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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 4  |  Issue : 1  |  Page : 18-24

Vitrification of In vitro-matured Oocytes: Effects of meiotic spindle morphology on clinical outcome


1 Shanghai Ji Ai Genetics and IVF Institute, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011, China
2 Center for Reproductive Medicine, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200120, China
3 Reproductive Medical Center, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
4 Department of Gynaecology and Obstetrics, Qianfoshan Hospital, Shandong University, Jinan 250014, China
5 Shanghai Ji Ai Genetics and IVF Institute, Obstetrics and Gynecology Hospital, Fudan University; Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Shanghai 200011, China

Date of Submission08-Jul-2019
Date of Decision11-Sep-2019
Date of Acceptance29-Nov-2019
Date of Web Publication2-Apr-2020

Correspondence Address:
Xiao-Xi Sun
Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, No. 588, Fangxie Road, Shanghai 200011
China
Yi-Juan Sun
Shanghai Ji Ai Genetics and IVF Institute, Obstetrics and Gynecology Hospital, Fudan University, No. 588, Fangxie Road, Shanghai 200011
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2096-2924.281854

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  Abstract 


Objective: The meiotic spindle controls chromosome movement and mediates various functions essential for fertilization and early postfertilization events. This study aimed to examine whether vitrification causes meiotic damage in vitro- matured metaphase II (MII) human oocytes, and whether the meiotic spindle morphology influences the subsequent developmental outcomes.
Methods: The spindle characteristics of MII human oocytesin vitro matured were studied before and after vitrification using PolScope imaging and immunofluorescence staining. The developmental competence of oocytes was also examined.
Results: A total of 419 human MII oocytes were obtained from 593 intracytoplasmic sperm injection cycles at our hospital. Of these oocytes, 54 were used for immunofluorescence staining, whereas the other oocytes were examined by PolScope imaging and classified into three groups according to the meiotic spindle morphology: (A) normal morphology, (B) weak refraction and short meiotic spindle, and (C) no detectable meiotic spindle. The three groups demonstrated statistically significant differences in terms of survival after vitrification. However, differences were not found in terms of oocyte chromosome structure and meiotic spindle morphology on immunofluorescence staining performed before and after vitrification. Oocyte survival, fertilization, and early embryonic development rates were significantly higher in Group A than in Groups B and C with or without vitrification. While vitrification had no effect on these metrics in Group A, Groups B and C demonstrated significantly lower fertilization and cleavage rates after vitrification/warming.
Conclusions: Screening for normal meiotic spindle morphology and chromosome configuration before vitrification may increase the yield of healthy viable oocytes for various assisted reproductive technologies.

Keywords: Immunofluorescence Staining, Meiotic Spindle, Oocytes, PolScope Imaging, Vitrification


How to cite this article:
Gu RH, Li ZC, Lang JW, Chen H, Feng Y, Guo S, Fu J, Sun XX, Sun YJ. Vitrification of In vitro-matured Oocytes: Effects of meiotic spindle morphology on clinical outcome. Reprod Dev Med 2020;4:18-24

How to cite this URL:
Gu RH, Li ZC, Lang JW, Chen H, Feng Y, Guo S, Fu J, Sun XX, Sun YJ. Vitrification of In vitro-matured Oocytes: Effects of meiotic spindle morphology on clinical outcome. Reprod Dev Med [serial online] 2020 [cited 2020 May 26];4:18-24. Available from: http://www.repdevmed.org/text.asp?2020/4/1/18/281854




  Introduction Top


Infertility affects at least 15% of couples desiring children.[1] The development of assisted reproductive technologies (ARTs), such as in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), plays an important role in the treatment of infertility due to female or male factors. Infertility treatment may also require preservation of sperm, oocytes, embryos, and/or reproductive tissues. Oocyte cryopreservation is a potentially important ART adjunct for clinical applications in humans and other mammals. For instance, women without a partner or with loss of ovarian function due to cancer can store their oocytes for future use or simply for increasing cumulative pregnancy rates. For patients undergoing IVF, oocyte freezing can provide a source for donating oocyte or avoiding the requirement of repeated oocyte retrieval. This is especially important to infertile couples in countries that authorize the donation of oocytes but not of embryos.

