In-vitro maturation of human oocytes from regularly menstruating women may be successful without follicle stimulating hormone priming

Anne Lis Mikkelsen1,3, Steven Dale Smith1 and Svend Lindenberg1,2

1 The Fertility Clinic, Herlev University Hospital, DK-2730 Herlev and 2 Ciconia Fertility Clinic, Frydendalsvej 5, DK-1809 Frederiksberg, Denmark


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This prospective randomized study investigated whether the developmental potential of in-vitro matured (IVM) human oocytes is improved by follicle stimulating hormone (FSH) priming before aspiration. Normally cycling women were recruited among couples referred for in-vitro fertilization or intracytoplasmic sperm injection because of male factor and/or tubal disease. In the first experiment, 20 women were randomly allocated to either no stimulation (n = 10) or stimulation for 3 days with rec-FSH (Gonal-F, Serono) at a fixed dose of 150 IU/day from day 3 (n = 10). The oocytes were aspirated when the follicle(s) was observed to be 10 mm and matured in vitro for 36 h. In experiment 2, 12 women received rec-FSH (150 IU/daily) from day 3. In five patients stimulation was given for 3 days and aspiration performed as in the first experiment; the remaining seven patients continued stimulation until follicles were 10 mm in diameter and aspiration was performed 72 h later. All the oocytes were cultured for 48 h. FSH priming did not increase the number of oocytes obtained per aspiration and did not improve on maturation to metaphase II, cleavage rate or embryo development. The implantation potential of IVM embryos may be improved by maturation for 36 h compared to 48 h. In the first experiment, five out of 20 patients (25%) obtained a clinical pregnancy with an implantation rate of 15% (5/33). One has delivered a healthy boy and three pregnancies are ongoing beyond 32 gestational weeks. In the second experiment, an implantation rate of 7% and a single pregnancy (8%) was obtained, with delivery of a healthy girl.

Key words: FSH priming/follicle size/human/immature human oocytes/in-vitro maturation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The ability of immature oocytes to resume meiosis spontaneously when removed from the follicle was first demonstrated by Pincus and Enzman (Pincus and Enzman, 1935). Human oocytes from small follicles can resume and complete meiosis in vitro, but to a lesser extent than in other mammals (Edwards, 1965Go). Successful maturation of unstimulated oocytes has been reported (Cha et al., 1991Go; Trounson et al., 1994Go; Barnes et al., 1996Go; Russell et al., 1997Go), and high rates of fertilization can be achieved when these oocytes are subjected to intracytoplasmic sperm injection (ICSI) (Russell et al., 1997Go), but their developmental potential has been reported to be low (Barnes et al., 1996Go; Cha and Chian, 1998Go). The in-vitro maturation (IVM) protocol is relatively simple with a shorter period of treatment and reduced costs compared to conventional in-vitro fertilization (IVF). In addition, the side-effects of stimulation, in particular ovarian hyperstimulation syndrome, are eliminated. Despite the poor pregnancy rate seen in human trials until now, the record of success in animals has been encouraging. In monkeys, priming with follicle stimulating hormone (FSH) has been shown to enhance nuclear and cytoplasmic maturation of oocytes in vitro (Schramm and Bavister, 1994Go). Increasing FSH concentrations within the follicle are coincident with the generation of a positive signal necessary to complete in-vivo oocyte maturation in humans (Gomez et al., 1993Go) and a short FSH stimulation has been shown to increase the percentage of oocytes that reach metaphase II after IVM (Wynn et al., 1998Go).

In this prospective randomized pilot study, we investigated whether the developmental potential of IVM immature oocytes may be improved by FSH priming before the aspiration.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Included in the study were couples referred for IVF or ICSI because of a male factor of infertility and/or tubal disease. The women were at least 18 and at most 38 years of age and had normal ovulatory cycles with a mean length of between 24 and 35 days and a body mass index (BMI) between 18 and 29 kg/m2. Excluded were all patients with infertility caused by endocrine abnormalities such as hyperprolactinaemia, polycystic ovary syndrome and previous ovarian stimulation cycles in which fewer than three oocytes were retrieved. Also excluded were all women with an elevated FSH concentration (>15 IU) on cycle day 3. The study was approved by the local ethical committee. All couples participated in the study after written consent.

