Luteal phase start of low-dose FSH priming of follicles results in an efficient recovery, maturation and fertilization of immature human oocytes

Anne-Maria Suikkari1,4, Maija Tulppala1, Timo Tuuri1, Outi Hovatta2 and Frank Barnes3

1 The Family Federation of Finland, Infertility Clinic, Helsinki, Finland, 2 Huddinge Sjukhus, Karolinska Institute, Stockholm, Sweden and 3 Pacific Fertility Medical Center, Westwood Medical Plaza, Los Angeles, California, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this prospective study we investigated whether the maturation and fertilization of immature oocytes can be improved by administration of recombinant follicle stimulating hormone (rFSH) starting in the late luteal phase in two groups of women: group 1 (n = 6) women with regular menstrual cycles; and group 2 (n = 6) women with irregular cycles and polycystic ovaries (PCO) on ultrasound examination. Low-dose (37.5 IU) rFSH was commenced 11 days after LH surge during a spontaneous menstrual cycle and on the ninth day of progesterone administration in an irregular cycle. Recombinant FSH was continued until the leading follicle was approximately 10 mm in diameter. The oocytes were retrieved after withdrawing rFSH for 2–5 days. In total, 136 oocytes were recovered (group 1, 67 oocytes; group 2, 69 oocytes). Nine of the oocytes from PCO women were atretic at retrieval. Oocytes complete with cumulus cells were cultured for 44 h in complex tissue culture medium supplemented with gonadotrophins and fetal calf serum. After maturation, the cumulus cells were removed and metaphase II oocytes were injected with spermatozoa. Respectively, the oocyte maturation and fertilization rates were 64 and 72% in group 1, and 78 and 57% in group 2 (not significant). After fertilization, the zygotes (group 1, n = 22; group 2, n = 11) and cleavage stage embryos (group 1, n = 9; group 2, n = 15) were frozen in propanediol. All women except one (11/12) had approximately five zygotes or cleaved embryos frozen. The viability of in-vitro matured frozen–thawed embryos was generally poorer than that (81%) seen after conventional intracytoplasmic sperm injection, with 61% survival in group 1 and 23% in group 2. Fifteen embryo transfers resulted in one miscarriage at 6 weeks gestation. The late luteal start of low-dose rFSH yielded a good number of immature oocytes in women with both regular and irregular cycles. Two out of three of these oocytes matured and fertilized. However, cryosurvival of the zygotes and cleaved embryos was unsatisfactory and thus cryopreservation of in-vitro matured embryos may not be an optimal procedure.

Key words: FSH/immature oocytes/in-vitro maturation/minimal stimulation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
There are several incentives to develop milder forms of ovarian stimulation. The idea to produce competent oocytes for in-vitro maturation and fertilization using minimal stimulation of the recruitable follicles would eliminate several problems of the high order ovarian stimulation used for conventional in-vitro fertilization (IVF) (Edwards et al., 1996Go). There would be no need for large amounts of expensive fertility drugs which often cause considerable and occasionally severe side effects such as ovarian hyperstimulation syndrome in otherwise healthy women.

A fully grown oocyte is competent to progress to maturity. Follicle size can be used as an indicator for the meiotic competence of an oocyte (Moor and Trounson, 1977Go). In animal studies, the developmental competence of the oocytes affects the ability of an early cleaved embryo to develop to the blastocyst stage (Carolan et al., 1996Go). Once a follicle has entered the growing phase, its survival depends mostly on follicle stimulating hormone (FSH). In a natural cycle, serum FSH concentration begins to increase in the late luteal phase (Christin-Maitre et al., 1996Go). This is thought to be essential for the recruitment of follicles for the following menstrual cycle, possibly by inhibiting apoptosis in the granulosa cells (diZerega and Hodgen, 1981Go).

