Pregnancy following fertilization of zona-free, coronal cell intact human ova: Case Report

James D. Stanger1, Karen Stevenson, Aaron Lakmaker and Robert Woolcott

Lingard Fertility Centre, Merewether, Newcastle, Australia


    Abstract
 Top
 Abstract
 Introduction
 Case report
 Results
 Discussion
 Note added in proof
 References
 
A pregnancy by intracytoplasmic sperm injection (ICSI) of a couple whose oocytes were recovered without a zona pellucida is reported. Previous IVF/ICSI cycles indicated all oocytes had either very thin or no zona pellucida at the time of cumulus removal prior to ICSI. As a result, many ova were very fragile and lysed upon handling. In the latest attempt, six ova were recovered. After coronal cell dissection of two ova where both lacked a zona pellucida and one lysed, it was elected to attempt to inject the remaining four ova without removal of their coronal cells. The zona pellucida was not visible through the cell layer and the position of the polar body in three ova was speculative. Two embryos possessed normal pronuclei on day 1, and displayed six blastomeres prior to transfer on day 3. During preparation for transfer, sufficient coronal cells fell away to confirm both embryos lacked a zona pellucida. Pregnancy was confirmed by ultrasound observation of a fetal heart 4 weeks later. This report documents that where the zona pellucida is absent, the coronal cells provide sufficient support to maintain blastomere interaction and embryo viability and the zona pellucida is not essential for early embryo development and pregnancy.

Key words: ICSI/IVF/pregnancy/zona-free


    Introduction
 Top
 Abstract
 Introduction
 Case report
 Results
 Discussion
 Note added in proof
 References
 
The zona pellucida is produced by the developing oocyte early in follicle development (Rankin and Dean, 1996Go). The functions of the zona pellucida include physical support for the blastomeres, inhibition of inappropriate tubal implantation, and protection from the maternal immune system. It acts as a barrier to sperm–oocyte interaction, polyspermy and to embryo uterine interaction prior to implantation. The size of the zona pellucida is known to vary in thickness with thick zona pellucida being associated with poor fertilization (Bertrand et al., 1995Go; Loret De Mola et al., 1997Go). Zonae of average but variable thickness are conversely associated with pregnancy presumably because of increased competency to hatch prior to implantation (Cohen et al., 1989Go; Palmstierna et al., 1998Go). There are no reports to reveal whether the zona pellucida itself is essential for embryo development. Studies on mouse knockout strains deficient in the zona protein ZP3 exhibit poor oocyte maturation suggesting dysfunctional coronal cell–oocyte contact (Paterson et al., 1992Go, 1998Go). Recently, the enzymatic removal of the zonae pellucidae from human embryos by day 3 have been reported to result in pregnancy (Mansour et al., 2000Go). By this time, however, some early adhesion between blastomeres may be anticipated and consequently the embryonic signals leading to compaction already produced. The critical period may involve the precompaction stages where each blastomere remains relatively independent. It is thought that during this stage, cell contact is critical for the E-cadherin-induced cytoskeleton redistribution to proceed (Neganova et al., 2000Go).

Occasionally, the ova of a woman presenting with unexplained infertility and treated with IVF do not possess a zona pellucida. Three such cases including the patient described in this report have been noted over 15 years at Lingard Fertility Centre. In such cases, either the ovum failed to fertilize or lysed on dissection. Typically the ova are very fragile without the zona's protection. In a previous treatment cycle of the current patient, two zonae-free ova, which survived coronal denuding, were injected by intracytoplasmic sperm injection (ICSI) (because of the husband's oligospermia). These ova fertilized and developed to the 4-cell stage but then arrested with no further development. The blastomeres of these embryos remained in contact, albeit tenuous, with each other as long as they were not handled. Even gentle aspiration caused some blastomeres to become isolated. Thus our earlier experience with embryos from this couple support the argument that the zona pellucida may act to maximize the degree of cell–cell contact. During the current attempt at ICSI, a similar experience was encountered when two ova also without a zona pellucida were denuded for ICSI. An alternative strategy was attempted where the coronal cells of the remaining ova were not removed. Rather, they were left in situ to act as a support for the zona pellucida. This case documents that pregnancy may be achieved after transfer of an embryo that lacked a zona pellucida at ovulation.


