Morphology of in-vitro matured oocytes: impact on fertility potential and embryo quality

Anne Lis Mikkelsen2,1 and Svend Lindenberg,1

The Fertility Clinic Herlev University Hospital, Fruebjergvej 3, DK-2100 Copenhagen, Denmark


    Abstract
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
BACKGROUND: The purpose of the present study was to investigate the morphology of in-vitro matured metaphase II (MII) oocytes and to observe if there was a difference in the morphology between polycystic and normal ovaries. Furthermore, the morphology of in-vitro matured MII oocytes was related to their subsequent fertilization and cleavage rates and to embryo quality. METHODS: This retrospective study included 264 MII oocytes obtained in 100 consecutive cycles. Oocyte retrieval was performed transvaginally and cumulus enclosed oocytes were matured for 28–30 h before evaluation. Prior to ICSI, all MII oocytes were graded into three groups according to the number of anomalies: grade I: oocytes without any anomaly (n = 144, 54%), grade II: oocytes with one anomaly (n = 87, 33%) and grade III: oocytes with at least two anomalies (n = 33, 12.5%). RESULTS: Oocyte grades did not differ between women with polycystic ovaries [grades I, II and III respectively: 58/94 (61.7%), 29/94 (30.9%) and 7/94 (7.4%)] and women with normal ovaries [grades I, II and III respectively: 86/170 (50.6%); 58/170 (34%); 26/170 (15.3%)]. Morphology was not related to fertilization rates. The cleavage rate was, however, affected by morphological anomalies (grade I [77/144 (53.5%) versus grade II 33/87 (37.9%) (P = 0.03)], although no significant decrease in cleavage rate could be demonstrated when all grade II and III oocytes were compared with normal oocytes. Significantly more embryos of good quality developed after grade I oocytes [54/144 (37.5%)] compared with those from grade II and grade III oocytes (22/120; P = 0.001). The presence of cytoplasmic abnormalities significantly decreased the cleavage rate (P = 0.04) and also the number of good quality embryos (P < 0.001). CONCLUSION: The in-vitro maturation of oocytes without anomalies yields higher quality embryos, with higher cleavage rates, than those with anomalies.

Key words: embryo quality/fertilization/in-vitro maturation/oocyte morphology


    Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Embryos resulting from in-vitro maturation have failed to demonstrate the same implantation potential as those derived from in-vivo matured oocytes (Trounson et al.1998Go; Cha et al.2000Go; Mikkelsen et al.2000Go). It has been postulated that the impaired embryo development of human oocytes obtained after in-vitro maturation might be due to impaired cytoplasmic maturation and/or asynchrony of nuclear and cytoplasmic maturation.

The relationship between metaphase II (MII) oocyte morphology and IVF outcome is well accepted in conventional IVF (Bedford and Kim, 1993Go; ,Veeck et al.1998), but it is controversial after intracytoplasmic sperm injection (ICSI). While two studies (Xia, 1997Go; Loutradis et al.1999Go) found that morphologic anomalies are related to fertilization rate and embryo quality after ICSI, others were not able to demonstrate this ( De Sutter et al.1996Go; Balaban et al.1998Go). Oocytes with cytoplasmic anomalies failed to demonstrate the same implantation rate as oocytes without anomalies ( Serhal et al.1997Go) and poor ongoing pregnancy rates have been reported even if fertilization, embryo quality and total pregnancy rates were normal ( Kahraman et al.2000Go). Extracytoplasmic anomalies such as first polar body morphology at the time of ICSI have been found to be suitable indicators of subsequent fertilization and embryo quality (Xia, 1997Go; Ebner et al.2000Go), and elective transfer of embryos selected on the basis of first polar body morphology is associated with increased rates of implantation and pregnancy ( Ebner et al.1999Go). These previous findings suggest that the presence of anomalies in MII oocytes is associated with a decreased potential of the oocyte to fertilize and cleave and develop into viable embryos. No study has previously focused on oocyte morphology of in-vitro matured MII oocytes.

The present paper reports the results of a study in which MII oocytes, obtained after in-vitro maturation, were evaluated morphologically by criteria previously used on MII oocytes obtained after in-vivo maturation. The purpose of the study was to observe if there was a difference in the morphology between polycystic and normal ovaries. Furthermore, the morphology of MII oocytes was compared with the outcome of ICSI (fertilization rate, cleavage rate and embryo quality).


