Case Report: Breaking barriers - Pregnancy achieved by intracytoplasmic sperm injection of a zona-free oocyte

Introduction

The zona pellucida is a thick acellular layer surrounding mammalian eggs and pre-implantation embryos.¹ The zona contributes to oocyte growth and the development of follicles. It plays a crucial role during fertilisation and has many other important functions in pre-implantation embryos like protecting it from infections, preventing its premature adherence, and also prevents premature implantation.² It contains certain glycoproteins including ZP1, ZP, and ZP3, that help in sperm binding and recognition, which in turn facilitates sperm penetration by acrosomal reaction; it also creates a barrier which prevents polyspermy by zona reaction.^1,3 In addition, after fertilisation, it protects the growing embryo before implantation by preventing aggregation of embryos and its premature adherence to the oviduct wall.⁴ It also hampers chimera formation and protects embryos against bacterial and viral infections. These functions make the zona essential for the fertilisation and development of the oocyte and embryo.^5,6

Absence of zona pellucida can be due to genetic, immunological or mechanical factors. Genetic and immunological factors can impair the zona production or cause defects in ZP1 expression, while mechanical factors cause trauma to zona pellucida, such as a fracture which can occur during the ovum pick-up procedure. Sometimes, the zona is removed intentionally.⁶ Absence of a zona makes oocytes very fragile.^7,8 It is said that composition, thickness, or shape of zona pellucida may affect optimal function and pregnancy outcome might be reduced.⁹

Zona-free oocytes are usually seen in the assisted reproductive laboratory, but are mostly considered unsuitable for fertilisation. There are not many studies and reports showing successful pregnancy outcomes with zona-free oocytes.⁸ This case report shows that zona-free oocytes can be successfully fertilised and lead to a positive outcome.

Using conventional in vitro fertilisation (IVF) on zona-free oocytes can increase the chances of polyspermy; thus, we used intracytoplasmic sperm injection (ICSI). Sometimes, the zona pellucida is damaged during denudation or ICSI. Those zona-free oocytes are mostly rejected. It is said that most of these oocytes are degenerated. However, it is possible that a few of them are still healthy and can be successfully fertilised. Those can be fertilized using ICSI and give successful pregnancy outcome.⁷

Case presentation

Treatment

Firstly, intrauterine insemination was done using the husband’s semen but sadly the results were unsuccessful. Then IVF was chosen as a treatment.

The female patient underwent controlled ovarian stimulation to increase the number of available eggs for fertilization. She received a GnRH antagonist to suppress natural ovulation. After that, we began ovarian stimulation using gonadotropins. The ovarian response was monitored by ultrasound scans and serum oestradiol measurements. The trigger was given when follicles reached the desired size of 18 mm and above. After thirty six hours of trigger, oocytes retrieval procedure was carried out.

During the oocyte retrieval procedure, two oocytes were retrieved, but one of them was a germinal vesicle (GV) and the other one lacked a visible zona pellucida, which is the outer protective layer surrounding the oocyte. The absence of the zona pellucida was confirmed through careful examination under high magnification after denudation (Figure 1).

Figure 1. Retrieved oocyte with absence of zona pellucida.

Given this unique situation, the couple was informed about the possibility of performing ICSI with a zona-free oocyte, as it was their only chance for fertilization in this particular cycle. After analysing potential outcomes, the couple decided to proceed with the ICSI procedure using the zona-free oocyte.

The ICSI procedure of the zona-free oocyte was performed (Figure 2). The zona-free oocyte was held in place using a holding pipette, while a single motile spermatozoon was aspirated into a needle. The sperm was then injected directly into the oocyte. Following ICSI, the injected oocyte was cultured in a suitable culture medium and assessed for fertilization after 16-17 hours.

Figure 2. Micropipette injecting a sperm into the zona free oocyte holded by a holding pipette.

The embryo progressed from the four-celled to eight-celled embryo stage (Figure 3).

Figure 3. (A) is showing four-celled embryo formed after fertilisation. (B) is showing eight-celled embryo.

On day three, fresh embryo transfer was carried out. Fourteen days after embryo transfer, a βhCG test was done and it was positive.

The couple was delighted with the successful outcome of the treatment and expressed gratitude. They were counselled on the importance of regular care during gestation and the need for appropriate follow-up post-delivery.

Discussion

Our report discusses the rare situation where not only successful fertilization occurred in a zona-free oocyte using ICSI, but also where this fertilized egg cleaved, implanted and led to a positive pregnancy outcome. As mentioned above, the zona pellucida is a highly important component of the oocytes and has multiple functions during fertilization, oocyte growth, follicles and preimplantation development. Despite these important purposes, our study demonstrates that an embryo can still survive without zona pellucida and pregnancy is very much possible. There are very few reports regarding this. Although zona-free oocytes have been shown to have fertilized after ICSI, cleavage and blastocyst formation is always challenging. ICSI is usually preferred when infertility is due to male factors, but when zona-free oocytes are encountered, then ICSI should be chosen because in the absence of zona, multiple sperms can enter the oocyte during conventional IVF.

In a report by Ebner et al, zona-free oocytes were successfully fertilised and cleaved, but pregnancy could not be achieved with zona-free embryos.⁹ Similarly, a study by Ding et al. reported two cases where oocytes without zona were fertilised and developed into blastocyst but implantation could not occur.⁷ Finally, Stranger et al. demonstrated ICSI of zona-free oocytes and successful pregnancy was achieved.¹⁰ Hu and Trolice presented the first case using repetitive autologous zona-free oocytes that underwent ICSI and fertilisation. When these reached at blastocyst stage, fresh embryo transfer was conducted, which resulted in a positive pregnancy and later, led to the live birth of twins.¹¹

In the future, zona-free culture have a potential to become a feasible alternative to the conventional approach in ART.⁶ Similarly, another case study by Watson et al. reported a live birth using zona-free oocytes. They used detailed protocols with time-lapse footage of blastocyst development and demonstrated that a careful improved approach to ovarian stimulation, oocyte pick-up, and culture of embryo may encourage successful blastocyst development and healthy live birth in patients with failed/impaired zona production.⁸ Lately, it has been observed that the absence of zona pellucida does not necessarily affect he normal development of preimplantation embryos. Zona-free embryo culture systems in animals also showed improvement in overall efficiency. Further studies are required to confirm this.⁴ Some even claim that zona-free embryos give better pregnancy outcome. Monsour et al. intentionally removed the zona pellucida. Their study compared the transfer of day 3 zona-free embryos and day 3 zona-intact embryos in two groups. Results showed improved pregnancy rates in a selected group of poor-prognosis women undergoing ICSI. There was no negative effect of removing zona on the pregnancy rate in either of the groups studied.¹²

Conclusions

One in five couples suffers from infertility. It can be either primary or secondary infertility. In this case report, the couple had primary infertility. Here, the probable causes of infertility were low AMH, poor ovarian reserve, the poor-quality oocytes of the female and oligoasthenozoospermia of the male patient.

The absence of zona in the oocyte could be due to various causes like genetic, immunological or physical damage during denudation. Since the zona pellucida is said to be essential for the fertilisation and early-stage embryo, zona-free are usually being considered ‘unusable’ in ART labs. Thus, this report illustrates that zona-free oocytes are equally advantageous as zona intact oocytes. Also, using ICSI for such oocytes is the most favourable choice, because it can be assured that only one sperm is fertilising the egg. ICSI should be carried out attentively and carefully. As rare as it is considered, zona-free oocytes can be successfully fertilised and implanted, giving a positive pregnancy outcome. This case report is significant in highlighting this major breakthrough in ART. ICSI in zona-free oocytes can pioneer advancement, paves the way for future research and offers a new hope for individuals seeking infertility treatment.

Consent

Written informed consent of the couple was taken for publishing their clinical details.