The Fate of Zona free Oocyte Following Intracytoplasmic Sperm Injection: A Case Report and Review of Literature.

A 44-year-old woman and her partner, both of similar age, presented with a history of failed fertilisation through intrauterine insemination (IUI) treatment. The patient was referred to Tiara IVF Clinic, Graha Amerta, Dr. Soetomo General Academic Hospital for further evaluation. The patient’s anti-Mullerian hormone level, which was notably low at 0.38 ng/L, was accompanied by asthenoteratozoospermia in the analysis of her partner’s semen. The patient underwent a GnRH antagonist protocol, initiated on day 3 of her cycle. Baseline hormonal levels were as follows: serum estradiol at 24.98 pg/mL, luteinising hormone (LH) at 8.18 mIU/mL, follicle-stimulating hormone (FSH) at 3.50 mIU/mL and prolactin at 21.99 mIU/mL. Upon reaching a diameter of 20.33 mm, the lead follicle was triggered with an administration of hCG (Ovidrel 250 mg, Merck). The endometrial thickness was 10.1 mm and serum estradiol levels before ovum retrieval were 348.98 pg/mL. Following oocyte retrieval, daily progesterone pessaries (Crinone, Merck) provided luteal support. Oocytes were collected 36 h post-trigger using transvaginal ultrasound-guided aspiration. The male partner provided a semen sample on the same day. Denudation was performed using a hyaluronidase solution (Hyadase; CooperSurgical, USA) and a fine denuding pipette with a diameter of 200 μm (RI EZ Strip, CooperSurgical, Målov, Denmark). During this process, the ZP detached, leaving behind a single ZFO. The oocyte oolemma radius was 10.17 µm and the area was 323.86 µm which were both smaller than a normal oocyte (radius – 13.85 µm; area – 595.39) [ Figure 1 a and e]. Embryo development from day 0 to day 3: (a) Day 0, oocyte with polar body extruding from the zona pellucida and the dimension, (b) Day 1, fertilised oocyte displaying two pronuclei, (c) Day 2, embryo cleavages into three cells, (d) Day 3, embryo cleavages into four cells, (e) Normal oocyte ooplasm dimensions Sperm preparation was performed using a simple wash method. Microinjection involves stabilising the oocyte with gentle suction on the lemma. Post-ICSI, the oocyte was cultured in Cleav™ Medium (Origio, CooperSurgical, USA). Fertilisation was assessed the following morning [ Figure 1 b], and the embryo was cultured for 48 h. Abnormalities such as multinucleation were observed at the three- and four-cell stages [ Figure 1 c and d]. Embryo transfer was conducted on day 3 using a K-JET catheter (Cook Medical) with ultrasound guidance. Following the transfer of a four-cell embryo, the patient’s β-hCG level was recorded as negative (7.9 mIU/mL) 14 days after the procedure. This case report was prepared following the CARE checklist to improve the quality of reporting and increase the accuracy, transparency and utility of case reports.

The oocyte itself synthesises and secretes the zona pellucida (ZP), an extracellular matrix that surrounds the human oocyte. The ZP has multiple roles, including protecting the oocyte and ensuring proper sperm selection. However, under certain conditions, the ZP may become damaged or absent due to genetic, immunological or mechanical factors. Such abnormalities result in zona-free oocytes (ZFOs). The ZP is known to play 11 essential functions in reproductive physiology, such as selecting spermatozoa for fertilisation, regulating species-restricted fertilisation, protecting the oocyte against sperm, preventing polyspermy, inhibiting blastomere separation in embryos and preventing blastomere attachment to the tube wall during the pre-compaction phase. While ZFOs are rare in clinical practice, their developmental potential remains underexplored.[ 1 2 ] This case report presents a 44-year-old patient receiving intracytoplasmic sperm injection (ICSI) treatment, emphasising the distinctive developmental potential of ZFOs, which are marked by a marginally reduced cytoplasm, an undetectable spindle due to microscope limitations and its consequences for assisted reproductive technologies. This is an uncommon occurrence, with merely four case reports globally and one associated study recorded.

