parameters.

Embryo culture Embryos were cultured in a CO2-incubator at 36.7 ° C in a humidified atmosphere with 7.3% CO2 and 20% O2. Embryos were cultured individually in Global microcaps (LifeGlobal) supplemented with 5 mg/ml protein (LifeGlobal) under a layer of mineral oil. On day 3, after fertilization, the culture medium was replaced with fresh medium. Evaluation of embryo development was performed daily. On days 5-6 of development, good quality blastocysts were cryopreserved for subsequent embryo transfer in the cryo-cycle.

Embryo quality assessment Development and quality of zygotes and embryos were individually evaluated under a microscope ~ 18, ~ 45, ~ 72 and ~ 96 h after fertilization. On days 5-6 of development, the quality of the formed blastocysts was assessed. For embryo transfer and cryopreservation, expanded blastocysts with a grade of AA, AB, BA, and BB by Gardner grading system were used.

Cryopreservation and thawing of embryos Cryopreservation and thawing of the blastocysts were performed with Cryotech Vitrification Kit (101) (Cryotech, Japan) and the Cryotech Warming Kit (102) (Cryotech, Japan), respectively, according to the manufacturer’s instructions.

Assisted hatching The assisted hatching was performed 5–15 minutes after thawing the embryos (while the blastocysts remained partially collapsed) on a Leitz Fluovert FU Inverted Microscope (Wild Leitz GmbH, Germany) equipped with Hoffman modulation contrast optics and a set of Narishige micromanipulators (Narishige, Tokyo Japan ).

Complete removal of the ZP was carried out on a heating table at 37°C in a Petri dish containing micro-droplets of HTF/HEPES medium (LifeGlobal) with the addition of 5 mg ml protein (LifeGlobal) under a layer of mineral oil.

A partial mechanical cutting (70–90% of the circumference) of the ZP was performed using a micropipette for the ZP dissection (Cook Medical). After the incision was made, the embryo was removed from the suction pipette, and the complete removal of ZP, that is, the complete hatching of the embryo from the ZP was carried out using a gentle mechanical pipetting through a 175-200 µ capillary. For each embryo, the whole procedure of complete ZP removal took no more than 3 minutes.

Blastocyst survival assessment After complete ZP removal, the blastocysts were washed several times from the handling medium and cultured in HTF medium (LifeGlobal) with the addition of 15 mg/ml protein (LifeGlobal) until the time of transfer.

Embryo viability was evaluated under a microscope within 1–3 hours after thawing, and blastocysts that quickly re-expand after warming were considered viable.

Preparation of endometrium, embryo transfer, and the assessment of the pregnancy onset The transfer of thawed embryos was carried out in natural or hormone replacement cycles. In natural cycles, embryo transfer was performed on day 5 after ovulation, regardless of the day (5 or 6) of the blastocyst cryopreservation. In cycles with hormone replacement therapy, the patient’s endometrium was prepared by the administration of 3.0 mg transdermal estradiol (Divigel; Orion Corporation, Finland). Progesterone administration (Utrogestan caps., 200-600 mg/day) was commenced when the endometrium thickness exceeded 8 mm. Embryo transfer was carried out after 5 days of progesterone treatment, regardless of the day (5 or 6) of blastocyst cryopreservation. The time from the moment of the embryo thawing to the time of transfer ranged from 2 to 4 hours. One or two viable blastocysts were transferred into the patient’s uterine cavity. The mean number of embryos per transfer was 1.1.

Clinical pregnancy was diagnosed by ultrasound visualization of a gestational sac, the parietal-coccygeal length of the fetus measuring 2–4 mm, and detection of fetal cardiac activity on transvaginal ultrasonography at 4–5 weeks after embryo transfer. The ongoing clinical pregnancy rate was determined by the ratio of the total number of clinical pregnancies for a period of 12 or more weeks to the total number of transfers.

Statistical analysis Statistical analysis was performed using the MS Excel (Microsoft, US). Median (Me) and quartiles Q1 and Q3 were used to describe the age of patients. Difference between groups in the mean age of the patients was assessed using Student t-test. Proportions of embryo implantations, the onset of clinical, ongoing clinical, and multiple pregnancies were compared using the χ2 test. Differences between the groups were considered statistically significant at p<0.05.

Results

No embryo degeneration was observed after complete removal of ZP. There were no statistically significant differences between the experimental and control groups regarding all clinical outcomes. When the patients in the experimental and control group were subdivided into < 36 and > 36 age subgroups, also, no statistically significant differences were found between these subgroups in the experiment and control group. Characteristics of patients in the study groups the study findings are presented in the table.

Discussion

Implantation rate per embryo transfer in human IVF programs ranges from 8% to more than 40%. Relatively low implantation rates in IVF programs, at least in part, may be associated with impaired hatching. To date, the hatching process of blastocysts is still not completely understood.

