1 Centre for Reproductive Medicine, Walsgrave Hospital, Coventry CV2 2DX, 2 Sir Quinton Hazell Molecular Medicine Research Centre, Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK, and 3 Laboratory of Reproductive Biology, Rigshospitalet, Copenhagen, Denmark
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Abstract |
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Key words: human/ICSI/in-vitro maturation/meiosis activating sterol/oocyte
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Introduction |
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Immature oocyte retrieval combined with IVM could offer an alternative to the current ovarian stimulation protocols used in IVF, however, efficient maturation, fertilization and embryo development are needed before IVM can be applied routinely in the treatment of human infertility. Recently, several groups have reported improved viability of human IVM oocytes using various protocols including partial, minimal or no exogenous hormonal stimulation (Wynn, et al., 1998; Chian et al., 1999
; Jaroudi et al., 1999
). The content of the medium used for IVM may also affect the outcome. A key experiment in the 1990s, which re-awakened interest in human IVM, used human follicular fluid to supplement the culture medium (Cha et al., 1991
) but, with the growing preference for defined media, the roles of individual factors are now being assessed and the inclusion of biological fluids is declining.
Various hormones included in the culture medium have been reported to promote oocyte maturation and subsequent embryo development, e.g. epidermal growth factor (EGF; Gómez et al., 1993a, b; Goud et al., 1998) or FSH (Barnes et al., 1996; Durinzi et al., 1997
) with or without human chorionic gonadotrophin (HCG; Jaroudi et al., 1997; Liu et al., 1997). In addition, intracytoplasmic sperm injection (ICSI) of IVM oocytes has increased the likelihood of normal fertilization (Barnes et al., 1996
; Cha and Chian, 1998
).
Meiosis activating sterols (MAS) have been purified from human follicular fluid (FF-MAS: 4,4-dimethyl-5-cholesta-8,14,24-trien-3ß-ol) and bull testicular tissue (T-MAS: 4,4-dimethyl-5
-cholesta-8,24-dien-3ß-ol) (Baltsen and Byskov, 1999
). FF-MAS is present in human pre-ovulatory follicular fluid at concentrations of ~1.3 µmol/l, and is an intermediary occurring naturally in the biosynthetic pathway between lanosterol and cholesterol (Byskov et al., 1995
). It activates meiotic resumption of both cumulus-enclosed and denuded mouse oocytes in vitro (Byskov et al., 1995
, 1999
; Hegele-Hartung et al., 1999
). MAS is synthesized by cumulus cells of intact oocytecumuluscomplexes in response to FSH stimulation (Byskov et al., 1998
) and the concentrations of MAS in pre-ovulatory follicular fluid samples are correlated with the ability of the associated oocyte to fertilize and cleave (Byskov et al., 1998
). A histological study of 81 human oocytes suggested that the completion of maturation in vitro might be promoted by FF-MAS (Grøndahl et al., 2000
). Our focus has been to assess functional maturation by obtaining data on the fertilization and embryonic competence of human oocytes after FF-MAS exposure during IVM. No data on this subject have been published previously.
The prevalence of polycystic ovaries (PCO) in `normal' adult women is ~20% (Polson et al., 1988; Clayton et al., 1992
). PCO may be associated with infertility and such patients may be hyper-responsive to the gonadotrophin stimulation normally used for IVF. A classical polycystic ovary has several antral follicles up to ~10 mm diameter around the periphery of the ovary. Although these follicles are not actively growing, they contain immature oocytes which may be capable of further development (Trounson et al., 1994
; Chian et al., 1999
). Women with PCO therefore represent a patient group most likely to benefit from IVM.
There may be additional potential benefits from IVM, for example, allowing rapid oocyte collection in women with cancer who cannot spend time undergoing a normal IVF cycle before sterilizing chemotherapy. The potential of an embryo largely reflects the competence of the oocyte and it might become possible, in future, to select immature oocytes with the greatest potential before fertilization, avoiding some of the ethical dilemmas of embryo selection.