ZFO is a rare occurrence in nature. During IVF, ZFO may be identified either at the time of oocyte retrieval or as a consequence of oocyte manipulation during cumulus cell removal. This case report and literature review highlight that ZFO possesses the potential for fertilisation, cleavage and successful live birth outcomes, whether through fresh or frozen embryo transfer, and should therefore not be discarded. The application of modified embryological techniques, as discussed, is recommended to optimise outcomes. The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient has given his consent for his images and other clinical information to be reported in the journal. The patient understands that his name and initials will not be published and due efforts will be made to conceal his identity, but anonymity cannot be guaranteed. There are no conflicts of interest. The data that support the findings of this study are available from the corresponding author, upon reasonable request.

This case highlights the developmental potential of ZFOs during an ICSI procedure. Despite successful fertilisation, it did not lead to successful implantation. This case occurred in a healthcare facility that lacked a specialised microscope to observe spindle fibres; however, this limitation was mitigated by the visibility of the polar body. The small size of the oolemma and the invisible spindle during the ICSI procedure presented additional challenges. In these circumstances, ICSI will have a favourable outcome if the ZFO exhibits a polar body, as this indicates oocyte maturity and safeguards the spindle from potential damage. ICSI performed on a ZFO without a visible polar body does not lead to fertilisation (unpublished data). The ZP is composed of relatively simple yet layered three-dimensional structures.[ 2 ] The glycoproteins surround the developing oocyte, establishing an interconnected network of cross-linked tissues.[ 3 ] The width of the ZP in human cells is approximately 22 µm. ZP-free oocytes can be obtained in the assisted reproductive laboratory and usually result from the loss or damage of the ZP, which may increase the risk of polyspermy. The anticipated challenges in this case include spindle positioning, oocyte maturation, mechanical vulnerability, mitochondrial function, the risk of polyspermy and embryo handling. The absence of a polarised light microscope restricts the capacity to visualise spindle positioning, increasing the risk of spindle damage during ICSI. The absence of a polar body complicates the assessment of oocyte maturity. The zona in oocytes serves as a protective barrier against multiple sperm penetrations; therefore, strict laboratory protocols are necessary when ZFOs occur. Excessive manipulation can harm ZFOs, which are particularly prone to fragmentation and damage. Damage may lead to abnormal cell division kinetics, thereby diminishing implantation potential. The ZP plays a significant role in protecting blastomeres during division; however, this function decreases as the blastocyst undergoes compaction. In addition, evaluating the maturity level and the positioning of the meiotic spindle in ZFOs is challenging due to the lack of a polar body.[ 4 5 ] Genetic defects related to mutations in the ZP genes (ZP1, ZP2, ZP3 and ZP4) may be causative factors in some patients resulting in ovarian endometriosis, empty follicle syndrome, oocyte maturation arrest, oocyte degeneration or presence of ZFOs. The genes are located on chromosomes 11, 16, 7 and 1, respectively.[ 4 6 7 ] Mutation in ZP genes also causes in vitro fertilisation failure, the oocytes failed to develop and implant,[ 8 9 ] forming 3 pronuclei after ICSI from ZFO.[ 10 ] This case underlines the necessity for specialised management strategies in the handling of ZFOs to achieve successful outcomes. Strategies include minimising mechanical manipulation, using advanced imaging techniques for spindle positioning, single embryo culture and adhering to strict laboratory protocols to reduce the risks of polyspermy and damage. Despite the unique challenges posed by ZFOs, their developmental potential requires further investigation to optimise assisted reproductive technologies. This case was supported by a 1999 case report published by Ding et al. , which described the first documented instance of fertilisation and in vitro development with ZFO. ZFO occurs due to the detachment of the ZP when the oocyte is freed from its cumulus. This report marked a significant milestone in reproductive medicine by demonstrating the viability of ZFOs in assisted reproductive technology, paving the way for further exploration of their clinical applications. This becomes one of the absolute indications for performing ICSI, either due to ruptured ZP during in vitro manipulation or the purposeful removal of the ZP.