Based on results of previous studies, we postulated that a low-dose FSH priming of follicles would be beneficial in supporting the growth of multiple follicles and increase the number of immature oocytes retrieved for in-vitro maturation (Trounson et al., 1994Go; Rombauts et al., 1998Go). It has been shown that early atresia is no impediment to the oocytes (Moor et al., 1998Go). Developmentally competent oocytes can be retrieved from atretic follicles from patients undergoing coasting during ovarian stimulation (Sher et al., 1995Go). We postulated that depriving the follicles of FSH for a few days would promote early atresia in the outer cumulus cells. The main purpose of this study was to investigate if stimulation of the follicles in late luteal phase with a low-dose recombinant FSH (rFSH) would produce competent oocytes for in-vitro maturation and fertilization in women with regular and irregular cycles. In addition, we wished to assess the cryosurvival of these embryos, and thus all zygotes and cleaved embryos were cryopreserved and transferred later in a natural or hormone replacement cycle.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects
Twelve involuntarily infertile women with a previously determined need for IVF or intracytoplasmic sperm injection (ICSI) were recruited. Six women had regular ovulatory cycles (26–32 days) and six women had irregular cycles with polycystic ovaries PCO (see Table IGo). All women had a baseline serum FSH concentration below 10 IU/l. A written informed consent to participate was obtained before entering in the study. The study protocol was approved by the Ethics Committee of The Family Federation of Finland.


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Table I. Patient characteristics, duration of infertility (mean ± SD), number of previous in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) treatment cycles, and main cause of infertility
 
Priming of follicles
Low-dose rFSH (37.5 IU, Gonal-F®; Serono Nordic, Stockholm, Sweden) was commenced 11 days after a positive urinary luteinizing hormone (LH) test (Clearplan®; Unipath Ltd, Bedford, UK), and was continued in the following cycle until a leading follicle of 9–11 mm diameter was seen on ultrasound scan. Recombinant FSH was then withdrawn for 2–5 days before the oocyte retrieval. In women with anovulatory cycles, a withdrawal bleed was induced by using 600 mg daily vaginal micronized progesterone (Lugesteron®; Leiras, Turku, Finland) for 10 days. Low-dose rFSH was commenced on the ninth day of progesterone administration.

Recovery of oocytes
Oocytes were recovered by ultrasound-guided transvaginal aspiration of visible (>=3 mm diameter) follicles. A 17 gauge aspiration needle (Cook, Eight Mile Plains, Queensland, Australia), a Hitachi EUB-415 ultrasound (Hitachi Medical Corporation, Tokyo, Japan) and a 5 MHz vaginal probe (Hitachi) was used for oocyte recovery. The aspiration pressure was approximately half (7.5 kPa) the pressure used for standard oocyte retrieval for IVF.

Immature oocytes were collected into pre-warmed 10 ml Falcon tubes containing 2 ml of Dulbecco's phosphate-buffered saline (PBS) (Gibco-BRLTM, Life Technologies, Paisley, UK) containing heparin. Isolation of oocytes was performed by washing the follicular aspirate in Dulbecco's PBS containing 2% heat-inactivated fetal calf serum, 0.05 mg/ml penicillin and 0.075 mg/ml streptomycin through an Em-Con embryo concentrating filter (Veterinary Concepts, Spring Valley, WI, USA). Oocytes were identified in the filter retentant under a stereo dissecting microscope in 100x15 mm Petri dishes on a warm stage (37°C).

Oocyte maturation and fertilization in vitro
Oocyte–cumulus masses were matured in Tissue Culture Medium 199 (Gibco-BRLTM, Life Technologies Inc.) supplemented with 10% heat-inactivated fetal bovine serum (Gibco-BRLTM; Life Technologies Inc., Rockville, MD, USA), 0.075 IU rFSH/ml (Gonal-F®), 0.5 IU recombinant LH/ml (Lhadi®; Serono), 0.29 mmol/l pyruvate, 0.05 mg/ml penicillin and 0.075 mg/ml streptomycin. Oocytes were cultured under oil in 60x15 mm Petri dishes for 44 h at 37°C in 5% CO2 in air. ICSI was used routinely to fertilize the mature oocytes.