These factors, together with the developing knowledge on mechanisms controlling follicle and oocyte growth and early embryology suggest that the time is right for IVM to be applied with caution in patient treatment. We have performed this preclinical research study to assess the effects of meiosis activating sterol (FF-MAS), a naturally occurring stimulator of meiosis, upon the maturation and preimplantation development of immature human oocytes from two different sources: (i) unstimulated patients with PCO and (ii) patients undergoing a fully stimulated cycle of ICSI treatment.
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Materials and methods |
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Inclusion criteria were: (i) PCO, diagnosed primarily by the ultrasound appearance of the ovaries, but additionally by a history of oligomenorrhoea or amenorrhoea, and blood hormone measurements; (ii) no drug therapy for infertility in the 4 months before surgery; and (iii) requiring diagnostic laparoscopy and/or laser drilling of the ovaries. Patients wishing to participate provided written consent. Laparoscopy was scheduled irrespective of the stage of the menstrual cycle.
Retrieval of immature oocytes
Oocyte retrieval procedures were carried out in the afternoon in theatres on the same site but separate from the main embryology laboratory. A previously described technique (Trounson et al., 1994) was followed using a purpose-designed 17 gauge needle (Cook IVF, Letchworth, UK) and a laparoscopic approach. All visible follicles were punctured and aspirated using 80100 mmHg suction. Measurement of follicular diameter was not possible by direct laparoscopic inspection since the follicles were relatively small (<10 mm) and the stroma was dense. The fluid was collected into 10 ml sterile plastic tubes (Fahrenheit Laboratory Supplies, Milton Keynes, UK) containing 2 ml pre-warmed, heparinized (3 IU/ml; Leo Laboratories Ltd, Buckinghamshire, UK) Ham's F10 medium (ICN Pharmaceuticals Ltd, Thame, UK) buffered with 20 mmol/l HEPES and supplemented with 0.5% human serum albumin (HSA; Immuno Ltd, Dunton Green, UK). The aspirates were maintained at 37°C in a thermostatic hot-block (Grant, Cambridge, UK) and transported to the embryology laboratory in a portable incubator (Cell Trans 4016 Transport Incubator; Labotech). The oocyte aspiration procedure took ~40 min and the journey <10 min.
Follicular aspirates were placed into an Em-Con filter of 75 µm pore size (Immuno Systems, Spring Valley, WI, USA) which had been pre-rinsed with warmed heparinized Ham's F10 medium as above. The aspirates were flushed with 250 ml of the same medium to remove contaminating blood cells and the filter retentate was transferred to sterile dishes (Falcon, Fahrenheit).
Oocytecumulus masses and free oocytes were identified using a dissecting microscope (Leica Microsystems, Milton Keynes, UK), equipped with a heated stage, and transferred to tissue culture dishes (Nunclon; Gibco, Life Technologies, Paisley, UK) containing 2 ml IVM medium. IVM medium comprised Medium 199 (Gibco) supplemented with 0.23 mmol/l sodium pyruvate (Sigma, Poole, UK), 50 IU/ml penicillin G and 50 µg/ml streptomycin (Gibco). The IVM medium was prepared on the morning of oocyte retrieval and dishes of medium were pre-equilibrated in a humidified incubator at 37°C, containing 5% CO2 in air. The preparation of MAS-containing IVM medium is explained below.
The level of cumulus cover surrounding the oocyte was graded subjectively from 03, where 0 = devoid of cumulus; 1 = partially covered zona; 2 = completely covered zona; and 3 = substantial multi-layered cumulus cover. Individual viable oocytes were randomly allocated to one of three concentrations of MAS using tables of random numbers.