[ 4 11 ] The ZP plays a significant role in protecting blastomeres during the division process; however, this protective function decreases as the blastocyst becomes compacted. Ding (1999) and Shu (2010) hypothesised that the ZP impacts cell-to-cell contacts during early division, facilitating the alignment of cells in a horizontal plane to optimise communication and structural integrity during early embryonic development. The presence of tight junction cell-to-cell contact is influenced by proteins that affect cell development. At the 4-cell stage, these proteins include catenins, E-cadherin and CXADR; at the pre-compacted 8-cell stage, they include catenins, E-cadherin, CXADR, occludin, claudin, JAM-A, Par3/Par6/aPKC, gap junction and ZO1α−; at the compacted 8-cell stage, they include catenins, E-cadherin, CXADR, occludin, claudin, JAM-A, gap junction, cingulin, ZO1α− and ZO2, and at the blastocyst stage, they include cingulin, occludin, claudin, ZO1α−, ZO2, ZO1α+, JAM-A, E-cadherin, catenins, desmosomal cadherin, PK and PKP, desmoplakin and gap junctions.[ 4 12 13 ] Shu (2010) claims that this case report marked the first time a vitrified zona-free embryo (ZFE) resulted in pregnancy and a live birth. ZFO occurs during the manipulation of oocytes detaching from their cumulus. Shu’s case demonstrated the development of four cells, which subsequently returned to the ZP. The goal of this step was to create an environment similar to the ZP’s natural protective environment. This would lower the risk of damage during embryo culture and promote structural integrity, which is important for future development.[ 12 ] The conclusion is that the ZP plays a role in blastocyst formation and the relationship between cells. Specialised management must be carried out with caution on ZFOs to prevent lysis.[ 14 ] Specific practices include minimising mechanical manipulation, ensuring an individual culture of ZFOs to avoid blastomere merging and carefully replacing the culture medium to maintain a stable environment. In addition, the use of advanced imaging techniques can help monitor cell health and spindle positioning during the procedure.[ 15 ] Stranger (2001) reported a case of ZFO in all oocytes obtained in one cycle. The oocytes were zona-free due to abnormal zona production following injection without removal of the corona cells. ZFE were transferred on day 3, with six blastomeres resulting in pregnancy and live birth.[ 16 ] Hu and Trolice reported a case of ZFO wherein the oocyte obtained did not have a ZP since the retrieval. ZFE results in twin births. The division of embryos into Groups 1 and 2 was intended to reduce potential damage associated with ICSI. This approach, utilising the intact corona, is derived from a case report by Stanger et al. that supports sperm injection.[ 15 16 ] Following the transfer of the zygote to a culture dish, the corona cells were noted to detach from the embryo spontaneously. This indicated that all oocytes obtained were possibly ZFOs due to disturbances in zona production.[ 15 ] Blastomeres found in some ZFEs can merge during the division phase when cultured in the same drop, necessitating individual culture. Individual culture prevents physical contact between blastomeres, thereby reducing the risk of unintentional fusion or abnormal development. This approach ensures that each blastomere develops independently, maintaining its structural and functional integrity throughout the division process. Minimisation of excessive manipulation of ZFEs is advised, with a recommended medium replacement of 90% of the original, ensuring the embryo remains undisturbed.[ 12 15 ] According to Shrivastava et al. , two oocytes were retrieved during ovum pickup: one at the germinal vesicle (GV), which subsequently degenerated, and the other without a ZP. Observations indicated that zona-free gametes are capable of undergoing fertilisation, developing into high-quality embryos and resulting in successful pregnancies.