The first experiment was a randomized prospective study including 20 patients recruited among couples scheduled for ICSI because of male factor. The women were randomly allocated to two groups. Group I (n = 10 cycles) received no stimulation and group II (n = 10 cycles) received rec-FSH (Gonal-F; Serono) 150 IU/day for 3 days initiated day 3. Aspiration was performed on the day after a leading follicle of 10 mm in diameter was observed at ultrasound. All the oocytes were matured in vitro for 36 h before ICSI.

In the second experiment, 12 consecutive couples were included. All the women received rec-FSH (Gonal F, Serono) priming before aspiration and 150 IU was given daily from day 3. Five patients received the same stimulation as group 2 in the first experiment, with a fixed dose of 150 IU/day for 3 days and aspiration the day after a leading follicle of 10 mm was observed. The remaining seven patients continued stimulation until the leading follicle was 10 mm and aspiration was performed 72 h later. All the oocytes were cultured for 48 h until inseminated with ICSI.

In all patients, transvaginal ultrasound was performed on cycle day 3 and baseline measurements of FSH, luteinizing hormone (LH), oestradiol, inhibin A and inhibin B were obtained. Hormone profiles were followed from day 3 until the day of aspiration. In the case of an ovarian cyst the cycle was cancelled. The second ultrasound examination was performed on day 6–7 and the following days ultrasound was performed daily or with an interval of 2–3 days depending on the size of the follicles.

Follicular diameters were measured by the same observer during transvaginal ultrasound scanning using a 7.5 MHz transvaginal transducer (B-K Medical, Gentofte, Denmark). The follicular diameter was calculated as the mean of the longest follicular axis and the axis perpendicular to it in the same scanning plane.

In both experiments, endometrial priming consisted of 17-ß-oestradiol started on the day of oocyte retrieval, and the women received 2 mg orally three times per day. If endometrial thickness was <6 mm at ultrasound on the day of aspiration, the cycle was cancelled. Two days after aspiration, treatment with intravaginal progesterone suppositories was initiated and continued until the pregnancy test. Oestrogen and progesterone were continued if the pregnancy test was positive until 50 days gestation.

Oocyte recovery was performed transvaginally with a 17-G Cook needle with a reduced aspiration pressure as described previously (Trounson et al., 1994Go). The follicular aspirates were transferred in tubes to the laboratory and washed on an embryo filter (Falcon 1060) with a pore size of 70 µm to remove erythrocytes and small cellular debris. The retained cells were then resuspended in equilibrated Ham's F-10 (Life Technologies, Copenhagen, Denmark) containing both bicarbonate and HEPES buffers and supplemented with 2 mg/ml human serum albumin (HSA) (Statens Serum Institute, Copenhagen, Denmark). The oocytes were matured in tissue culture medium (TCM 199; Sigma, Roedovre, Denmark) supplemented with sodium pyruvate 0.3 mmol/l, 1500 IU/ml penicillin G, 50 mg/ml streptomycin sulphate, oestradiol 1 µg (all from Sigma), rec-FSH 0.0751 IU/ml (Gonal-F; Serono, Geneva, Switzerland), human chorionic gonadotrophin (HCG) 0.5 IU/ml (Profasi; Serono) and 10% serum from the patients was obtained on the day of aspiration. Oocytes were cultured separately in 25 µl drops of IVM medium (Medi-Cult, Copenhagen, Denmark) under paraffin oil at 37°C in 5% CO2.

Oocytes were denuded with hyaluronidase (IVF Science, Sweden) and mechanical pipetting and the oocyte was classified as having undergone germinal vesicle breakdown when the nuclear membrane was absent and as a mature metaphase II (MII) oocyte when the first polar body was extruded. Motile spermatozoa were prepared by either PurespermTM (Cryos, Denmark) gradient separation or by swim-up. For ICSI, denuded oocytes were placed individually into 5 µl drops of sperm preparation medium (Medi-Cult) and 2 µl of sperm suspension was placed into a 10 µl drop of PVP (IVF Science).

Fertilization with ICSI was performed on all MII oocytes. The oocytes were then placed into 10 µl droplets of IVF medium (Medi-Cult) and cultured under oil in Falcon Petri dishes to day 2 or 3 after fertilization. Embryos were scored on a scale of 1–4, where types 1 and 2 (<10% fragmentation) were considered to be transferable (Deschacht et al., 1988Go). A maximum of two embryos was transferred.

