Fertility and Sterility
Volume 95, Issue 1 , Pages 333-337, January 2011

Peroxisome proliferator-activated receptor gamma and early folliculogenesis during an acute hyperandrogenism condition

  • Monica Faut

      Affiliations

    • Laboratorio de Fisio-patología Ovárica, Centro de Estudios Farmacológicos y Botánicos, Buenos Aires, Argentina
    • ,
  • Evelin Mariel Elia

      Affiliations

    • Laboratorio de Fisio-patología Ovárica, Centro de Estudios Farmacológicos y Botánicos, Buenos Aires, Argentina
    • ,
  • Fernanda Parborell, Ph.D.

      Affiliations

    • Instituto de Biología y Medicina Experimental, Consejo Nacional de Investigación Científica y Técnica, Buenos Aires, Argentina
    • ,
  • Noelia Melina Cugnata

      Affiliations

    • Laboratorio de Fisio-patología Ovárica, Centro de Estudios Farmacológicos y Botánicos, Buenos Aires, Argentina
    • ,
  • Marta Tesone, Ph.D.

      Affiliations

    • Instituto de Biología y Medicina Experimental, Consejo Nacional de Investigación Científica y Técnica, Buenos Aires, Argentina
    • Facultad de Ciencias Exactas, Universidad de Buenos Aires, Buenos Aires, Argentina
    • ,
  • Alicia Beatriz Motta, Ph.D.

      Affiliations

    • Laboratorio de Fisio-patología Ovárica, Centro de Estudios Farmacológicos y Botánicos, Buenos Aires, Argentina
    • Corresponding Author InformationReprint requests: Alicia Beatriz Motta, Ph.D., Centro de Estudios Farmacológicos y Botánicos, Consejo Nacional de Investigación Científica y Técnica, Paraguay 2155, Buenos Aires, Argentina (FAX: 00-54-11-4508-3680).

Received 9 May 2010; received in revised form 1 July 2010; accepted 26 July 2010. published online 02 September 2010.

Article Outline

Acute hyperandrogenism decreases serum P levels and induces early apoptosis of antral follicles by a mechanism mediated by the peroxisome proliferator-activated receptor gamma system and independent of the steroidogenic acute regulator protein.

Key Words: Folliculogenesis, steroidogenic acute regulatory protein (StAR), peroxisome proliferatoractivated receptor gamma (PPARγ), dehydroepiandrosterone

 

The levels of fuel sensors such as metabolites (e.g., glucose, fatty acids, amino acids) and hormones (e.g., adiponectin, insulin, leptin, ghrelin) are involved in the regulation of fertility (1). The treatment of polycystic ovary syndrome (PCOS) patients with insulin-sensitizing agents such as thiazolidinediones or the biguanide metformin restores reproductive functions by modulating these molecules 2, 3. To exert their actions, thiazolidinediones bind to the nuclear peroxisome proliferator-activated receptor gamma (PPARγ) and metformin activates the 5'adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway 4, 5. PPARs are a family of transcriptional nuclear factors with three isoforms—α, β, and γ—that regulate gene expression 6, 7. The three PPAR isotypes are detected in developing follicles of several species 7, 8, 9, 10, 11, 12, 13. The activation of PPARγ regulates the synthesis of steroid hormones in the granulosa cells (14), and the disruption of PPARγ in the ovary leads to female subfertility (15). We previously reported that hyperandrogenization of BALB/c mice prevents ovulation 5, 16, 17, 18, 19, 20 by modulating AMPK (5). These findings, together with the fact that AMPK and PPARγ control the energy balance in the ovary (21), have led us to study whether hyperandrogenism interferes with early folliculogenesis by modulating the PPARγ pathway. For this purpose, we investigated the in vivo acute effects of hyperandrogenism on the expression of the steroidogenic acute regulator (StAR) and PPARγ and the production of P. We also studied whether acute hyperandrogenism induces follicular apoptosis during early-induced folliculogenesis.

The animal model consisted of immature (22–25 days old) female Sprague Dawley rats injected intraperitoneally with 25 IU of chorionic gonadotropin (eCG; Sigma-Aldrich, St. Louis, MO) in 0.1 ml saline solution (eCG group). The hyperandrogenized group consisted of rats injected intraperitoneally with 25 IU/rat eCG together with as SC injection of 60 mg/kg body weight DHEA (Sigma-Aldrich) in 0.1 mL oil (eCG+DHEA group). The control group consisted of rats injected with both vehicles. Rats were housed under controlled temperature (22°C) and illumination (14 h light: 10 h dark; lights on at 05:00 h) and were allowed free access to Purina (Buenos Aires, Argentina) rat chow and water. All procedures involving animals were conducted in accordance with the Animal Care and Use Committee of Consejo Nacional de Investigaciones Científicas y Técnicas 1996.