    Case report
 Top
 Abstract
 Introduction
 Case report
 Results
 Discussion
 Note added in proof
 References
 
The 36 year old female and 37 year old partner were registered for treatment at Lingard Fertility Centre using ICSI because of the husband's severe oligospermia. The woman exhibited grade 2 endometriosis, had a body mass index of 35 and regular menses. The couple subsequently undertook five ICSI attempts (including the current cycle). The stimulation for all cycles employed a long down-regulation protocol using the intra-nasal gonadotrophin-releasing hormone (GnRH) agonist, Nafarelin® (Syneral, Abbotts, Sydney, Australia) and follicle stimulation with recombinant FSH [either Gonal-F® (Serono, Sydney, Australia) or Puregon® (Organon, Sydney, Australia)] at doses between 250 IU/day and 450 IU/day. Gonadotrophin stimulation commenced when basal endocrinology was documented and continued at a constant dose until two to three follicles >18–20 mm were observed on ultrasound. Ova were recovered by vaginal ultrasound follicle aspiration under local anaesthetic 36 h later (Woolcott and Stanger, 1997Go).

The male partner produced a semen sample 1–2 h after ova collection and a motile subfraction was recovered using discontinuous density gradient centrifugation at 500 g for 20 min (Puresperm®; Nidacon, Gothenburg, Sweden). The semen sample was washed in human tubal fluid (HTF) media (Irvine Scientific, Chemtec, Melbourne, Australia) containing 10 mg/ml human serum albumin (IVF Sciences, Gothenburg, Sweden). All ova were washed after oocyte recovery and cultured in open culture in 1.0 ml HTF culture containing 10 mg/ml human albumin incubated at 37°C and 6% CO2, 5% O2 and nitrogen balance until micro-injection. Normally ova were denuded immediately prior to micro-injection in a heated workstation in 4-well Nunc dishes using various hand-drawn Pasteur pipettes calibrated down to ~250 µm for coarse dissection of the cumulus cells. Routinely, all ova were prepared for ICSI by incubation in HEPES medium (Irvine Scientific) containing 150 IU hyaluronidase (Hylase®; Fisons, Sydney, Australia) for 2–3 min until the cumulus cells fell away with gentle aspiration. In previous cycles, individual ova–coronal complexes were then aspirated with a micropipette until most of the coronal cells were removed and the ova could be visualized. After the ova were denuded, each ova was injected using micro-injection needles (RML, Adelaide, Australia) using 1 mg/ml polyvinylpyrrolidone (RML) in HTF + HEPES + albumin. When completed, the ova were washed in HTF without HEPES and transferred to 5–10 µl microdrops of HTF (Quinns basal X1 + 10 mg/ml albumin; SART 1007; Sage Biopharma, Bedminister, NJ, USA) under embryo tested mineral oil (Ovoil®; IVF Sciences). The embryos remained in this medium until transfer on day 3. The embryos were transferred in 20 µl medium using three French soft catheters (K-PETS-2031; Cook, Brisbane, Australia).

Luteal support of 1500 IU human chorionic gonadotrophin (HCG) (Organon) was provided every 3 days from day 2 after oocyte recovery. Pregnancy tests were performed on or after day 16 post-oocyte recovery. Ultrasound confirmation of pregnancy was made 3 weeks after diagnosis of pregnancy.

Modification during last procedure
The stimulation regimen for the current cycle utilized a dose regimen of 450 IU Gonal-F® daily for 13 days. Ovulation was induced with 5000 IU HCG (Pregnyl®; Organon) when two follicles reached 18 mm or greater. All other aspects of the last treatment cycles resulting in pregnancy were the same as described above except for the denuding and injection of the oocytes. In this cycle, six ova were recovered and denuded of cumulus cells. Two ova were initially denuded of the coronal cells. Since both ova did not have a zona pellucida, the remaining four ova were not denuded of the coronal cells. Each oocyte was held by the coronal-cell layer and the micro-injection needle passed through it and into the oocyte. The coronal cells formed a tight layer around the oocyte such that the cells were not displaced with gentle handling. The cytoplasm, when drawn into the needle prior to the injection of the spermatozoon, was clearly visible. The distortion of the oocyte was apparent during the injection process but the transfer of the spermatozoa was not. On withdrawal of the needle, it was evident the spermatozoa had been transferred on each attempt. There was no loss of cytoplasm and all oocytes remained intact after the injection. The coronal cell layer provided support for the oocyte both when held by the holding pipette and during the initial passage of the needle into the oocyte.


    Results
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 Abstract
 Introduction
 Case report
 Results
 Discussion
 Note added in proof
 References
 
In the previous four cycles of ICSI, 27 ova were recovered (six, twelve, three and four ova respectively). When these ova were denuded, 8/28 were without a zona pellucida (2/6, 2/12, 0/3 and 4/4 respectively) and all other ova had very thin zonae pellucidae. In the first three cycles six embryos with thin zonae pellucidae were transferred without pregnancy. In the fourth attempt, all ova were without a zona pellucida, three had a polar body attached and were injected with great care. It was proposed to delay embryo transfer until day 4 when compaction may have provided sufficient adhesion not to lose cells at transfer or prior to implantation. Two of these fertilized and developed to the 4-cell stage over 2 days after which they arrested and failed to develop further. Since the embryos failed to develop beyond the 4-cell stage the transfer was abandoned.