An oocyte was considered to be viable if it had an intact oolemma, a light coloured cytoplasm and a regular-appearing spherical shape. In previous experiments using the fluorescent vital stains, carboxyfluorescein diacetate (CFDA) and propidium iodide (PI), these features had been confirmed as identifying viable oocytes. Where possible, it was recorded whether a germinal vesicle (GV) was present in the ooplasm, and any other features of oocyte morphology. Oocytes obscured by tightly surrounding cumulus cells (cumulus enclosed; CE) were presumed to contain a GV. The initial assessment of maturity was performed ~2 h after oocyte retrieval.
Images of oocytes were collected daily via a video link on an inverted high power microscope (Olympus IX 70) employing Hoffman optics and Image Pro-plus software. Oocytes which were clearly atretic on collection, e.g. dark cytoplasm and irregular shape, were noted before discarding.
ICSI patients
Immature oocytes were donated by women undergoing ICSI at the Centre for Reproductive Medicine, Walsgrave Hospital. Women wishing to participate gave written consent prior to the oocyte retrieval. Patient preparation, oocyte retrieval and embryological procedures were performed using the centre's standard protocols (Garello et al., 1999). Any immature oocytes identified when oocytes were stripped of cumulus in preparation for ICSI were randomly allocated to MAS treatment groups using random number tables; thereafter, they were treated identically to oocytes from PCO patients. Oocytes were considered immature if they contained a GV or had undergone germinal vesicle breakdown (GVBD) but had not released a polar body.
Meiosis activating sterol (FF-MAS)
FF-MAS was purified from human follicular fluid as described (Byskov et al., 1995; Baltsen and Byskov, 1999
). MAS was stored at 20°C in n-heptane under N2 in glass vials. Before use, a stream of nitrogen gas was used to evaporate the heptane from a known quantity of MAS which was dissolved in a known small volume of absolute ethanol (EtOH) and added to the prepared IVM media in glass tubes to final concentrations of 10 and 30 µg/ml (24.4 and 73.2 µM). EtOH alone, treated in the same manner, was added to controls. All wells contained a final concentration of 0.5% EtOH. MAS/EtOH was added to the cultures ~1 h before oocyte retrieval.
Volumes of 100 µl IVM medium containing the various concentrations of MAS were set up in a sterile 96-well plate (Nunclon) which also contained medium in the surrounding wells to increase humidity and reduce evaporation. Cultures were housed in a humidified incubator (37°C, 5% CO2 in air). An oil overlay was not used, thus avoiding oil phase extraction of MAS. Control experiments demonstrated that the osmolarity of cultures maintained under these conditions varied by <1% after 24 h.
Up to four oocytes were co-incubated in each well. The oocytes were examined after 2324 h and their maturational stage recorded. Oocytes which had extruded a polar body were considered mature and removed for ICSI. 4048 h after initiation of culture, any remaining cumulus cells were removed by pipetting and all oocytes were assessed microscopically. Mature oocytes were injected with known fertile donor spermatozoa according to the Centre's established ICSI protocol (Garello et al., 1999). Frozen stored spermatozoa from two fertile donors were used for the entire study.
After injection, oocytes were transferred individually to S1 or G1 medium (Scandinavian IVF Science, Sweden) in 100 µl drops under oil (Ovoil; Scandinavian IVF Science). Dishes were pre-equilibrated at 37°C in an atmosphere of 5% CO2 in air for 1 day before use. The oocytes were checked for fertilization 1418 h post-injection using standard procedures of pronuclear assessment. Zygotes and cleaving embryos were observed and moved to fresh drops daily. Computerized photographic images were collected each day until the embryo arrested or degenerated.
Statistical analyses
The proportions of oocytes in groups exposed to different concentrations of MAS were compared using p x q 2 test (Campbell, 1989
). Scatter plots were assessed for correlation using regression analysis. P < 0.05 was considered to be statistically significant. Student's t-test was used to compare the numbers of viable oocytes in the two patient groups.