The embryo development rate was defined as the number of transferable embryos out of the total number of oocytes injected. The implantation rate was defined as the number of gestational sacs seen on ultrasound examination out of the total number of embryos replaced.

Statistically analysis was done by the Student's t-test. Because none of the hormone variables displayed a normal distribution the non-parametric Mann–Whitney U-test was used to analyse statistically significant differences between unpaired data and Pratt's test (Pratt, 1974Go) for paired data. Values were considered significant when P < 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
In experiment 1, the median age of the women was 31 years (range 28–36 years) in the group without FSH priming and 32 years (range 28–36 years) in the group that received FSH. This did not differ from the age of the women in experiment 2 (median 32 years, range 27–37 years). Other clinical characteristics including duration of infertility, cause of infertility and number of previous IVF cycles were similar in all the groups.

First experiment
Maturation and embryo development
In the first experiment including 20 women, 77 oocytes could be used for IVM, and 62 (81%) matured to MII after 36 h. After fertilization with ICSI, 2PN fertilization was seen in 49 (79%) and of these 45 (72%) cleaved. The embryo development rate was 40/62 (65%) and implantation rate 5/33 (15%). The clinical pregnancy rate per started cycle was 5/20 (25%). FSH priming did not have any effect on oocyte maturation, fertilization rate, cleavage rate or embryo development (Table IGo). In total, five pregnancies were obtained. One has delivered a healthy boy, one miscarried in gestational week 8 and the remaining are ongoing beyond 32 weeks of gestation.


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Table I. The numbers of oocytes obtained for in-vitro maturation (IVM), maturation, fertilization, cleavage and pregnancy rates in experiment 1
 
Hormone profiles
There were no differences between the two groups in the concentrations of oestradiol, FSH, LH, inhibin A and inhibin B on day 3 and the day of aspiration respectively (Table IIGo).


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Table II. The hormone concentrations in serum on day 3, day 6–7 and the day of aspiration in the two groups in experiment 1
 
In the non-stimulated group, the FSH levels during the follicular phase did not differ from the values in the stimulated group, although a large inter-individual variation was observed.

The level of serum oestradiol remained constant from day 3 to day 6–7 in the group without stimulation. On the day of aspiration a significant increase was seen. In the stimulated group the concentration of oestradiol increased earlier (day 6–7) with a plateau phase before aspiration. No premature LH peak was observed in the two groups.

The levels of inhibin A showed the same pattern as oestradiol, with an early increase in the stimulated group and a late increase in the non-stimulated group. In the unstimulated group the level of inhibin-B increased earlier than inhibin A (day 6–7). At this time an increase was also observed in the stimulated group but the level was 3-fold that in the non-stimulated group and a significant decrease in inhibin-B was observed from day 6–7 until aspiration.

Second experiment
In the second experiment, including 12 women, 38 oocytes could be used for IVM, and after 48 h 27 (71%) had matured and were inseminated by ICSI. The fertilization rate and cleavage rates were 61 and 53% respectively, and this did not differ from those obtained in experiment 1. Fifteen embryos were transferred in 10 patients; none was cryopreserved. One pregnancy was obtained with the delivery of a healthy girl. The embryo development rate in this group was 56% and the implantation rate 7%.

By prolongation of the stimulation period from a fixed dose of 150 IU/day for 3 days up to 6 days until follicles were 10 mm, we obtained an increased size of the follicles on the day of aspiration and an increased level of oestradiol in serum, but the number of oocytes obtained per aspiration did not increase (Table IIIGo).


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Table III. The number of oocytes obtained for in-vitro maturation (IVM), the maturation and cleavage rates in low dose versus high dose gonadotrophin stimulation in experiment 2
 
No hospitalization due to infection, bleeding or abdominal pain referred to as gynaecological was observed in this study. No extrauterine pregnancy was observed.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
To our knowledge, this is the first study reporting a pregnancy rate of 25% (5/20) per aspiration with an implantation rate of 15% after IVM of oocytes. The immature oocytes in this study appeared to perform almost as well as those recovered after superovulation and maturation in vivo (Meldrum et al., 1998Go).