In the current pregnancy cycle, six ova were recovered. The cumulus mass surrounding each ovum was removed with hyaluronidase leaving the coronal cell layer intact. Two ova were initially completely denuded with disappointing results. Both ova lacked a zona pellucida. One ovum was at the germinal vesicle stage and the second had resumed oocyte maturation but had not extruded a polar body (or had lost it during denuding). At this point, given the experience during the previous cycle, it was elected to inject the ovum directly without denuding the remaining ovum. Each ovum–coronal cell mass (OCC) was inspected under the inverted microscope (x200) for a zona pellucida and a polar body. Not surprisingly, the image made identification of the polar body difficult but no zona pellucida could be seen in any OCC. In one OCC, a polar body was clearly visible while in the remaining three ova the polar body was difficult definitively to identify but was discernible through the coronal cells.

In all four ova, the coronal cell layer remained intact. Consequently, the injection pipette was passed through the coronal layer without any attempt at separating a pathway to the oocyte. The cells did not adhere to the needle and did not appear to be carried into the oocyte. The needle was passed into the ovum with the presumed polar body at 6 o'clock with distortion clearly visible. The cytoplasm was drawn into the oocyte such that it was visible beyond the cell layer before being reinjected with the spermatozoon. It was not possible clearly to identify the passage of the spermatozoon into the ooplasm but the spermatozoon was not in the pipette on its withdrawal.

The ova were gently washed and placed in individual 5 µm microdrops of Quinn's basal X1 medium containing 10 mg/ml albumin and left untouched for 3 days prior to transfer. On day 1, two ova, when examined under an inverted microscope, each contained two pronuclei clearly visible through the coronal cell layer, which remained in place around the zygote. The remaining two ova did not have pronuclei and these failed to cleave. On day 2, 42 h post-injection, one embryo was a 4-cell embryo and the other a 2-cell embryo. On day 3, 66 h after injection, both were thought to contain six blastomeres with no fragmentation. Up to this point, the embryos had not been handled and the coronal cells continued to adhere to the embryo. During handling prior to transfer, some coronal cells fell away allowing partial visualization of the embryos. Unfortunately, no photomicrographs could be taken to demonstrate the embryo development. This confirmed that each embryo lacked its zona pellucida. A concentration of 282 IU HCG was reported on day 17 and an ultrasound scan 3.5 weeks later demonstrated a single intrauterine pregnancy with a normal fetal heart. The pregnancy is due in early 2001.


    Discussion
 Top
 Abstract
 Introduction
 Case report
 Results
 Discussion
 Note added in proof
 References
 
All mammalian ova are encased within a soft pliable coating that has many functions (Rankin and Dean, 1996Go). It has been proposed these could include encapsulation until implantation, creation of a micro-environment allowing for autocrine stimulation of blastomeres by embryo-derived growth factors and protection from the maternal immune system. While its existence is a major barrier to fertilization, it does act as a barrier to polyspermy and as a medium to process spermatozoa. It is currently proposed that only acrosome intact spermatozoa bind to the zona pellucida, which acts as a trigger to induce the acrosome reaction. The equatorial region of the acrosome reacted spermatozoa is able to bind to the oocyte plasma membrane. Thus the zona pellucida has a positive role in facilitating fertilization. Consequently, fertilization in vitro of zona-free ova can occur but only with spermatozoa which have spontaneously undergone the acrosome reaction (Laws-King et al., 1987Go). The role of the zona pellucida during cleavage is less clear with some suggestions that oviductal proteins may bind to the zona pellucida (Bleau and St-Jacques, 1989Go).

This report confirms that the zona pellucida is not an essential component of human embryo development since the absence of the zona pellucida did not inhibit embryo development. It is interesting that in the previous cycle, where embryos were created and cultured without a zona, they failed to progress beyond the 4-cell stage, while in the current cycle, where the coronal cells continued to surround the embryo during the early stages, they were able to continue growth. While the removal of the zona pellucida by pronase treatment prior to blastocyst transfer is reported to increase the pregnancy rate (Fong et al., 1998Go), there appear to be no reports of zona-free embryos developing to pregnancy from fertilization of zona-free oocytes with ICSI. Recently, Mansour et al. (2000) demonstrated that zona removal on day 3 also resulted in pregnancies indicating that after initiation of compaction, the zona is not required. Interestingly, the removal of the zona on day 3 had no impact for `good' prognosis cases but appeared beneficial for `poor' prognosis cases.

