Oolemma invagination of fresh and cryopreserved human oocytes during in vitro fertilization by ICSI

Development of novel prognostic criteria for evaluation of in vitro fertilization of fresh and cryopreserved oocytes is an important component of modern assisted reproductive technologies. There was compared a degree of oolemma invagination during intracytoplasmic sperm injection into fresh and cryopreserved oocytes. The classification of the gamete turgor state based on oolemma degree invagination during intracytoplasmic sperm injection was developed. This state of oocyte membrane is a prognostic characteristic of zygotic formation rate. Oocytes with normal turgor were characterized by a high fertilization rate higher than 80%. Rapid oolemma puncture during oocyte micromanipulation showed structural and functional damage of membrane and led to further oocyte degeneration. After cryopreservation by a standard method the ratio of different types of oolemma invagination has been changed. A relative number of cells with normal turgor was decreased down to 54% due to an increase of oocyte number with higher membrane elasticity. The four-stage method of cryoprotective media removing after oocyte freeze-thawing is more effective for preserving gametes with normal turgor contrary to the standard two-stage one.

Plasma membrane, the mechanical characteristics of which are determined by the presence of cholesterol and structural proteins of cytoskeleton is the primary target of the action of cryopreservation factors [13].In combination with microtubules and microfilaments of cytoskeleton, it provides volume regulation under the effect of osmotic factor.Carbohydrate components of oolemma, being in combination with proteins (glycoproteins) and lipids (glycolipids), ensure the stability of protein molecules [5].
The research aim was to compare the degree of oolemma invagination during ICSI and frequency of zygotic formation during fertilization of fresh and cryopreserved oocytes using standard and modified vitrification protocols.

Materials and methods
The findings represent a retrospective analysis of the data of embryological protocols of the patients In the study there were analyzed 106 oocytes obtained after induction of superovulation in 14 women aged of 21-38 years, mean age made (29.9 ± ± 4.8) years.The oocytes were divided into three groups: 1 -freshly isolated oocytes (n = 40); 2oocytes cryopreserved according to the standard protocol (n = 30) and 3 -oocytes cryopreserved according to the modified protocol (n = 36).
Superovulation was induced using a long midluteal protocol with agonists of gonadotrophin releasing hormone and recombinant follicle-stimulating one.Controlled superovulation was performed under ultrasound and hormonal monitoring.Recombinant chorionic gonadotrophin was used as a trigger for the final maturation of follicles.
The oocytes obtained after aspiration of follicles were cultured in Global Total for Fertilization medium (LifeGlobal, USA).After 4 hours of in vitro culturing, the oocytes were placed into 80 IU/ml of hyaluronidase solution (LifeGlobal, USA) for 30 sec, thereafter they were denuded (cumulus cells were removed by pipetting).Two hours after the described procedure, oocytes evaluated as mature (stage MII) were cryopreserved according to a standard protocol: the oocytes were equilibrated for 12 min in 7.5% dimethyl sulfoxide (DMSO) and 7.5% ethylene glycol (EG) and then transferred to a vitrification solution containing 15% DMSO and 15% EG [10].
The modification of the method consisted in step-by-step washing of oocytes from cryoprotective solution.Herewith the oocytes after thawing were transferred to solutions with a gradually decreasing concentration of sucrose (0.75, 0.5, 0.25, 0.125 and 0 M) and kept in each of them for 2 min.
Intracytoplasmic injection of sperm into the oocyte was carried out in accordance with the generally accepted procedure [14].The fact of zygote formation was established in 16-18 hrs after ICSI by the presence of two pronuclei in oocyte ooplasm.
During sperm injection into ooplasm, the following types of invagination of oolemma were observed: A -normal invagination, with penetration of injection needle to a depth of up to 1/2 of oocyte diameter; B -low with penetration of injection needle to a depth of less than 1/3 of oocyte diameter; C -without oolemma invagination -the oocyte puncture occured immediately after injection; Dincreased membrane integrity, complicated puncture, the needle penetrated to a depth of more than 3/4 of oocyte diameter (Fig. 1).
The groups were compared using the χ 2 test.The data were presented as (M ± m) and compared using two-tailed unpaired t-test.The value of p < 0.05 was considered as statistically significant.
During ICSI a normal invagination of oolemma was observed in (83 ± 7.3)% of cases in native oocytes (Fig. 2, group 1).Herewith the frequency of their fertilization was quite a high, namely above 80% (Table ).After cryopreservation with the standard technique, the number of oocytes with normal invagination of oolemma decreased, but the use of modified method allowed to augment the oocyte number with the mentioned characteristics up to (69 ± 4.9)% (Fig. 2, groups 2 and 3).
After cryopreservation, the oocyte number with an increased membrane strength significantly augmented (Fig. 2, group 2).However, this had no effect on their fertilization frequency.During ICSI, a higher pressure should be applied in the oocytes with an increased turgor of oolemma to penetrate the needle into ooplasm for passing through intracytoplasmic structures, when meiotic apparatus and cytoskeleton may be damaged [11].Therefore, this behavior of oolemma needs special attention in order to avoid further cultivation and transfer into uterine cavity of the embryos with chromosomal pathologies.
In the embryos, procured from oocytes with a low turgor, the fragmentation of cytoplasmic sites appears more frequently, being associated with a low implantation potential [9].The degree of oolemma invagination rate was shown to depend on ZP state.For example, the oocytes with ZP have the Young's modulus of oolemma, more than 85 times higher than ZP-free ones [4].This may partly explain an important role of ZP in oolemma cryoprotection, i. e. the resistance to mechanical stress due to a possible ice formation during cryopreservation.
Cryopreservation and further injection of ZPdeprived oocytes are questionable.There are the reported data on their successful survival, fertilization,  embryo development and pregnancy onset after embryo transfer into uterine cavity [19].

Conclusions
1.The type of oolemma invagination may be a prognostic index in fertilization of fresh and cryopreserved oocytes after ICSI.The oocytes with normal oolemma invaginations are characterized by a high fertilization frequency, and if it is absent the fertilization frequency decreases, and the oocyte degeneration is observed.
2. After cryopreservation according to the standard protocol, the number of oocytes with increased membrane strength augments.A four-step method for cryoprotectant removal out of oocytes with 2-minute intervals is more efficient for preserving a normal oocyte turgor than a two-step one.
undergoing an infertility treatment by ART at the ART-Clinic of Reproductive Medicine, Kharkiv.All the manipulations with gametes and embryos were performed according to 'The Convention on human rights and biomedicine of the Council of Europe' and the report of the Steering Committee On Bioethics (Oviedo, 1997) on 'The Protection of the human embryo in vitro' (Strasbourg, 2003) with an informed patient consent as well as the decision of the Committee in Bioethics of the Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine.