1 Department of Obstetrics and Gynecology, Abant Izzet Baysal University, Izzet Baysal Faculty of Medicine Bolu, 2 Centrum Clinic, 3 Department of Biological Sciences, Middle East Technical University, 4 Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility and 5 Department of Hematology, Gülhane School of Medicine and 6 Zekai Tahir Burak Women's Health and Educational Hospital, Ankara, Turkey
7 To whom correspondence should be addressed at: Centrum Clinic, Nenehatun, No. 59, Ankara, Turkey 06520-8063. Email: gogsenonalan{at}yahoo.com
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Abstract |
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Key words: apoptosis/IVF/metformin/PCOS/sFas
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Introduction |
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Diverse hormonal signals by gonadotrophins, growth factors, steroid hormones and death factors, such as Fas ligand (FasL), converge on selective intracellular pathways to regulate follicular apoptosis (Kaipia and Hsueh, 1997). Fas and Fas ligand are membrane proteins that exist in both transmembrane and soluble forms. The former triggers apoptosis when bound by FasL, whereas the latter inhibits Fas-mediated apoptosis by preventing death signal transduction (Ueno et al., 1999
). Soluble Fas (sFas) is detected in human sera and fluids of the reproductive system, including seminal plasma, oviductal fluid and follicular fluid (Srivastava and Fichorova, 1998
). Presence of apoptotic and proliferative factors in both follicular fluid and serum samples of women undergoing IVF cycles further confirms apoptosis as a regulatory mechanism for oocyte maturation and survival (Sarandakou et al., 2003
).
Among women with PCOS, 80% of obese and 3040% of women with normal weight present with hyperinsulinaemia (Dunaif et al., 1989). Insulin modulates insulin-like growth factor (IGF)-binding protein and biologically active IGF-I levels which are important factors in ovarian cellular mitosis, steroidogenesis and apoptosis. Metformin, an oral biguanide, improves hyperandrogenism by decreasing insulin levels. Clinical outcomes of metformin therapy prior to IVF are controversial with few studies on this subject (Stadtmauer et al., 2001
; Fedorcsak, 2003
; Kjotrod et al., 2004
). Metformin is suggested to be beneficial in PCOS patients undergoing gonadotrophin therapy and IVF as well as ovulation induction (Stadtmauer et al., 2002
).
Analysis of follicular fluid in combination with serum reflects autocrine and paracrine factors regulating ovarian folliculogenesis along with systemic endocrine dynamics (Lambert-Messerlian et al., 1997). To our knowledge there are limited data in the literature about the levels of soluble apoptotic factors in IVF cycles and whether they have any prognostic value on the clinical outcome. In this prospective, randomized, placebo-controlled study, we investigated the serum and follicular fluid levels of sFas and sFasL in patients with PCOS undergoing IVF/ICSI cycles and compared them with those of infertility due to male factor. We also investigated whether metformin therapy prior to IVFICSI cycles alters any of these parameters and luteinized granulosa cell apoptosis among patients with PCOS.
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Materials and methods |
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Individual follicular fluid and serum samples were collected from 40 women undergoing IVF treatments. Patients with PCOS (n=28) had the diagnostic criteria of oligomenorrhoea or amenorrhoea as a surrogate for oligo-anovulaton since menarche. These patients also had at least one of the following indications of hyperandrogenism: (i) a hirsutism score of >7, according to Ferriman and Gallway classification (Ferriman and Gallway, 1961); (ii) an elevated level of serum testosterone (
3.15 nmol/l). All other causes of hyperandrogenism were ruled out before the diagnosis of PCOS. Diagnosis of PCOS follows the criteria approved by the Rotterdam Consensus Conference since oligo- and/or anovulation, clinical and/or biochemical signs of hyperandrogenism fulfil two out of three criteria for the revised diagnosis of PCOS by the Conference (Rotterdam ESHRE/ASRM-sponsored PCOS consensus workshop group, 2004
). PCOS patients were divided into two groups, one group receiving metformin (n=14), and the other receiving placebo (n=14) for 8 weeks before IVFICSI cycle. Subjects treated with hormonal medications or insulin-lowering agents within 3 months were excluded from the study. Twelve had infertility due to male factor which was described as <500 000 sperm with progressive motility and >90% abnormal sperm. Ovulation was confirmed by mid-luteal progesterone measurements. Progesterone levels <3 ng/ml were accepted as evidence for anovulation.
IVFICSI stimulation procedure
All patients underwent controlled ovarian stimulation (COS) with a combination of GnRH agonist and recombinant (r)FSH, using a long protocol (n=40) for IVFICSI treatment. During the standard long protocol for pituitary suppression, GnRH agonist (0.1 mg Decapeptyl; Ferring, Germany) was administered s.c. from day 21 of the previous cycle followed by FSH (Gonal F; Serono, Turkey) for ovarian stimulation. The standard initial dose of gonadotrophin was 24 ampoules of rFSH (150300 IU) per day and doses were adjusted based on individual responses. hCG (10 000 IU, Pregnyl; Organon, Turkey) was administered when a minimum of three leading follicles 18 mm mean diameter and serum levels of E2 <5500 pg/ml were detected. Oocyte retrieval was performed by vaginal ultrasound-guided follicular puncture, 3536 h following hCG administration. In addition to patients with infertility due to male factor, patients with PCOS were offered ICSI, a procedure which has the advantage of bypassing the barriers that may be of oocyte origin, and may thus overcome the lower fertilization rates in PCOS patients during their IVF cycles (Hwang et al., 2005
). Oocytes were inspected for the presence of pronuclei 1618 h after ICSI procedure. The embryos were evaluated according to blastomere size equality and the relative proportion of anucleate fragments. A maximum number of two embryos was transferred on the third day. Pregnancy was confirmed when serum hCG concentrations were rising on at least two separate occasions between the 12th and 14th days after the embryo transfer. Clinical pregnancy was diagnosed by ultrasonography during the 7th week of pregnancy.
In PCOS patients, for the randomization process, women were given an envelope labelled as either metformin or placebo according to the code provided by computer-generated randomization in blocks. Patients in the metformin group received metformin (Glucophage Retard; Ilsan-Ilta Pharmaceuticals, Istanbul, Turkey) 850 mg twice a day if they had a body mass index (BMI) of <28 kg/m2 or three times a day (850 mg) for a BMI >28 kg/m2 for 8 weeks before their first IVFICSI cycle. Placebo identical to the metformin capsule was obtained from the same company. Patients were instructed not to modify their usual eating habits throughout the study. All patients received their treatments until a positive pregnancy test.
Serum and follicular fluid collection and measurement of sFas, sFasL levels
Blood samples were drawn just before follicle aspiration. Follicular fluid aspirates from all mature follicles (14 mm diameter) were collected at the time of transvaginal ultrasound-guided oocyte retrieval and immediately centrifuged at 4°C. The cell-free supernatants were aliquoted and stored at 80°C until assay. As a reflection of follicular asynchrony during ovarian stimulation for IVF, wide interfollicular variations in steroid and cytokine concentrations have been reported (Barak et al., 1992
). Therefore, we collected pooled follicular fluid aspirations from each patient in an attempt to assess whole ovarian production as previously recommended, rather than to evaluate each follicle separately (Orvieto et al., 1995
).
Follicular fluid and serum levels of sFas and sFasL were analysed by a sandwich enzyme-linked immunosorbent assay (human sFasL and sFas; Biosource International, CA, USA). Sensitivities, intra- and inter-assay coefficients of variation were 0.1 ng/ml, 6.1 and 7% for sFasL and 20 pg/ml, 4.4 and 5.9% for sFas respectively.
Serum and follicular levels of sFas, sFasL and the parameters participating in IVF outcome, including serum levels of E2 on day 3 of the menstrual cycle and on the day of hCG injection, number of antral follicles and oocytes >17 mm in diameter, total number of oocytes retrieved, metaphase I and II oocytes, rate of fertilization, number of embryos transferred, rates of biochemical and clinical pregnancies were compared between the study groups.
DNA isolation and fragmentation analysis
Cellular DNA was extracted from luteinized granulosa cells, precipitated and dissolved as described previously with some modifications (Sanbrook et al., 1989). Briefly, granulosa cells were collected by centrifugation at
(100xg) for 5 min at room temperature. All cells were treated with 3 ml lysis buffer (400 mmol/l NaCl, 10 mmol/l TrisHCl, 2 mmol/l EDTA, pH 8.0), 200 µl 10% sodium dodecyl sulphate and 150 µl proteinase K (10 mg/ml), mixed gently and incubated overnight at 37°C. The next day 1 ml of ammonium acetate (saturated) was added onto the mixture and incubated for 15 min at room temperature. The mixture was then centrifuged at
(2500xg) for 30 min at room temperature. Supernatant was transferred into another tube and 2.5 vols of absolute ethanol was added. DNA was dissolved in TrisEDTA buffer (pH 8.0). Extracted DNA fragments were electrophoretically separated on 2% agarose gel containing ethidium bromide (0.5 mg/ml) in Tris Acetate-EDTa buffer at 90 V for 1 h. DNA samples were visualized in a UV transilluminator.