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Results |
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Figure 1 shows the results of MAS exposure of oocytes obtained from PCO patients. MAS at a concentration of 30 µg/ml significantly increased oocyte survival (P < 0.01, p x q
2 test, Campbell, 1989); 90% survival in 30 µg/ml compared with 62 and 63% for 10 µg/ml and control respectively. The increased survival of oocytes in 30 µg/ml MAS was not associated with an alteration in the proportion of surviving oocytes which matured, which remained at ~45%.
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Discussion |
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MAS significantly (P < 0.01) improved the survival of immature oocytes collected from unstimulated women with PCO. This is in contrast with the results obtained culturing immature oocytes from stimulated ICSI patients in whom FF-MAS did not affect oocyte survival (which was >90% in this patient group), but maturation in vitro was significantly increased (P < 0.05). The variations in embryo development in both patient groups with MAS supplementation of IVM media are also interesting, particularly in view of the compromised developmental competence which is widely believed to be associated with immature oocytes collected from PCO patients, although statistical analysis was precluded by the limited numbers of oocytes reaching the later stages in our study.
There are some difficulties of working with steroids in vitro. We avoided the use of oil, which would extract the steroidal MAS, but we cannot exclude the possibility of it adhering to the plastic culture vessel or being affected by albumin or other constituents in the culture medium. Similarly, the method of dissolving steroids in EtOH is widely used, but recent evidence has shown that even concentrations <1% EtOH can adversely affect bovine IVM and subsequent embryo development (Avery and Greve, 2000). Bovine oocytes contain more lipidic yolk-like granules than human oocytes; however, it is possible that some of the limited development observed in our study may relate to our use of EtOH in the cultures. This would not, however, affect the validity of the results observed, since EtOH was included at a similar concentration in all cultures.
MAS is produced in cumulus cells (Leonardsen et al., 2000). Its effects are likely to be mediated via mechanisms employed by other follicular steroids, perhaps in a manner similar to progesterone stimulation of meiosis in Xenopus oocytes (Byskov et al., 1998
). MAS receptors have not yet been identified; however, MAS is known to have direct actions upon mouse oocytes and does not depend upon mediation by cumulus cells (Byskov et al., 1995
), unlike, e.g. gonadotrophins. The steroid environment of follicles in PCO is disturbed (e.g. Almahbobi and Trounson, 1996; Pierro et al., 1997), and so the influence of MAS in this environment might differ from other circumstances.
Immature oocytes retrieved from unstimulated PCO patients may or may not have cumulus cells covering them. The presence or absence of cumulus cells provides some indication of the follicle from which the oocyte originated, denuded oocytes being more likely to have arisen from atretic follicles. The known roles of cumulus cells include supporting oocyte growth and maintaining meiotic arrest throughout all immature stages of oocyte development. They are steroidogenically active and also produce hyaluronic acid during cumulus expansion to facilitate the approach of spermatozoa to the zona pellucida at fertilization. Oocytes surrounded by cumulus cells may therefore be at an advantage during IVM culture. Most enclosed oocytes had compact corona cells and contained a GV, and many of those without had progressed to GVBD, probably as a result of incipient atresia in the follicle (Gougeon and Testart, 1986; Anderson et al., 1997
). Surprisingly, similar proportions of oocytes from PCO and ICSI patents had a GV or had undergone GVBD; however, these oocytes differed substantially in their predispositions towards atresia and their ability to mature to MII. The removal of cumulus cells from immature oocytes collected from ICSI cycles might have affected some aspects of their development.
The mean number of viable oocytes collected from PCO patients was 7.0, which was lower than the means of 13.8 and 13.1 obtained from anovulatory and ovulatory PCO patients respectively (Trounson et al., 1994), but comparable with the average of 8.1 per patient reported in a later study (Coskun et al., 1998
). We did not find any specific characteristics of the PCO patients which were predictive of the numbers of oocytes retrieved; specifically, patient age, time since last menstrual period, and weight showed no significant correlations. The number of oocytes collected would be expected to reduce as the ovarian reserve declines with advancing age (Cha et al., 1991
; Cha and Chian, 1998
; Whitacre et al., 1998
); however, this was not our experience in this study. One group (Barnes et al., 1996
) has previously reported the retrieval of a larger number of oocytes from irregularly cycling and anovulatory patients compared to regularly cycling women (16.5 and 4.9 respectively) which relates to the relative ease of aspiration from the peripherally located antral follicles in PCO. Extremes of weight are known to affect fertility; in obese women with PCOS, fertility may be enhanced by a loss of weight. In this study, which included a range of obese and non-obese women, we observed no association between weight and oocyte yield.