Currently, two types of cryopreservation protocols are employed, i.e., slow freezing and vitrification. Of the two, vitrification has become a more popular approach because it results in less oocyte damage. Nevertheless, there is limited consensus on the use of this technique due to the large size, high volume/surface area ratio, and low membrane permeability of oocytes. Indeed, compared with 30%–40% of noncryopreserved oocytes, a much lower percentage of vitrified MII oocytes (only 7%–8%) results in pregnancy and live birth.[2] The probable causes of reduced oocyte viability following vitrification are disruption of cell organelles [3] and premature release of cortical granules.[4]

The meiotic spindle of oocytes is composed of microtubules constructed by polymerization of α-/β-tubulin dimers. Spindle microtubules originate at the organizing centers of both spindle poles and anchor chromosomes at kinetochores.[5] In oocytes, the meiotic spindle controls chromosome movement through the different stages of meiosis and mediates various functions essential for oocyte fertilization and early postfertilization events.[6] The meiotic spindle is a dynamic structure highly sensitive to temperature fluctuations and cryoprotectants. Cryopreserved oocytes analyzed immediately after warming exhibited severe disorganization or spindle disappearance when subjected to slow freezing or vitrification. Incubation for 1−3 h at 37°C has been shown to result in variable spindle recovery depending on the time intervals after freezing and warming.[7],[8],[9],[10] The specific effects of oocyte cryopreservation on the meiotic spindle depend on species, maturation stage, and methodology.

PolScope imaging, an orientation-independent polarized light microscopy, is more effective than the invasive techniques that provide static pictures and prevented the subsequent use of gametes. PolScope imaging has facilitated the study of the dynamic behavior of the meiotic spindle, permitting repetitive observations in vivo. Using this technique, Ciotti et al. demonstrated that spindle recovery is faster after vitrification than after slow freezing.[11]

Approximately 15%–20% of all retrieved oocytes during IVF or ICSI are classified as immature. Immature oocytes are classified as germinal vesicle (GV) or metaphase I (MI) depending on the presence of a GV or absence of both GV and the first polar body, respectively. In general, these immature oocytes are discarded; hence, they provide a valuable research tool to investigate the effects of cryopreservation on oocyte viability and reproductive potential. The aims of the present study were to examine whether vitrification/warming influences the meiotic spindle characteristics of metaphase II (MII) human oocytes in vitro matured using PolScope imagining and immunofluorescence staining and to assess if meiotic spindle morphology before/after vitrification/warming affects clinical outcome.


  Methods Top


Females and oocyte collection

This prospective study enrolled 477 females aged 20–38 years who underwent infertility treatment between November 2017 and December 2018. All patients were treated with gonadotropin-releasing hormone agonists (Serono, Geneva, Switzerland) and then with recombinant follicle-stimulating hormone (rFSH; Serono, Geneva, Switzerland). Oocytes were retrieved 36 h after administration of human chorionic gonadotropin (Livzon Pharmaceutical Group Inc., Zhuhai, China). The oocytes were cultured for 2–3 h; then, the cumulus − oocyte complex was isolated. Oocytes were collected from 593 ICSI cycles, and 564 MI oocytes were used in this study.

Males and sperm collection

Fresh sperms were collected from males aged 20−35 years. The basic clinical features, including age, body mass index, and sperm quality, showed no significant difference among the patients (data not shown). The swim-up method was used to isolate motile sperm from semen.

Ethical consideration

All patients provided written informed consent, and the study was approved by the Ethics Review Board of Shanghai Ji Ai Genetics and IVF Institute, Obstetrics and Gynecology Hospital of Fudan University, Shanghai, China (kyy2019-20).

In vitro oocyte maturation

Immature MI oocytes were cultured with the G-IVF medium (Vitrolife, Sweden AB) supplemented with 10% (v/v) human serum albumin (HSA, Vitrolife, Sweden AB), 0.075 IU/mL rFSH, 0.075 IU/mL rLH, and 20% human mature follicular fluid at 37°C under a 5% O2/6% CO2 atmosphere. After 24−25 h, MII oocytes were obtained, and those with a normal morphology were selected for subsequent experiments.

PolScope imaging

The OCTAX enhanced polarizing microscope (EyeWare 1.5.0.0°CTAX Microscience GmbH) was used for the analysis of oocyte spindle morphology. The MII oocytes were individually observed during the culture phase and both before and 2–4 h after vitrification/warming. The oocytes were rotated using a holding needle until a correct position for spindle visualization was found.

Vitrification and warming procedure

The vitrification reagent (Irvine Scientific, USA) included an equilibration solution (containing 7.5% ethylene glycol (EG), 7.5% dimethyl sulfoxide (DMSO), and 20% dextran serum supplement) and vitrification solution (containing 15% EG, 15% DMSO, 0.5 mol/L sucrose, and 20% dextran serum supplement). The warming reagents included 1 mol/L sucrose, 0.5 mol/L sucrose, 0.25 mol/L sucrose, and base solution.