Embryo cryopreservation and transfer
All zygotes and cleaved embryos were frozen using 1,2-propanediol (PROH) and sucrose as cryoprotectants in PBS, following a previously described procedure (Lassalle et al., 1985Go). The embryos were first placed in PBS supplemented with 20% human serum for evaluation of the embryo quality and then immediately transferred to 1.5 mol/l PROH solution for 10 min. After incubation, embryos were placed in 1.5 mol/l PROH and 0.1 mol/l sucrose solution, and loaded into straws (1–>=3 embryos per straw). The straws were sealed and placed into a programmable freezer (Kryo 10; Planar Biomed, Sunbury-on-Thames, Middlesex, UK). The straws were cooled at a rate of 2°C/min down to –8°C, and held for 10 min. After seeding, the straws were cooled to –30°C at a rate of 0.3°C/min, and then to –150°C at 50°C/min, and finally plunged into liquid nitrogen. For thawing, the straws were taken from liquid nitrogen, held at room temperature for 30 s, and then placed into a 30°C water bath for a further 30 s. The embryos were expelled into 1.0 mol/l PROH and 0.2 mol/l sucrose for 5 min at room temperature, and transferred to 0.5 mol/l PROH and 0.2 mol/l sucrose for 5 min, whereafter the embryos were first placed into 0.2 mol/l sucrose and then into PBS + 20% human serum solution for 10 min at room temperature. Before transferring the embryos to pre-equilibrated culture media (MediCult, Jyllinge, Denmark), they were incubated for a further 5 min in PBS + 20% human serum at 37°C.

In women with regular menstrual cycles, the embryo transfer was timed by monitoring for spontaneous ovulation using vaginal ultrasound scan and urine LH concentration. Hormone replacement therapy in a down-regulated cycle was used for women with irregular, anovulatory cycles to provide an optimal endometrium for implantation. Buserelin (Suprecur®; Hoechst Marion Roussel, Frankfurt am Main, Germany) nasal spray 150 µg every 8 h was used to down-regulate endogenous ovarian function. When down-regulated, 4–6 mg of 17ß-oestradiol valerate (Progynova®; Schering, Berlin, Germany) was administered orally until the endometrial thickness was at least 7 mm on ultrasound scan. Vaginal micronized progesterone (Lugesteron®) 400 mg daily was commenced 2–3 days before embryo transfer depending on the age of the embryos. The zygotes were thawed the day before transfer to confirm cleavage. The cleaved embryos were thawed on the day of transfer. The cryosurvival of 107 zygotes and 399 cleaved embryos thawed in our IVF programme, and 57 zygotes and 162 cleaved embryos thawed in our ICSI programme during 1998 was analysed.

Statistical analysis was carried out using the {chi}2 and Mann–Whitney U-tests where appropriate. Data in the tables were expressed as the mean ± SD. A difference of P < 0.05 was considered as significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In total, 136 oocytes were retrieved from 12 women, six women with regular (67 oocytes) ovulatory cycles and six with irregular cycles and PCO on ultrasound examination (69 oocytes including nine atretic oocytes at retrieval). The characteristics of the subjects and the cause and duration of their infertility are shown in Table IGo. There were no significant differences between the two groups.

The average duration, pre- and post-menstruation, of low-dose rFSH priming prior to immature oocyte retrieval was 7.8 days in the women with regular cycles and 8.8 days in the women with irregular cycles (Table IIGo). One woman stopped the injections on day 1 of her menstrual cycle. Despite the early withdrawal of rFSH, 12 oocytes were retrieved on day 5 of her menstrual cycle, eight oocytes were viable and of these, seven matured and fertilized. In the other women, rFSH was continued for approximately 4 days of the following menstrual cycle.


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Table II. The duration of follicle stimulating hormone (FSH) administration, coasting, and the timing of immature oocyte retrievala
 
Once the follicles were growing, they were coasted for approximately 3 days to promote early atresia (Table IIGo). At the immature oocyte retrieval, the largest follicle was 11.1 ± 1.8 mm in women with regular cycles and 10.3 ± 4.0 mm in women with irregular cycles. In one woman in each group the leading follicle reached 14 mm in diameter. The number of oocytes retrieved, maturation and fertilization rates were comparable with values achieved with leading follicles <14 mm (results not shown). There were no significant differences between groups.

The majority (71%; 90/127) of the viable oocytes retrieved was capable of resuming meiosis and reaching maturity after 44 h in culture. Some 64% (58/90) of these mature oocytes fertilized after ICSI (Table IIIGo). One zygote degenerated before freezing. All normally fertilized zygotes (n = 33) and cleaved embryos (n = 24) were frozen to be transferred in an optimally timed natural or hormone replacement cycle (Table IVGo). All women except one had zygotes or cleaved embryos frozen. Again, there were no significant differences between the groups.