One physical function of the zona pellucida in early cleavage is to ensure maximum contact between blastomeres prior to compaction. In a recent study, Neganova et al. (2000) reported contact between mouse blastomeres was essential to promote E-cadherin signalling to the microtubule cytoskeleton and mitochondrial distribution. E-cadherin appears about the 2-cell stage in the mouse but sufficient cell contact is required to promote cell adhesion. It would appear that a similar process may occur in the human given the failure of non-enclosed 4-cell embryos to continue development. In this case, the surrounding coronal cells effectively achieved the same function of the zona pellucida. Two previous studies have suggested that compaction and early blastulation could occur in vitro with zona-free oocytes (Veeck, 1998Go; Ding et al., 1999Go). In these cases, the oocytes escaped from the zona pellucida during cumulus removal and were not transferred. It remains unclear whether there are differences in the capacity of blastomeres to adhere and compact when freed from the confines of, or when unable to synthesize, a zona pellucida.

There have been several reports relating variability in the thickness of normal zona pellucida to the establishment of pregnancy in humans (Cohen et al., 1989Go). Excessive zona thickness has also been linked to age and high dose regimens of gonadotrophins (Bertrand et al., 1995Go; Loret De Mola et al., 1997Go) and is thought to be a cause of poor fertilization. However, the reason why a woman may produce an incomplete or absent zona pellucida is more likely to have a genetic rather than environmental cause (Rankin and Dean, 1996Go). Knockout studies in mice have revealed that all three zona proteins are required for complete zona formation (Paterson et al., 1992Go, 1998Go). Mouse knockouts missing a functional ZP1 gene have thinner zona pellucida. ZP2 and ZP3 appear to be strongly related to oocyte development since knockouts for these two proteins produce significantly fewer ova and no 2-cell embryos in vivo. ZP3-/- mice characteristically produce disorganized oocyte–cumulus complexes with the oocyte remaining at the germinal vesicle stage or becoming dissociated from the cumulus cell matrix. It would seem from these mouse knockout models that the woman in this case might have a genetic related problem with the expression of ZP1 protein. In future cases, the couple should be counselled that, like Y chromosome microdeletions, any female offspring might inherit a genetic predisposition to infertility. An alternative explanation might involve the development of anti-zona pellucida antibodies, which have the potential to interfere with the structural assembly of the zona proteins into the zona pellucida (Sacco and Yurewicz, 1989Go). Regardless of the aetiology of the production of zona-free ova, ICSI using an intact coronal cell layer appears a practical method of achieving a pregnancy in such rare cases.

There have been several other concerns about injection of the oocyte directly through the coronal layer. The first related to the difficulty in visualizing the polar body and therefore orientating the ovum correctly to avoid the cytoplasm containing the condensed chromosomes. In one ovum, the polar body was visible while a clear view of the polar body in the remaining ova was not possible. Manipulation of the OCC by rolling across the ICSI drop revealed a flattening of the ooplasm membrane and an impression of the polar body. In cases where the polar body is small or compressed, it may well be difficult to orientate the oocyte correctly. In a recent study, the position of the polar body was adjacent to the meiotic spindle in only 37% of ova in oocytes matured in vivo (Hardarson et al., 2000Go). The alignment was better in immature ova cultured in vitro until polar body expulsion, suggesting the denuding of mature oocytes may contribute to the dislocation of the polar body relative to the spindle. The spindle, identified by immunostaining of tubulin, was always in the animal pole of the oocyte. Injection of mature oocytes directly through the coronal cell layer without denuding could reduce concerns about the correct position of the spindle and polar body of denuded oocytes.

Another concern relates to the risk of including somatic DNA into the ovum. Recent experiments have demonstrated that foreign DNA attached to spermatozoa can be included and expressed in Rhesus monkey births. In this case, the concern may be raised that injection through the coronal cells at least raised the possibility that somatic coronal DNA could be dragged into the ooplasm (Chan et al., 2000Go).

In summary, while the incidence of a genetically impaired or dysfunctional zona pellucida are very rare among infertile couples, using the coronal cells as a de facto zona enclosure appears to be beneficial to the early cleaving embryo and not detrimental to the advanced stages of preimplantation embryology or to implantation and pregnancy.


    Note added in proof
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 Abstract
 Introduction
 Case report
 Results
 Discussion
 Note added in proof
 References
 
The pregnancy is progressing normally at 35 weeks.


    Notes
 
1 To whom correspondence should be addressed at: Lingard Fertility Centre, 23 Merewether Street, Merewether, NSW 2291, Australia. E-mail: Jim.Stanger{at}lingard.maynick.com.au Back


    References
 Top
 Abstract
 Introduction
 Case report
 Results
 Discussion
 Note added in proof
 References
 
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Chan, A.W.S., Luetjens, C.M., Dominko, T. et al. (2000) Foreign DNA transmission by ICSI: injection of spermatozoa bound with exogenous DNA results in embryonic GFP expression and live Rhesus monkey births. Mol. Hum. Reprod., 6, 26–33.[Abstract/Free Full Text]

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Submitted on June 20, 2000; accepted on October 6, 2000.