[ 17 ] The previous case report demonstrates that normal embryonic development is possible. In this case report, this did not occur. This may be due to the small size of the oolemma, which could not support normal development. A typical oocyte contains several hundred thousand mitochondria, dispersed randomly throughout the cells during the first 6 days of embryo development. This distribution is crucial for supplying the energy required for embryonic cells to execute biological processes, such as cell division. Elevated levels of mitochondrial DNA (mtDNA) have been linked to lower implantation rates in euploid embryos. For instance, embryos with elevated mtDNA often experience reduced energy production efficiency, leading to impaired cellular functions required for implantation. In addition, increased mtDNA levels are associated with higher oxidative stress, which may disrupt normal embryonic development and compromise the integrity of cellular processes during critical implantation stages. It is suggested that the increased mtDNA in embryos results from higher oxidative stress associated with aneuploidy. However, variations in mitochondrial load might also result from differences in cell division kinetics during embryo development. mtDNA is measured as a ratio to nuclear DNA (nDNA), referred to as mitochondrial content (MC). Abnormal or slow cell division can lead to higher mtDNA readings compared to normal cell division.[ 18 19 20 ] A retrospective study in Japan found that embryos without an intact ZP had the same rates of implantation and development as embryos with an intact ZP. This is one of only a few studies that have compared the outcomes of embryos with and without an intact ZP. This suggests that while the ZP provides structural and protective roles, its absence does not necessarily compromise the developmental competence of the embryo in certain clinical conditions. The results showed no significant differences between the two types of embryos. The ZP serves to protect the oocyte during its passage through the oviduct and acts as a physical barrier against pathogens, thereby safeguarding the oocyte and early embryo from infection. The ZP possesses antimicrobial properties that enhance its protective function protecting the oocyte and embryo during the critical stages of early development. Furthermore, it maintains the structure of blastomeres during pre-implantation development.[ 21 ] After fertilisation, ZP undergoes changes that significantly hinder sperm adherence. Before uterine implantation, an enlarged blastocyst must break free from the ZP. If the ZP thickness exceeds 16 µm, implantation becomes challenging. While the ZP is not essential for the development of human oocytes post-fertilisation, it can help ensure the safety of cultured embryos in vitro . Therefore, not everyone recommends a ZP-free culture method. Implantation occurs when hydrostatic pressure from the fluid within the blastocyst during its extended phase and the process of proteolysis cause the ZP to rupture and attach to the endometrial wall. However, in cases with ZP rupture or zona-free oocytes, this does not happen, as the blastocyst or cleavage directly implants into the endometrial wall.[ 22 ] Embryo transfer without the zona offers advantages such as the reduction of fragmentation in embryos exhibiting severe fragmentation at the pronuclear stage, which enhances blastocyst development and clinical outcomes.[ 18 23 ] Assisted hatching (AH) is utilised in IVF-embryo transfer procedures to promote spontaneous hatching and improve embryo implantation. Growth oocyte synthesis ZP, in the absence of ZP, the stability of gap junctions between the oocyte and surrounding follicular cells is diminished, which in turn impairs the transfer of nutrients, metabolites and other essential molecules required for the growth and development of both the oocyte and follicle.[ 24 ] Growth oocytes surrounded by cumulus granulosa cells (CGS), where the growth and development of the oocytes and granulosa cells are synchronised.[ 25 ] mtDNA plays a crucial role in this process. Variations in the oocyte microenvironment lead to distinct mitochondrial modifications, including changes in mtDNA. The replication of mtDNA in the oocyte is completed at the GV stage. mtDNA levels of CGCs declined from GV to metaphase I (MI) and did not change from MI to MII. The minimum threshold of mtDNA copy number plays a crucial role in determining oocyte quality and subsequently influences key processes such as fertilisation, implantation and embryonic development.[ 9 26 ] Insufficient mitochondrial biogenesis or cytoplasmic maturation may result in low oocyte mtDNA content.[ 26 ] The absence of ZP leads to abnormalities in the oocyte that may result in the lack of mtDNA, and further studies are needed to see the association between ZP and mtDNA.