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Table III. Number of oocytes retrieved, matured and fertilized after intracytoplasmic sperm injection (ICSI)
 

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Table IV. Frozen–thawed zygotes and cleavage stage embryos in patients with regular cycles and irregular cycles with polycystic ovaries (PCO)
 
The frozen–thawed embryo survival was better in women with regular menstrual cycles compared with women with irregular cycles (61% and 23% respectively; P = 0.007; Table IVGo). Most (59%) of the zygotes and cleaved embryos degenerated after thawing. To date, 15 embryo transfers have been performed, and on 10 occasions only one embryo was available for transfer. Eight embryos are still frozen to be transferred later. One pregnancy was achieved, but this resulted in miscarriage.

During 1998 the cryosurvival of IVF zygotes and cleaved embryos was 92% and 66% respectively, and that of ICSI zygotes and cleaved embryos 84% and 80% respectively. The clinical pregnancy rate of freeze–thaw IVF cycles was 24%, and that of ICSI cycles 20%.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Here, we describe a new method for minimal stimulation using late luteal start of low-dose FSH for efficient recovery of immature oocytes for in-vitro maturation and fertilization. The development of immature oocyte collection techniques for in-vitro maturation and fertilization opens new possibilities for assisted reproduction (Trounson et al., 1994Go). First, the risk of ovarian hyperstimulation and other side effects related to fertility drugs would be reduced. Second, the costs and inconvenience of the treatment cycle would be reduced. Milder ovarian stimulation and in-vitro maturation of oocytes would make oocyte donation more feasible and attractive to altruistic women.

The ability of oocytes to complete all stages of development is known to be related to the follicle size, and consequently to the size of the oocyte (Moor and Trounson, 1977Go; Durinzi et al., 1995Go). Animal and human studies have suggested that administration of FSH for a few days in the early follicular phase would increase the number of competent oocytes retrieved (Schramm and Bavister, 1994Go; Wynn et al., 1998Go). Furthermore, unintentionally recovered immature oocytes from fully stimulated ovaries show good developmental capacity even when stripped of cumulus cells or cryopreserved before maturation (Veeck et al., 1983Go; Nagy et al., 1996Go; Jaroudi et al., 1997Go; Tucker et al., 1998Go).

The presence of small antral follicles responsive to FSH in the luteal phase is thought to determine the number of recruitable oocytes in the following menstrual cycle (Gougeon and Testart, 1990Go). Thus, an increase in FSH concentration in the late luteal phase would allow more follicles to continue their growth in the following menstrual cycle. In a previous study, however, in which late luteal start of FSH was used in combination with gonadotrophin releasing hormone (GnRH)-agonist suppression for ovarian stimulation in IVF, there was no increase in the number of oocytes retrieved compared with conventional early follicular FSH administration (Rombauts et al., 1998Go). In an unpublished randomized pilot study, we used the late luteal FSH protocol without GnRH-agonist for IVF/ICSI cycles. A low FSH dose (37.5 IU) was used in the luteal phase, and FSH was increased to 150 IU in the early follicular phase. We found that the oocyte recovery, fertilization and pregnancy rates were similar in women with low FSH and GnRH-agonist stimulation protocols. These results suggest that although the late luteal phase FSH administration may not be a benefit when a GnRH-agonist stimulation protocol is used, it is possible to acquire a reasonable number of good quality oocytes using a low-dose luteal phase FSH stimulation without down-regulation.

There are conflicting results on the benefits of using FSH priming of follicles for in-vitro maturation of oocytes in women with regular menstrual cycles. In a recent study, more oocytes matured and cleaved in patients receiving FSH in early follicular phase compared with women not receiving FSH. However, the investigators could not find any differences in either the numbers of oocytes recovered, fertilized or pregnancies achieved between the two groups (Mikkelsen et al., 1999Go). In women donating oocytes to research, FSH administration in the early follicular phase improved oocyte maturation but had no effect on the number of oocytes retrieved (Wynn et al., 1998Go). In this study, we have shown that using the low-dose late luteal start FSH in women with regular menstrual cycles, it is possible to recover two to three times more oocytes per patient than reported in other studies (Trounson et al., 1994Go; Mikkelsen et al., 1999Go). Furthermore, in this study, the number of oocytes recovered, matured and fertilized were comparable with the results of Russell et al. who used no in-vivo FSH priming (Russell et al.. 1997Go). The reasons for conflicting results on the effect of in-vivo FSH on the developmental competence of immature oocytes may be due to the timing of oocyte collection and the culture conditions used for oocyte maturation. The developmental stage of the oocyte at the time of retrieval may be crucial for its further development. The use of FSH priming of follicles may enhance some of the maturational processes in the oocyte necessary to achieve full developmental capacity.