    Material and methods
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Patients
The study included MII oocytes (n = 264) obtained after in-vitro maturation in 100 consecutive oocyte retrieval from October 1999 until June 2000. A total of 481 cumulus enclosed oocytes were retrieved, out of which 264 (55%) were at metaphase II after maturation in vitro for 28–30 h. In-vitro maturation was performed in two groups of women, one with regular menstruation and normal ovaries detected by ultrasound, the other with polycystic ovarian syndrome (PCOS). The regularly menstruating women (n = 71 aspirations) were referred to the in-vitro maturation programme for either male factor (n = 32) tubal factor (n = 34) or a combination of these factors (n = 5). The PCOS patients (n = 29 aspirations) were characterized by (i) detection of polycystic ovaries by ultrasonography, defined as >10 follicles 2–8 mm in diameter, in one plane, located either around or through an echodense thickened dense stroma (Adams et al.1985Go); (ii) oligomenorrhoea or amenorrhoea for >6 months; and (iii) endocrine abnormalities (hormone analysis showed elevated LH/FSH ratio or elevated androgens).

The ages of the women were 18–37 years and they had a body mass index (BMI) between 18 and 29 kg/m2. Excluded were patients with possible poor quality of the oocytes, which meant low (<20%) cleavage rate in previous IVF/ICSI and at least four previous unsuccessful IVF/ICSI treatments. Furthermore, cycles in which no MII oocytes were obtained (n = 18 cycles in the study period) were excluded.

In all patients ultrasound examination was performed and the size of the follicles was measured on day 3, on day 6–7 and daily/every second day until the day of aspiration. In regular menstruating women (n = 170 cycles) the aspiration was performed the day after a leading follicle of 10 mm was observed (Mikkelsen et al.2000Go). The women with PCOS received rFSH (Gonal-F, Serono) 150 IU/day for 3 days, initiated on day 3 after withdrawal bleeding, and the aspiration was performed on day 8–9.

Embryo transfer was performed on day 3 following ICSI. To support the luteal phase the women were given 17-ß-oestradiol (6 mg/day) initiated on the day of aspiration. Micronized progesterone (300 mg/day) was initiated 2 days later. Oestrogen and progesterone were continued until the pregnancy test and if the pregnancy test was positive until 50 days gestation. Transvaginal ultrasound visualization of a gestation sac with evidence of heart activity 5 weeks after embryo transfer indicated clinical pregnancy.

The in-vitro maturation/IVF–embryo transfer procedure was approved by the local ethics committee, and written informed consent was obtained from all participants.

Oocyte recovery, maturation, fertilization and embryo culture
All procedures were performed as previously described (Mikkelsen et al.2000Go; Smith et al.2000Go). In brief, oocyte retrieval was performed transvaginally with a single lumen 17 G needle. The follicles were punctured, not flushed. After aspiration the needle was flushed with prewarmed Ham F-10 with heparin (Life Technologies, Copenhagen, Denmark). Follicular aspirates were filtered (Falcon 1060; 70 µm mesh size) to remove erythrocytes and small cellular debris. Healthy appearing cumulus enclosed oocytes were matured in TCM 199 (Sigma, Roedovre, Denmark) for 28–30 h. The culture medium was supplemented with 0.3 mmol/l Na pyruvate, 1500 IU/ml penicillin G, 50 mg/ml streptomycin sulphate, 1 µg/ml oestradiol (all from Sigma), rec-FSH 0.075 IU/ml (Gonal-F; Serono, Geneva, Switzerland), 0.5 IU/ml HCG (Profasi; Serono) and serum from the patient (10%). After maturation oocytes were exposed to hyaluronidase to facilitate mechanical removal of the surrounding cumulus cells. Thus, accurate assessment of metaphase oocytes was possible.

After ICSI, the oocytes were incubated separately in order to avoid mixing of the different oocytes. Fertilization rate and embryo grade (Steer et al.1992Go) per oocyte were assessed 18–20 h and 68–70 h after ICSI respectively. The criterion for accepting fertilization as normal was the presence of two pronuclei as well as two polar bodies. The criterion for good quality embryos was <10% fragmentation plus development to at least a 2-cell embryo on day 2 and a 4-cell embryo on day 3 after ICSI.