Vitrification was performed at room temperature. In the equilibration solution, oocyte size first decreased and then gradually recovered, suggesting the completion of equilibration. The equilibration time was defined as the time required for oocytes to recover their original size. After equilibration, oocytes were incubated in the vitrification solution for 35 s, loaded on a Cryotop device, and stored in liquid nitrogen for a minimum of 1 week.

For warming, the oocytes were incubated with prewarmed WS1 at 37°C for 1 min, WS2 for 3 min, WS3 for 5 min, and WS4 for 5 min. The warmed oocytes were cultured in G-IVF medium +10% HSA at 37°C for 1 h under a 6% CO2/95% air atmosphere. Oocytes with a translucent appearance, cell rupture, or karyopyknosis and without cytoplasmic discoloration and fracture of the zona pellucida were defined as nonsurviving oocytes. The number of surviving oocytes was recorded and compared with that of freshly isolated oocytes.

In vitro fertilization and embryonic development

After culturing for 2–4 h, surviving MII oocytes subjected to vitrification/warming and freshly isolated oocytes were inseminated with fresh sperm using standard ICSI protocols and individually cultured on 50 μL G-IVF. At post-ICSI 16–18 h, oocyte fertilization was evaluated by the presence of pronuclei and two polar bodies. After fertilization, the embryos were cultured in G-I (Vitrolife, Sweden, AB) +10% serum substitute supplement (SSS, Irvine Scientific, Daimler St., Santa Ana, CA, USA) for 3 days. Embryo quality, including the degree of fragmentation and number and degree of blastomere equality, was evaluated daily.

On day 3 (D3), the culture medium was replaced with pre-equilibrated G-2 (Vitrolife) +10% HSA medium. On day 5 or 6, the morphology of blastocyst-stage embryos was evaluated. Blastocoel cavity expansion (EH stage) and the number and cohesiveness of the inner cell mass (grade) and trophectodermal cells (grade) were evaluated according to the Gardner and Schoolcraft grading system.[12]

Immunofluorescence staining

Immunofluorescence staining of tubulin and chromatin was performed as previously described.[13] Briefly, oocytes were fixed in 4% paraformaldehyde for 20 min, washed with Dulbecco's phosphate-buffered saline (DPBS), and incubated with 0.01% Triton X-100 in DPBS at room temperature for 40 min. The fixed oocytes were then washed and blocked at room temperature for 1 h in DPBS supplemented with 0.02% NaN3, 2% bovine serum albumin, and 0.1 mol/L glycine. After washing again with DPBS, the oocytes were incubated with β-tubulin monoclonal antibody at 4°C overnight, washed twice with DPBS, and then incubated with fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin G (Molecular Probes/Invitrogen) at room temperature for 60 min in the dark. After washing again with DPBS, chromatin was counterstained with 4,6-diamidino-2-phenylindole. Finally, oocytes were observed under a fluorescence microscope (Leica DMR; Manufac, Wetzlar, Germany). Examples of normal and abnormal meiotic spindle morphology and chromosome configurations are shown in [Figure 1].
Figure 1: The normal or abnormal character of meiotic spindle and chromosome alignment in human oocytes in vitro matured. (a-c) MII oocytes with normal meiotic spindle and chromosome alignment. (d-f) MII oocytes with abnormal meiotic spindle and chromosome alignment

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  Results Top


A total of 419 MII oocytes (74.3%) were obtained from 564 immature oocytes isolated from 477 consenting patients. The general characteristics of the donors (n = 477) are summarized in [Table 1]. In total, 365 of 419 Mll oocytes were observed with PolScope imaging and classified into three groups according to the meiotic spindle morphology: (A) normal morphology, (B) weak refraction and short meiotic spindle, and (C) no detectable meiotic spindle. Additional 54 oocytes were used for immunofluorescence staining. [Figure 2] summarizes the meiotic spindle characteristics of the three groups. 230 of 365 MII oocytes were vitrified/warmed and examined by PolScope imaging before and after vitrification and were inseminated after vitrification; 135 of 365 MII oocytes were inseminated with fresh sperm using standard ICSI protocols without vitrification. Fifty-four oocytes from 419 MII oocytes were used for observing spindle meiotic by immunofluorescence staining, 16 oocytes were in the fresh group and 38 oocytes were in the vitrification/warming group.
Table 1: General characteristics of the patients in the study

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Figure 2: The characteristic of meiotic spindle in human oocytes among three groups. (a) Normal morphology meiotic spindle; (b) weak refraction, short meiotic spindle; (c) no meiotic spindle appear