Women with PCO in particular, are at a high risk of ovarian hyperstimulation syndrome after conventional ovarian stimulation for IVF/ICSI, and therefore the use of immature oocytes from unstimulated ovaries is an attractive one. There are several studies reporting on the use of in-vitro matured and fertilized oocytes in women with irregular cycles and PCO (Trounson et al., 1994Go; Barnes et al., 1995Go; Mikkelsen et al., 1998Go; Cha et al., 1999Go). Mikkelsen et al. showed better results using a short-term FSH priming in the early follicular phase combined with 72-h delay after the last FSH injection before oocyte retrieval compared with a 24-h delay (Mikkelsen et al., 1998Go). A longer time between the last FSH injection and oocyte aspiration would allow early atresia to take place in the granulosa cells, and this would enhance the meiotic competence of the oocytes. On the other hand, in other studies with similar results, no in-vivo FSH was used (Trounson et al., 1994Go; Barnes et al., 1995Go; Cha et al., 1999Go). In one of these studies (Trounson et al., 1994Go), an average number of 15 oocytes recovered and 81% maturation rate was reported, but poor fertilization and cleavage rates (34% and 19% respectively) when no in-vivo FSH was used. The results of the present study showed that the number of oocytes retrieved, and the maturation and fertilization rates in women with irregular cycles and PCO, were comparable with those in regularly menstruating women.

There are several reports on cryopreservation of human immature oocytes followed by in-vitro maturation and IVF and live birth (Toth et al., 1994Go; Son et al., 1996Go; Tucker et al., 1998Go). We do not know of any previous reports on the survival of cryopreserved zygotes/cleaved embryos following in-vitro maturation and fertilization of human oocytes. Our rationale for using freeze–thaw embryo replacement cycles was a concern for the condition of the endometrium in the immature oocyte collection cycle (Barnes et al., 1995Go; Russell et al., 1997Go). Thus, we chose to transfer the embryos during a natural or a hormone replacement cycle to optimize the uterine receptivity. The cryosurvival of both zygotes and cleaved embryos in our IVF and ICSI programmes has consistently been good, and we expected the most competent in-vitro matured zygotes and embryos to withstand the freezing procedure. However, the cryosurvival of the in-vitro matured zygotes and cleaved embryos was disappointingly low compared with embryos generated from in-vivo matured oocytes. The majority (59%) of the zygotes and cleaved embryos degenerated after thaw. In patients with irregular cycles and PCO, 36% of zygotes and two out of 15 cleaved embryos, survived after thawing; for the regularly cycling women, taking zygotes and cleaved embryos together, approximately two out of three survived after thawing. It is possible that the in-vitro-produced embryos have a reduced capacity to withstand stress such as freezing, as shown in animal studies (Moor et al., 1998Go).

The full developmental competence of an oocyte and an embryo can ultimately be evaluated by live births. There are several studies reporting acceptable maturation, fertilization and embryo cleavage rates, but only occasional successful pregnancies (Cha et al., 1991Go; Barnes et al., 1995Go; Russell et al., 1997Go; Jaroudi et al., 1999Go). The explanation for this relatively low pregnancy rate of human in-vitro maturation followed by ICSI may be asynchrony between nuclear and cytoplasmic maturation with inadequate signal transduction and molecular reorganization of the oocyte, which is expressed later by the cessation of embryo development. The reasons for the lack of full developmental capacity of the in-vitro matured and fertilized human oocytes have been thought to depend on the size of follicles, the timing of immature oocyte collection, culture conditions and the endometrium (Moor et al., 1998Go; Mikkelsen et al., 1999Go). However, recent studies have shown improved pregnancy rates of 20–25% per embryo transfer (Cha et al., 1999Go; Mikkelsen et al., 1999Go). It is important to continue to find ways to improve both clinical and laboratory procedures to make the use of immature oocytes more efficient.


    Acknowledgments
 
We would like to thank all personnel at The Infertility Clinic of Family Federation of Finland in Helsinki for their help and technical assistance in looking after the patients and freezing the embryos. The study was supported by Cook Australia and Ares-Serono Nordic.


    Notes
 
4 To whom correspondence should be addressed at: Infertility Clinic, The Family Federation of Finland, PO Box 849, 00101 Helsinki, Finland Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on October 29, 1999; accepted on January 20, 2000.