Morphological evaluation
The morphological images of all MII oocytes were recorded in a computerized database (FQC, Fercom, Denmark) with a colour video camera (Panasonic) mounted on an inverted microscope with Hoffmann modulation contrast (Nikon). The setting for microscopic observations (x200 magnification) and bright field were kept constant during the study (Gabrielsen et al.2000Go).

Using previous published protocols (Xia, 1997Go; Ebner et al.2000Go) the anomalies were classified as (i) normal, (ii) extracytoplasmic anomalies and (iii) cytoplasmic anomalies. Oocytes without any anomaly showed clear cytoplasm with uniform texture and homogeneous fine granularity, a round or ovoid first polar body with smooth surface and perivitelline space of normal size. Oocytes with extracytoplasmic anomalies showed rough or fragmented first polar body and/or a large perivitelline space. Cytoplasmic anomalies included dark cytoplasm, granular cytoplasm or refractile bodies. Granular oocytes were dark with granularity either homogeneous affecting the whole cytoplasm, or concentrated as a dark mass in the central portion of the oocyte with a clear peripheral ring (Van Blerkom and Henry, 1992Go). Cytoplasmic inclusions comprised vacuoles or refractile bodies (Van Blerkom and Henry, 1992Go; Serhal et al.1997Go). The oocytes were graded into three groups according to the number of anomalies: grade I: oocytes without any anomaly, grade II: oocytes with one anomaly and grade III: oocytes with at least two anomalies.

Statistical analyses
Data were compared with the use of {chi}2 and Fisher's exact test. Statistical significance was defined as P < 0.05.


    Results
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
In-vitro maturation outcome and MII oocyte grading according to cause of infertility
The fertilization rate, cleavage rate and pregnancy rate were similar for PCOS patients and regular menstruating women, irrespective of the cause of infertility (male factor or tubal disease) (Table IGo).


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Table I. Fertilization rate and embryo quality according to cause of infertility
 
Morphological grading of all MII oocytes was as follows: 144 oocytes (55%) were morphologically normal, whereas 87 oocytes (33%) showed one anomaly and 33 oocytes (13%) showed more than one anomaly (Table IIGo). Oocyte grading did not differ between women with polycystic ovaries and normal ovaries (Table IIGo).


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Table II. Oocyte grading according to cause of infertility
 
Oocyte morphology and the outcome after ICSI
Oocyte morphology was not related to fertilization rates (Table IIIGo). A significantly reduced cleavage rate was found in grade II compared with grade I oocytes (P = 0.03), whereas no significant difference could be demonstrated when all oocytes with anomalies were compared with oocytes without anomalies. A significantly reduced quality of embryos was observed when embryos developed from oocytes with one or more anomalies were compared with embryos developed from normal oocytes (P = 0.001). Subdivision of morhological abnormal oocytes according to the appearance of cytoplasmic abnormalities is illustrated in Table IVGo. The cleavage rate and the number of good quality embryos was significantly higher in the group with extracytoplasmic anomalies compared with cytoplasmic anomalies (P < 0.05 and P < 0.001 respectively).


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Table III. Fertilization rate and embryo quality according to morphological grading of MII oocytes
 

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Table IV. Fertilization, cleavage and embryo quality according to cytoplasmic and extracytoplasmic abnormalities
 
We cannot draw conclusions with regard to implanted embryos because the transferred embryos originated from MII oocytes of different grading in the majority of the patients.


    Discussion
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
The gross morphology of MII oocytes includes variations such as shape of the oocyte, colour, granularity and homogeneity of the cytoplasm, size of the perivitelline space, debris in the perivitelline space, vacuolization, inclusions and abnormalities of the first polar body or of the zona pellucida. It is generally accepted that good quality MII oocytes should have a clear, moderately granular cytoplasm, a small perivitelline space, an ovoid or round first polar body with a smooth surface and a clear colourless zona pellucida ( Alikani et al.1995Go; de Sutter et al.1996Go; Serhal et al.1997Go; Xia, 1997Go; Balaban et al.1998Go; Ebner et al.2000Go). Many oocytes, however, exhibit a number of variations and the so-called ideal oocyte has been found to represent about one-third to one-half of all oocytes retrieved in an ICSI programme ( de Sutter et al.1996Go; Xia, 1997Go; Balaban et al.1998Go; Ebner et al.2000Go). In the present study, one half of the in-vitro matured oocytes appeared to be without anomalies.