The number of oocytes recovered from non-stimulated regularly cycling women in our study did not differ from the yield of oocytes recovered and matured in a previous study (Barnes et al., 1996Go). Pretreatment of patients with recombinant FSH did not increase the number of oocytes recovered. Previous studies have given conflicting results. An increased number of oocytes was recovered in regular menstruating women with FSH priming compared to women without FSH priming (Wynn et al., 1998Go), while a second study (Trounson et al., 1998Go) did not demonstrate any difference in the number of oocytes obtained. Previous observations have suggested that the number of recruitable follicles is determined as early as the preceding luteal phase (Gougeon and Testart, 1990Go) and brief, early FSH stimulation does not alter the number of recruitable immature oocytes. This is in accordance with the results in the present study. With an increasing amount of FSH and increasing size of the follicles in experiment 2, we observed that the chance of obtaining oocytes at aspiration did not increase. This is in agreement with previous publications (Templeton et al., 1986Go), where the chance of obtaining oocytes from these large follicles in the absence of HCG was very low.

The maturation, fertilization and cleavage rates observed in the present study are consistent with the findings in previous studies, but we observed a higher pregnancy rate and a higher implantation rate indicating an improved embryo development of the oocytes. A number of factors may account for this. The embryo development of IVM oocytes may be improved by prior FSH priming. In rhesus monkeys a short FSH priming improved the maturation and cleavage rate (Schramm and Bavister, 1994Go). In humans, pregnancies have been obtained following the transfer of immature oocytes from stimulated cycles (Nagy et al., 1996Go; Edirisinghe et al., 1997; Jaroudi et al., 1997Go; Liu et al., 1997Go).

We know from previous studies in humans that 80% of immature oocytes show nuclear maturation (extrusion of a polar body) and will be at MII by 48–54 h of culture (Trounson et al., 1994Go; Russell et al., 1997Go). However, a considerable asynchrony of maturation has been observed and at least 20% of the oocytes will be at MII after 24 h. Furthermore, those oocytes first reaching MII were shown to be the most competent to develop into blastocysts (Barnes et al., 1996Go). Therefore, we wanted to compare the developmental competence of oocytes matured for 36 h to that of oocytes matured for 48 h.

Recently, an increased maturation rate of oocytes after pretreatment with FSH has been reported (Wynn et al., 1998Go). In our first experiment, however, FSH priming of the women before oocyte retrieval did not improve the maturation rate in vitro. There are two possible explanations for the difference observed: (i) the culture conditions used or (ii) the timing of aspiration. The culture conditions we used have been described above, but briefly Wynn et al. used a defined serum-free medium (Wynn et al., 1998Go) while we supplemented our culture medium with serum from the patient. The timing of aspiration differed, as Wynn et al. performed the aspiration on a fixed day (day 7 in the menstrual cycle) (Wynn et al., 1998Go), while the day of aspiration in the present study depended on the size of the follicles. In the stimulated group, the day of aspiration was performed earlier (mean day 9) compared to the non-stimulated group (mean day 10) (Table IIGo), but the day of aspiration was fixed in the same way in the two groups. The size of the follicles was monitored by ultrasound and oocyte collection was performed the day after a leading follicle of 10 mm was demonstrated.

Wynn et al. did not perform fertilization of the oocytes (Wynn et al., 1998Go). In the present study, FSH priming did not have any effect on fertilization or cleavage rates. This is consistent with previous data (Trounson et al., 1998Go). They have reported that treatment of natural cycling women with recombinant FSH for 3 days had no effect on the maturation rate and fertilization rate in vitro. In the present group of regularly menstruating women, their endogenous FSH stimulation seemed to be sufficient to obtain oocytes that are competent to mature in vitro. We know from previous studies that a wide interindividual difference in follicular phase FSH on day 3 and marked interindividual variation of maximum FSH concentration has been demonstrated (Schipper et al., 1998Go). This interindividual variation in the levels of FSH may reflect differences in FSH threshold and differences in ovarian sensitivity to FSH and this may be one of the possible reasons for the lack of difference in maturation rate and fertilization rates, although the level of FSH differed.