Several studies have shown that oocytes aspirated after ovarian stimulation may be in various stages of maturation (Veeck et al., 1983; De Vos et al., 1999
). Oocytes that remain immature have failed to respond to ovarian stimulation in vivo and may be of inherently reduced quality, they may have come from small follicles with reduced gonadotrophin sensitivity or may never have reached meiotic competence. Alternatively, they may have arisen from otherwise normal follicles which did not receive the same hormonal stimulus as others, perhaps due to their relative position in the ovary or limited blood supply. Moreover, the artificial conditions of gonadotrophin stimulation may have affected some aspects of their metabolism or development (Johnson et al., 1991
). Nevertheless, it has been reported that these oocytes are capable of maturing in vitro, fertilizing and developing normally (Cha and Chian, 1998
). Our results demonstrate their responsiveness to stimulation with MAS, despite the absence of cumulus cells and their prior exposure to ovarian stimulation.
The importance of the endocrine environment in ensuring normal cytoplasmic maturation and subsequent fertilization is well known from work in animals (Moor and Trounson, 1977; Anderiesz and Trounson, 1995
). The differing results obtained in this study in patients with differing endocrine profiles underline the potential for IVM success rates to be affected by the oocytes' prior exposure to physiological or pathological hormonal environments. In vitro, 2830 h is required for the maturation of human oocytes to MI and 3637 h to MII (Edwards, 1965a
, b
). After HCG injection to simulate a LH surge, the results are similar, the majority of the oocytes having extruded the first polar body by 36 h after HCG injection (Janssenswillen et al., 1995
). One group (Jamieson et al., 1991
) reported significantly lower IVF and cleavage rates in oocytes retrieved <36 h after the luteinizing stimulus, demonstrating the importance of appropriate timing of insemination. Previous studies have shown that immature oocytes obtained from stimulated cycles are more likely to undergo maturation than unstimulated oocytes and that the time required for their maturation is reduced (Gomez et al., 1993a
; Cha and Chian, 1998
). Our results concur with these prior observations.
IVM can result in pregnancy (Cha et al., 1991; Trounson et al., 1994
; Barnes et al., 1995
; Russell, 1998
; Jaroudi et al., 1999
) but rates remain lower than those of in-vivo stimulated cycles, indicating that optimization of IVM remains a challenge (Goud et al., 1998
). The primary problem in oocytes matured in vitro is reduced developmental competence, particularly cleavage and development beyond the 4-cell stage (Trounson et al., 1994
). This was apparent in our study: the most advanced embryo observed reached 6 cells with most embryos arresting around the second cleavage division (34 cells). However, it appears that if the initiation of maturation is triggered in vivo, then developmental potential increases (Chian et al., 1999
) although the value of priming with low-dose FSH remains unclear (Wynn et al., 1998
; Mikkelsen et al., 1999
). Others have shown the relative effects of follicular versus luteal phase retrieval of oocytes (Cha and Chian, 1998
; Whitacre et al., 1998
), underlining the importance of the endocrine environment. In our study, PCO patients were not given any form of hormonal manipulation prior to oocyte retrieval; however, it is likely that induction of a withdrawal bleed with or without late follicular administration of HCG (Buckett et al., 1999
; Chian et al., 1999
) may improve the maturation rates achieved in the present study. These methods still have the benefit of avoiding the major element of the drugs normally administered for ovarian stimulation.