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The meiotic spindle morphology in human MII oocytes before and after vitrification by PolScope imaging

We have studied the spindle meiotic by PolScope every half hour after warming based on 20 MII human oocytes in vitro matured; about 20% (4/20) oocytes were not found spindle meiotic after 4 h of warming. No more changes later. At 2, 3, and 4 h after warming, oocytes were examined by PolScope. A total of 230 oocytes were vitrified/warmed and examined by PolScope imaging before and after vitrification; of these, 181 were classified as Group A, 35 as Group B, and 14 as Group C. A total of 163 (90.1%) oocytes survived after vitrification/warming in Group A; of these, 155 remained in Group A and 8 moved to Group B. Further, 25 (71.4%) oocytes survived vitrification/warming in Group B; of these, 23 remained in Group B and 2 moved to Group C. Finally, 7 (50%) oocytes survived vitrification/warming in Group C (50%) and demonstrated no detectable spindle. The survival rate significantly differed among the three groups [P < 0.05, [Table 2]. There was no significant difference in terms of the proportion of groups B and C oocytes before and after vitrification (49/230 [21.3%] vs. 40/195 [20.5%], P > 0.05).
Table 2: Meiotic spindle morphology of oocytes evaluated by the PolScope before and after vitrification

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Effects of vitrification on fertilization and developmental competence

While there was no significant change in terms of the rate of abnormal meiotic spindle after vitrification in any group, the rates of fertilization, cleavage, good-quality D3 embryo yield, and blastocyst formation were significantly higher in Group A than in Groups B and C [P < 0.05, [Table 3]. In contrast, fertilization and cleavage rates were significantly lower in Groups B and C after vitrification than those before vitrification (fertilization rate: 37.5% vs. 62.1%, cleavage rate: 26.1% vs. 61.1%; P < 0.05). Similarly, the abnormal fertilization rate was 73.3% for vitrified/warmed oocytes and 33.3% for freshly isolated oocytes in Groups B and C (P < 0.05). There were no significant differences in terms of the main outcome measures including good-quality D3 embryo yield and blastocyst formation rate between freshly isolated and vitrified/warmed oocytes with abnormal meiotic spindle morphology (P > 0.05). Furthermore, there were no significant differences in terms of oocyte developmental competence among all groups with or without vitrification.
Table 3: The developmental competence of fresh or vitrified/warmed oocytes with different meiotic spindle

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Effects of vitrification on meiotic spindle morphology and chromosome configuration as revealed by immunofluorescence staining

To prove the result of the PolScope, chromosome configuration and meiotic spindle morphology were also compared between freshly isolated and vitrified/warmed oocytes using immunofluorescence staining. In total, 47 oocytes, including 16 freshly isolated and 31 vitrified/warmed oocytes (survived vitrification among 38 oocytes), were evaluated. First, 47 oocytes were examined by PolScope before immunofluorescence staining. Of these, 14.9% (4 were vitrified/warmed oocytes and 3 were fresh oocytes) belonged to Group B+C. However, 12.8% (4 were vitrified/warmed oocytes and 2 were fresh oocytes) exhibited abnormal meiotic spindle morphology as revealed by immunofluorescence staining. The percentage of Group B+C was no significant difference between PolScope and immunofluorescence staining [P > 0.05, [Table 4]. Further, 26 of 31 oocytes surviving vitrification/warming exhibited normal meiotic spindle morphology. There were no significant changes in the proportion of oocytes with abnormal meiotic spindle morphology or aberrant chromosome structure with or without vitrification [P > 0.05, [Table 5].
Table 4: Comparison of meiotic spindle in fresh oocytes and vitrified/warmed oocytes by PolScope and immunofluorescence staining

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Table 5: Comparison of Meiotic spindle in fresh oocytes and vitrified/warmed oocytes

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  Discussion Top


The major advantage of vitrification is the elimination of mechanical damage caused by intra- or extracellular ice crystals and potential reduction of chilling damage by shortening the exposure period to suboptimal temperatures. However, there are concerns over vitrification due to high cooling rates and high concentrations of cryoprotectants, which could be detrimental to the meiotic spindle. Indeed, many studies have reported that oocyte cryopreservation may cause depolymerization and disorganization of spindle microtubules.[14],[15] In contrast, vitrification did not cause substantial damage to spindle integrity and chromosome configuration. The characteristics of the meiotic spindle before vitrification are associated with oocyte survival and embryonic development rates.