To determine the time of maximal follicular development, serum P levels were measured by radioimmunoassay (22) and determined at different intervals (0–12 h after treatment). Because the peak of P was obtained at 8 h after eCG treatment, ovaries and then follicles were isolated at 8 h for additional assays. A total of 20 rats for each group (control, eCG, and eCG+DHEA) was used as follows: five rats for Western blotting, five for reverse transcription polymerase chain reaction (RT-PCR), five for annexin V–iodide propidium, and five for DNA fragmentation. Healthy antral follicles (200–450 mm in diameter) were obtained by dissecting ovaries microscopically using fine needles, collected, pooled (60 healthy antral follicles per point, 10 points per group) and frozen at –70°C until assays were performed as previously described (23).

The content of StAR protein from antral follicles was evaluated by Western blotting. Each sample was applied to a 15% SDS-polyacrylamide gel, and the separated proteins were transferred onto nitrocellulose membranes. After blocking, the membranes were incubated with rabbit polyclonal anti-StAR 1:2000 (Cayman, Ann Arbor, MI). Individual bands were quantified directly from membranes by densitometry using the Image J (Softonic Intershare, S.L., Barcelona, Spain). We found that both eCG and CG+DHEA treatments significantly increased the expression of follicular StAR protein compared with the nonstimulated controls (Fig. 1A). In addition, the administration of eCG+DHEA resulted in an even higher protein content of follicular StAR compared with the eCG group (Fig. 1A).

  • View full-size image.
  • View full-size image.
  • Figure 1 

    (A) Protein expression of follicular StAR from control, eCG-treated, and eCG+DHEA-treated rats, a representative Western blot, and the graph corresponding to the integrated optical density of the bands. Each column represents the mean ± SEM of 10 measurements from different animals: a vs. b, a vs. c, and b vs. c. ∗P < 0.0001 by analysis of variance. (B) MRNA expression of StAR of the same three groups and the graph of integrated optical density bands. Each column represents the mean ± SEM of 10 measurements from different animals. ∗P < 0.001 was significantly different from control value by analysis of variance. (C) Protein expression of follicular PPARγ from control, eCG-treated, and eCG+DHEA-treated rats. A representative Western blot and the corresponding graph is shown. Each column represents the mean ± SEM of 10 measurements from different animals. ∗P < 0.0001 was significantly different from control value by analysis of variance. (D) The mRNA expression of PPARγ of the three groups and the corresponding graph. Each column represents the mean ± SEM of 10 measurements from different animals: a vs. b and a vs. c, P < 0.001; b vs. c, P < 0.05 by analysis of variance. (E) A representative dot plot and the quantitative estimation of viability and apoptosis. Each column represents the mean ± SEM of 10 measurements from different animals: a vs. c, P < 0.05; b vs. d, P < 0.001 by analysis of variance. (F) Agarose gel showing DNA fragmentation and the quantitative estimation of DNA cleavage. Data points represent the mean ± SEM of four independent gel runs: a vs. b, P < 0.01.

To determine whether the effect of eCG and eCG+DHEA in the StAR protein was a reflection of StAR gene expression, the StAR mRNA levels were measured by RT-PCR analysis. Total mRNA from each group of antral follicles was extracted using TriReagent (Molecular Research Center, Cincinnati, OH). The products were separated on 2% agarose and visualized with ethidium bromide staining. The analysis was performed by densitometry scanning using an Image Quant RT-ECL (Softonic Intershare, S.L., Barcelona, Spain). Bands were compared with internal control using Image J. For amplification of StAR cDNA, the primers were sense 5′-GGC CTT GGG CAT ACT CA-3′, antisense 5′-TCC TTG ACA TTTGGG TTC C-3′. L30 protein gene was used as an internal control. Figure 1B shows that StAR mRNA levels were detectable in follicles in the control group. StAR mRNA levels significantly increased after both eCG and eCG+DHEA treatment compared with controls (Fig. 1B).

The content of PPARγ protein was measured by Western blotting as described previously, using a rabbit polyclonal 1:2,500 anti-PPARγ (Cayman). We found that eCG increased follicular PPARγ protein expression compared with controls (Fig. 1C). The administration of eCG+DHEA led the expression of PPARγ to that of controls levels (Fig. 1C).

To determine whether the regulation in the PPARγ protein content induced by eCG and eCG+DHEA was a reflection of PPARγ gene expression, the PPARγ mRNA levels were measured by RT-PCR analysis as described previously with the primers were sense are 5′-TGA CAC AGA GAT GCC ATT CTG G-3′, antisense 5′-GAG CTA GAC CCA ATG GTT GCT GAT TAC-3′. We found that PPARγ mRNA levels were detectable in antral follicles beforeto eCG administration (Fig. 1D). eCG treatment significantly increased PPARγ mRNA levels compared with controls (Fig. 1D). Acute hyperandrogenism (eCG+DHEA) significantly decreased PPARγ mRNA levels, compared with the eCG group, although they were higher compared with controls (Fig. 1D).