Recently, the influence of a cytoplasmic factor, which might negatively influence the outcome in PCOS patients, has been suggested ( Ludwig et al.1999Go). The present study, however, did not demonstrate any difference in morphology between in-vitro matured oocytes obtained from normal ovaries and in-vitro matured oocytes obtained from polycystic ovaries.

When in-vivo matured oocytes obtained after hyperstimulation are evaluated, the extent to which their morphology, at the light microscopy level, correlates with the subsequent fertilization rate and embryo development after ICSI is controversial ( Alikani et al.1995Go; de Sutter et al.1996Go; Serhal et al.1997Go; Xia, 1997Go; Balaban et al.1998Go; Loutradis et al.1999Go; Ebner et al.2000Go; Kahraman et al.2000Go).

In the present study, a significantly higher number of good quality embryos developed after oocytes without anomalies compared to embryos developed after oocytes with anomalies. Oocyte maturation in mammals includes nuclear maturation and cytoplasmic maturation. Studies in mouse have suggested that oocytes can acquire competence to undergo cytoplasmic maturation independently of competence to complete nuclear maturation (Eppig, 1996Go). The impaired embryo development of human oocytes obtained after in-vitro maturation may be due to impaired cytoplasmic maturation and/or asynchrony of nuclear and cytoplasmic maturation. The observation that the highest proportion of best-grade embryos in the present study was derived from oocytes without anomalies, suggests that these oocytes were the most synchronous with respect to nuclear and cytoplasmic maturation. The results of the present study, regarding the correlation between morphology and embryo quality, are in accordance with previous studies (Xia, 1997Go; Loutradis et al.1999Go), in which 903 and 1333 respectively in-vivo matured oocytes were evaluated before ICSI, but in contrast to a previous study (de Sutter et al., 1995) including 528 oocytes before ICSI. The different results may be related to different criteria used to evaluate oocyte morphology. While in a prior study (Xia, 1997Go) and the present study described the status of the first polar body, size of the perivitelline space and cytoplasm, de Sutter et al. evaluated the perivitelline space and the cytoplasm but not the first polar body ( de Sutter et al.1996Go), and Loutradis et al. described the cytoplasm ( Loutradis et al.1999Go).

Recently, extracytoplasmic anomalies such as first polar body morphology at the time of ICSI have been found to be a suitable indicator of subsequent fertilization and embryo quality of in-vivo matured MII oocytes ( Ebner et al.2000Go). In the present study we found an association between first polar body morphology and embryo quality, although the difference between normal oocytes and oocytes with extracytoplasmic anomalies was not significant. Degeneration of the first polar body may be due to asynchrony of nuclear and cytoplasmic maturation ( Eichenlaub-Ritter et al.1995Go). It is possible that oocytes with fragmented polar bodies and enlarged space have been over-mature at the time of fertilization.

In the present study, cytoplasmic abnormalities appeared to parallel decreased cleavage rate and poor embryo quality. This is in accordance with a previous study (Xia, 1997Go). However, other studies have reported, that ICSI results are not influenced by cytoplasmic anomalies ( De Sutter et al.1996Go; Balaban et al.1998Go; Ebner et al.2000Go). Recently ( Kahraman et al.2000Go) poor ongoing pregnancy rates were achieved in couples with centrally located granular cytoplasm even if fertilization, embryo quality and total pregnancy rates are normal. These conflicting results may be due to the selection of patients in the various studies. More oocytes with cytoplasmic anomalies are obtained from women older than 35 years and couples diagnosed with female factor infertility compared to couples with male factor and women <35 years (Xia, 1997Go). The origin of most of the cytoplasmic anomalies is unknown. Oocytes with abnormal cytoplasmic morphology have been found to have a high frequency of aneuploidy (Van Blerkom and Henry, 1992Go; Kahraman et al.2000Go). However, it is not known which morphological anomalies are associated with a higher incidence of chromosomal aberrations. Furthermore, there is no study comparing genetics of oocytes with normal and abnormal morphology.

In conclusion, the highest proportion of best grade embryos was derived from in-vitro matured oocytes without anomalies, suggesting that these oocytes were the most synchronous group with respect to nuclear and cytoplasmic maturation.


    Notes
 
1 To whom correspondence should be addressed. E-mail: alm{at}ciconia.dk Back


    References
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
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Submitted on December 29, 2000; accepted on April 4, 2001.