Normal fertilization and subsequent embryonic development of MII oocytes require proper MII spindle assembly and chromosome configuration for correct sister chromatid segregation.[16] The meiotic spindle is highly sensitive to environmental changes. When oocytes are retrieved from follicles and exposed to an in vitro maturation medium, some damage is unavoidable. In this study, 78.7% of in vitro-matured oocytes exhibited normal meiotic spindle morphology, higher than that was reported previously.[17] This higher preservation rate may be attributable to differences in patient characteristics, media, and drugs used.

The extreme vulnerability of human MII oocytes to cryoinjury is related to the meiotic spindle. During the vitrification procedure, the spindle configuration may be preserved if oocytes are exposed to cryoprotectants, and its structure is completely lost in the thawing procedure.[18] Several reports have detailed the disappearance and reappearance of the meiotic spindle during MII in both mammalian and human oocytes subjected to slow freezing and vitrification.[19],[20] In vitrified mouse oocytes, Chen et al.[21] observed that open pulled straws, closed pulled straws, and electron microscopy grids showed better preservation of meiotic spindle morphology and chromosome pattern than conventional straws. Gook et al.[22] reported that 60% of human oocytes subjected to slow freezing exhibited normal meiotic spindle morphology at postwarming 1 h compared with 81% of freshly isolated specimens, and Ciotti et al.[11] reported that spindle recovery was faster after vitrification than after slow freezing. Above all, the effect of vitrification on the oocyte spindle is related to freezing technology, carrier, and other factors. In the current study, vitrification alone did not disrupt spindle integrity and chromosome configuration. Similarly, Cao et al.[23] found that the rates of spindle integrity and chromosome configuration abnormalities were similar between vitrified and freshly isolated oocytes. In our study, the percentage of the abnormal character of spindle meiotic was similar between PolScope and immunofluorescence staining, which was consistent with the previously reported study from the Wang's laboratory.[17] Hence, spindle meiotic with the PolScope can be applied to human IVF to help predict chromosomally normal oocytes for insemination. This may provide indirect evidence that the live birth rates obtained with vitrified oocytes should be comparable to those achieved with freshly isolated oocytes. Nevertheless, in the current study, cryopreservation damage of oocyte meiotic spindle morphology was observed before vitrification, with significantly decreased survival rate of oocytes with abnormal meiotic spindle morphology being observed after vitrification. On the other hand, the damage tolerance of oocytes to vitrification/warming reduced. If the spindle meiotic in fresh human MII oocytes is weak refraction, short, or no detectable meiotic spindle, it is more sensitive to cryoinjury than those oocytes with normal spindle meiotic. Therefore, the survival rate and developmental competence are lower in Group B and C.

In mammals, the meiotic spindle appears highly sensitive to temperature fluctuations,[24] with microtubules undergoing rapid depolymerization when they are exposed to conditions even a few degrees below the physiological temperature. Indeed, several reports have suggested that abnormal meiotic spindle morphology is a predictor of aneuploidy.[5] Spindle disorganization can also disrupt the sequence of events that lead to the completion of meiosis and fertilization, thus possibly contributing to low pregnancy rates.[25],[26] Wang and Keefe reported that oocytes without spindles demonstrated lower fertilization and early development rates than oocytes with spindles.[27] Our results confirm the previously reported findings that the meiotic spindle has negative effects on oocyte developmental competence with or without vitrification, whereas oocytes with normal meiotic spindle morphology before vitrification demonstrated fertilization rates and developmental outcomes similar to freshly isolated oocytes. If oocytes with abnormal meiotic spindle morphology or no meiotic spindles on PolScope imaging are sensitive to vitrification, lower survival and development rates can be obtained. Alternatively, we found no differences in terms of good-quality D3 embryo yield and blastocyst formation rates among the study groups, possibly due to the small sample size. However, oocytes with abnormal meiotic spindle morphology before vitrification showed numerically reduced developmental competence compared with oocytes with normal meiotic spindle morphology before vitrification.

In conclusion, meiotic spindle integrity is crucial for vitrification/warming tolerance and subsequent fertilization of MII oocytes. The survival and developmental potential of Mll oocytes with normal spindle morphology are not influenced by vitrification; vitrification greatly reduces the survival of Mll oocytes with abnormal meiotic spindle morphology. These findings suggest that meiotic spindle morphology should be checked before vitrification to enhance the yield of potentially viable oocytes for ART.

Financial support and sponsorship

Natural Science Foundation of China (No. 81601342, 81901558) from Dr. Yi-Juan Sun and Rui-huan Gu, Shanghai Municipal Planning Commission of Science and Research Fund (General Program, No. 201740075), Huangpu District, Shanghai Municipal Planning Commission of Science and Research Fund (No. HKW201659).

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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  In this article
Abstract
Introduction
Methods
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