Statistical analysis
Serum and follicular levels of sFas, sFasL and the parameters participating in IVF outcome were not normally distributed. MannWhitney U-test, KruskalWallis H-test and Fisher's exact tests were used where appropriate. SPPS for Windows 10.0 software was used for statistical analysis. P<0.05 was considered statistically significant.
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Results |
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Significantly lower serum levels of sFas were detected in women with PCOS compared to those in the male factor group (P<0.05) (Table I).
When we compared the serum and follicular fluid levels of both sFas and sFasL among PCOS patients receiving either metformin or placebo, the metformin group had decreased levels of sFasL in follicular fluid and increased levels of sFas in serum (P<0.05) (Table II).
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Discussion |
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Metformin reduces insulin resistance, insulin secretion and hyperinsulinaemia by increasing insulin sensitivity (Dunn and Peters, 1995). Although there are controversial data about the outcomes of metformin, the rationale for this treatment modality is to reduce insulin resistance and sequelae of hyperinsulinaemia including hyperandrogenism (Crave et al., 1995
). There are only limited studies reporting the effects of metformin on IVF outcomes (Stadtmauer et al., 2001
; Onalan, 2005
).
In the current study we demonstrated decreased levels of sFasL in follicular fluid and increased levels of sFas in serum samples among PCOS patients receiving metformin compared to those receiving placebo. Patients with PCOS on placebo had luteinized granulosa cell DNA fragmentation in follicular fluid samples. Similar DNA fragmentation was not observed in PCOS patients on metformin therapy, which may imply an antiapoptotic effect of metformin on soluble factors both in follicular fluid and serum. This effect can be a result of reduced androgen production by metformin that may inhibit apoptosis; however, the role of androgens in follicle development, whether atretogenic or trophic, is debatable probably due to interspecies differences in ovarian follicular development with different experimental conditions (Jonard and Dewailly, 2004). As in the
-cell in the pancreas (Kefas et al., 2004
), metformin can also inhibit apoptosis in granulosa cells, which may result in the induction of biosynthesis and release pathways preventing the functional exhaustion.
There was a tendency for higher number of retrieved oocytes, increased implantation and clinical pregnancy rates among PCOS patients on metformin; however, the differences were not statistically significant in comparison to those of PCOS patients on placebo in the current study. Apoptosis is suggested to predominate in the granulosa cell layer of preantral and antral follicles rather than the dominant follicles (10 mm) in normal ovaries with androgen-dominant follicles uniformly displaying characteristics of apoptosis (Yuan and Giudice, 1997
). The FasFasL system may play a role in preventing oocyte atresia during folliculogenesis. Since levels of sFas and FasL may not be critical in controlling the quality of oocytes once they overcome meiotic arrest and achieve the metaphase II stage of development (Jose de los Santos et al., 2000
), the FasFasL system may not have negative impact on IVF outcomes including fertilization of mature oocytes, embryo quality or pregnancy rate. In vitro and in vivo studies investigating the direct or indirect effects of metformin on ovarian cell apoptosis may further delineate its mechanism of action and studies with larger samples may shed light on the consequences for clinical outcomes.
To our knowledge this is the first study demonstrating serum and follicular fluid levels of sFas and sFasL in patients with PCOS undergoing IVF cycles and elucidating the effects of prior metformin therapy on these parameters and apoptosis of luteinized granulosa cells in follicular fluid.
In conclusion, the current study suggests that sFas and sFasL are detected in both serum and follicular fluid samples from IVF cycles. Patients with PCOS undergoing IVF cycles have lower serum levels of sFas. Metformin therapy in PCOS patients preceding IVF cycles decreases follicular fluid levels of sFasL, increases serum levels of sFas and prevents luteinized granulosa cell DNA fragmentation, demonstrating an antiapoptotic effect.
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Submitted on January 21, 2005; resubmitted on March 9, 2005; accepted on April 13, 2005.
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