Evidence is accumulating that MAS is an important endogenous factor involved in promoting oocyte maturation. The data presented here show positive effects of MAS upon survival and maturation of human immature oocytes collected from unstimulated PCO patients and stimulated ICSI patients respectively. The limitations of a morphological assessment of viability are acknowledged, but the choice of criteria was supported by earlier experiments using vital stains. Clearly, the use of an invasive method in this study would have precluded the collection of data on further development. This is an important area since it is not yet known whether embryos resulting from IVM in the presence of MAS have the potential to develop further or implant. In mouse cumulus cells, a heat-stable meiosis activating substance is stimulated by FSH (Byskov et al., 1997; Yding Andersen et al., 1999
) and artificial elevation of MAS in cumulus cells using inhibitors of specific enzymes on the steroid synthesis pathway promotes maturation of mouse oocytes in vitro (Leonardsen et al., 2000
). MAS was shown to improve immature oocyte function in mice by supporting microtubule assembly and delaying the release of cortical granules (Hegele-Hartung et al., 1999
).
Results were recently presented (Grøndahl et al., 1999, 2000
) of IVM of 81 human oocytes allocated into seven groups, with or without MAS, for histological analysis. These data showed a significant (P < 0.05) increase in the proportion of immature oocytes completing maturation after 30 h in vitro in the presence of 20 µmol/l MAS; however, at 22 and 40 h, the difference was not significant. Grøndahl's study differed from ours in several respects. His patient group had polycystic ovaries, but received oral contraception for 2 months, to which was added a gonadotrophin-releasing hormone (GnRH) agonist for pituitary down-regulation, followed by recombinant FSH for 3 days. Follicles of 812 mm were aspirated on days 79 and all oocytes were cumulus-enclosed throughout culture. These patients therefore underwent stimulation cycles. Also, half their aspirated oocytes were used for infertility treatment, although HCG was not administered. The medium used by Grøndahl et al. for the research oocytes was similar to ours, being M199 supplemented with 0.29 mmol/l pyruvate, antibiotics and 0.8% HSA, with or without MAS which was prepared in EtOH. None of their research oocytes was inseminated.
In this study, we have demonstrated oocyte maturation with developmental competence as far as the second cleavage division. The effects of MAS on embryo development could not be analysed statistically in view of the low numbers of embryos generated. The lower maturation rate observed in the oocytes originating from unstimulated PCO was probably due to compromised follicle development as a result of the abnormal endocrine environment, which could also account for the higher rate of atresia observed in the GVBD oocytes, compared with the ICSI patient group. In PCO patients, GVBD oocytes were probably retrieved from partially atretic follicles, where granulosa cells have dissociated from the oocyte and factors controlling meiotic arrest have been lost. Oocytes in atretic follicles may often be found to have progressed further than meiotic prophase I (Anderson et al., 1997).
In our study, the effects of MAS alone have been studied, although addition of FSH to the medium might augment MAS production in the cumulus (Byskov et al., 1997) and the inclusion of FSH, HCG and/or growth factors in vitro has already been demonstrated to stimulate IVM by others (Durinzi et al., 1997
). We chose to omit these other factors so that the effects of MAS alone could be established in the absence of confounding influences. We felt that this was important, even though a higher overall maturation rate might be gained by a combination of supplements in the medium.
In conclusion, oocytes derived from stimulated ICSI patients and unstimulated PCO patients constitute different populations, probably due to their differing endocrine and intrafollicular environments. MAS exerts positive effects upon different aspects of oocyte function in both groups. In view of its role as an intrafollicular steroid, and its ability to promote maturation, its potential for further development as an in-vitro meiotic stimulant should be evaluated.
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Acknowledgments |
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Notes |
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4 Present address: Assisted Reproduction Unit, University Hospital of Wales, Heath Park, Cardiff CF4 4XW, UK
5 To whom correspondence should be addressed at: Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK. E-mail: ghartshorne{at}bio.warwick.ac.uk
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References |
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Submitted on August 31, 2000; accepted on November 20, 2000.