In the non-stimulated group, we observed an increase in serum concentrations of oestradiol and inhibin A on the day of aspiration and this is known to be concomitant with the selection of the dominant follicle (Schipper et al., 1998Go). The concentration of inhibin-B increased earlier than inhibin A, and the level of both hormones was maintained until the day of aspiration. This is in accordance with previous studies (Groome et al., 1996Go; Schipper et al., 1998Go) and in accordance with our ultrasound imaging as the day of aspiration was just after the dominant follicle at 10 mm had been identified. This implies that the oocytes selected for IVM in these cases were obtained from follicles destined to go into atresia, and we observed that the embryo development of oocytes was not affected adversely by early stages of atresia. This has previously been demonstrated in cattle (Smith et al., 1996Go).

Production of both inhibin-A and inhibin-B is dependent on gonadotrophin (Lockwood et al., 1996Go) and in the gonadotrophin-stimulated group in the present study a 3-fold rise in the levels of both inhibin-A and inhibin-B was observed during stimulation with FSH. From day 6–7 until aspiration, the levels of both inhibin A and inhibin B significantly decreased and this observation is in accordance with non-growing follicles (Price et al., 1995Go).

The timing of aspiration and selection of oocytes for IVM is based on experience with other mammalian species (Edwards 1965Go; Eppig et al., 1992Go) but also human oocytes appear to have a size-dependent ability to resume meiosis and complete maturation (Durinzi et al., 1995Go). A decreased maturation rate of oocytes from small follicles (3–4 mm) compared to oocytes from larger follicles (9–15 mm) was found (Tsuji et al., 1985Go). A decline in fertilization rates of oocytes from hyperstimulated follicles of decreasing size has been observed (Dubey et al., 1995Go). While 58% of the oocytes from follicles 10–14 mm fertilized, 74% of oocytes from follicles 22–26 mm did so. In the present study, we observed that oocytes from large follicles in the second experiment with high dose gonadotrophin stimulation were competent to mature and cleave (Table IIIGo), but also oocytes from follicles at the size of 8–10 mm were developmentally competent when aspiration was performed after the leading follicle was demonstrated by ultrasound. A dramatic decrease in the rates of maturation and fertilization has been shown when immature oocytes were aspirated when the size of the dominant follicle exceeded 14 mm (Russell, 1998Go). This indicates that there may be a critical point where the selection process may have a negative effect on the existing follicles. Monitoring the diameter of the follicles may prove to be useful as a practical means of selecting oocytes competent for IVM.

Factors other than the size of the follicle appear to limit the efficiency of the IVM system. Priming of the endometrium is another important factor. In an immature oocyte cycle adequate endogenous oestradiol from the dominant follicle is lacking. Therefore exogenous priming is needed and one must synchronize the window of implantation with the embryo development. An increased maturation rate and fertilization rate when mid-follicular priming with oestradiol was initiated compared to early endometrial priming has been found (Russell et al., 1997Go). We know from hormone replacement in recipients of donor oocytes that 2-day-old embryos are best transferred to the endometrial cavity on day 3 or 4 of progesterone exposure (Rosenwaks et al., 1987Go). We aimed to imitate the normal priming as closely as possible and initiated oestradiol on the day of aspiration and supplemented with progesterone 2 days later. One might expect that in cycles with FSH priming the elevated concentration of oestradiol from day 6–7 might prepare the endometrium better than in non-stimulated cycles, where oestradiol increased just prior to the aspiration. We were unable to demonstrate any difference in the implantation rate between the two groups. This is in accordance with previous studies as no correlation between follicular phase serum oestradiol concentration and implantation rate has been found (Younis et al., 1996; Remohí et al., 1997Go).

These results need to be confirmed in a larger number of patients. They could lead to an attractive alternative to ovarian stimulation for IVF. Besides the clinical benefits of lowering side effects, especially elimination of hyperstimulation syndrome, this treatment may reduce the costs of IVF. Recently the extensive use of drugs for ovarian stimulation has been questioned (Edwards et al., 1997Go). Many attempts have been made to perform IVF in the natural cycle without the use of exogenous gonadotrophins. The combination of immature oocyte retrieval and IVM may enhance the success of natural cycle IVF. In IVF/ICSI because of male factor, most of the women are producing their own FSH to assist the stimulation of ovarian follicles, and this may be utilized in IVM. No benefit of low dose FSH priming compared to the natural cycle on embryo development could be demonstrated in this study. However, more studies are needed to elucidate this topic.


    Acknowledgments
 
The study was supported in part by Medi-Cult AS.


    Notes
 
3 To whom correspondence should be addressed Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on November 5, 1998; accepted on March 22, 1999.