Hatching requires a considerable energy-consuming effort from blastocysts to expand and mobility to escape the embryo through ZP. Re-expansion and collapse of the blastocyst can deplete the embryo’s energy reserve below the threshold, which is necessary for successful hatching and further development. Complete removal of ZP, presumably, may improve the probability of embryo implantation due to the preservation of this energy reserve in the absence of losses during unsuccessful attempts to hatch. Some authors reported in vivo rodent studies, showing a low proportion of expanded blastocysts with a high proportion of successful hatching. These observations have suggested that the mechanism of hatching in vivo and in vitro may differ. This phenomenon can be explained by a higher resistance of the ZP in embryos obtained in vitro. Time-lapse photography studies in vitro demonstrated that a high proportion of mouse blastocysts hatched without preliminary collapse-expansion cycles, that is, without the pulsatile activity of blastocysts. Pulsation of the blastocyst before the hatching process may be an artifact or a sign of difficulty associated with the hatching process, due to the rupture of the intercellular contacts of trophectoderm cells and the fluid leakage from the blastocyst.

From 2.8 to 25% of human blastocysts hatch from the ZP by day 6 of in-vitro development. However, a large proportion of morphologically normal blastocysts are experiencing difficulties with hatching, and more than half of them fail to hatch even after eight days of in-vitro culture and remain trapped in the ZP (trapping phenomenon).

It could be that blastocysts with low viability are unable to complete hatching after thinning or opening of ZP, therefore a complete removal of the zona pellucida may be recommended.

Vaita G. et al. [4] suggested that the complete removal of the ZP may lead to higher success rates after embryo transfer compared with other types of assisted hatching.

Initial studies investigating the effect of complete removal of the ZP on clinical outcomes compared the results of IVF programs after the transfer of blastocysts with removed and intact ZP. Four prospective randomized trials investigated the effect of complete removal of the blastocyst ZP using enzymatic treatment with pronase or acidified Tyrode’s solution before the embryo transfer [8-11].

All authors reported improved outcomes of ART programs with assisted hatching using a complete ZP removal, especially in patients with low quality blastocysts (25 cases; 4.3 embryos per transfer) [11], patients with a high risk of ovarian hyperstimulation syndrome (11 cases; 1.0 embryo per transfer) [9] or performed on embryos with developmental delays (48 cases; 2.2 embryos per transfer) [8]. In a study investigating outcomes after transfer of ZP-free day 3 embryos in patients below the age of 40 undergoing their first IVF attempt (27 cases; 3.2 embryos per transfer), no statistical differences in the rates clinical pregnancy and spontaneous miscarriages were found compared with ZP-intact group. However, in the poor prognosis group with patients over 40 and/or having two or more failed IVF attempts (30 cases; 3.9 embryos per transfer), removal of the ZP resulted in a significantly higher clinical pregnancy rate when compared with controls [12].

Also, the effect of complete removal and thinning/opening of the ZP on the clinical outcomes of IVF programs was evaluated in 2 retrospective studies. Lan K. [13] showed that, in patients undergoing fresh blastocyst stimulation cycles, chemical removal of the ZP on day 5 (104 cases; 2.6 embryos per transfer) and laser-assisted hatching at the cleavage stage with embryos cultured up to day 5 (104 case; 2.7 embryos per transfer), resulted in similar implantation rates. Hiraoka K. compared assisted hatching of vitrified and thawed blastocysts undergoing either a complete ZP removal using a laser and mechanical pipetting (57 cases; 1.5 embryos per transfer) or partial ZP opening using acid Tyrode’s solution (45 cases; 1.7 embryos per transfer). The clinical pregnancy and implantation rates were higher in the complete ZP removal group than in the partial opening group (67 vs. 42%, p = 0.014; 55 vs. 30%, p = 0.001, respectively), although multiple pregnancy rates did not differ between the groups [14].

To our knowledge, there are currently no published randomized controlled trials comparing clinical outcomes of IVF programs performing an embryo transfer using blastocysts with either a mechanically removed ZP or with ZP intact controls.

In general, the above data and the available meta-analyzes [15, 16], as a rule, report a superiority of complete removal of the ZP over other methods of assisted hatching. However, small sample sizes and a large number of embryos per transfer in previous studies make the evidence of the benefit of a complete ZP removal inconclusive.

Limited utilization of complete removal of the blastocyst’s ZP in the practice of IVF clinics can be explained by the concerns regarding the vulnerability of ZP-free embryos during routine procedures. The major disadvantages of ZP-free culture of embryos seem to be the lack of mechanical protection resulting from attachment to glass and plastic surfaces and the risk of infection and disease transmission. On the other hand, according to some authors, these concerns may be disregarded due to the evident benefit of a zona-free culture system [4].

A study by Ueno S. showed that complete ZP removal promoted blastocyst adhesion and outgrowth in fibronectin-coated dishes in the blastocyst outgrowth assay compared with partly removed or intact ZP [17]. However, one should not forget that the hatching process may vary between in-vitro and in-vivo conditions, which can have a significant impact on the blastocyst expansion, hatching, adhesion, and nidation.

Our findings are consistent and complement the above results reported in the international literature.

Conclusion

The findings of this study suggest that the complete mechanical removal of the ZP of human vitrified– thawed blastocysts 1-3 h before embryo transfer has no impact on clinical outcomes of IVF programs. All IVF outcomes including the embryo implantation rate, clinical and ongoing clinical pregnancy rate, and multiple and ectopic pregnancy rate were comparable without significant differences between the experimental and control groups, including subgroups of patients below and above the age of 36 years.