Cellular viability and apoptosis was determined by a kit containing annexin V conjugated to fluorescein isothiocyanate (FITC) and propidium iodide (PI; Calbiochem, Gibbstown, NJ). Quantification was done by flow cytometry as previously described (24). Viable cells do not bind FITC–annexin V and do not stain nuclear formation with PI, whereas apoptotic cells bind FITC–annexinV and also stain their nucleus with PI. Antral follicles were enzymatically dissociated with trypsin-free collagenase (740 IU/100 mg tissue) and suspensions were applied to a Ficoll hystopaque gradient 1.077 (Sigma-Aldrich) to remove blood cells. We found that the eCG treatment did not modify viability and cellular apoptosis when compared with the control group (data not shown), whereas eCG+DHEA administration significantly decreased follicular viability and increased apoptosis when compared with the control and eCG groups (Fig. 1E).

DNA fragmentation was also analyzed. Sixty healthy antral follicles per ovary for each treatment were incubated for 24 h at 37°C in 500 μL DMEM:F12 (1:1) culture media, supplemented with amphotericin B (250 μg/mL) and gentamicin (10 mg/mL; five ovaries per group) in 95% O2, 5% CO2 atmosphere. This model keeps the integrity of the follicle and allows the exhibition of the typical apoptotic DNA ladder: presence of internucleosomal fragments of 180-bp multiples as described previously (25). Densitometry analysis was performed with an Image Scanner (Genius, Miami, FL). We found that eCG+DHEA increased apoptosis compared with the control and eCG groups (Fig. 1F).

Although it is well known that in PCOS, follicles fail to mature and then enter atresia, the mechanisms involved remain unknown. PPARγ is strongly expressed in granulosa and theca cells 7, 26, 27. A direct association of polymorphisms in the genes encoding PPARs and PCOS has been reported recently (28). In fact, insulin resistance in patients with PCOS has been treated largely with synthetic ligands for PPARγ 29, 30, 31, 32, 33, 34. In the present study, we investigated whether acute hyperandrogenism regulates early folliculogenesis by modulating PPARγ system.

As we have described during the periovulatory period 5, 16, 18 and the luteal phase (35), acute hyperandrogenism decreases serum P levels to those of controls. The process of steroidogenesis requires the active delivery of the substrate cholesterol 36, 37, 38, 39, and then, as we expected, the stimulation of steroidogenesis by eCG treatment was mediated by an increase in both the protein and mRNA expression of StAR in antral follicles. Hyperandrogenism increased expression of StAR protein when compared with the eCG treatment. Until now, it was known that the gene expression for StAR was hormonally regulated by LH (40). Our data are the first demonstration that the gene expression for StAR also can be upregulated by acute hyperandrogenism.

In this study, we demonstrated that the eCG treatment increased the protein and mRNA expression of PPARγ, whereas acute hyperandrogenism led the expression of PPARγ protein to that of control levels. These data, together with the fact that hyperandrogenism did not modify the stimulation of StAR by eCG and even increases protein expression of StAR, led us to postulate that acute hyperandrogenism decreases serum P by a StAR-independent pathway. Reaffirming this suggestion, the profile of the PPARγ pathway is coincident to that of P production. In addition, Yazawa et al. (41) recently reported that the PPAR system regulates P production in ovarian cultured cells.

Apoptosis is a fundamental process during early folliculogenesis. The relationship between PPAR and apoptosis is controversial. It has been reported that the enhancement of PPARγ can prevent 42, 43, 44, 45 and mediate apoptosis 46, 47. Our data lead us to suggest that the PPARγ pathway seems to prevent apoptosis of antral follicles. In summary, our results demonstrate a StAR-independent novel pathway in the regulation of steroidogenesis by acute hyperandrogenism during early folliculogenesis played by the PPARγ system. Because the PPARγ system is impaired on PCOS-follicles, and because glitazones by improving ovarian insulin resistance normalizes the functions of granulosa cells (48), further experiments are being designed to discern which actions of PPARγ are regulated by insulin resistance and which are regulated by the hyperandrogenism.

Back to Article Outline

References 

  1. Fernandez-Fernandez R, Martín AC, Navarro VM, Castellano JM, Dieguez C, Aguilar E, et al. Novel signals for the integration of energy balance and reproduction. Mol Cel Endocrinol. 2006;254–255:127–132
  2. Iuorno MJ, Nestler JE. The polycystic ovary syndrome: treatment with insulin sensitizing agents. Diabetes Obes Metab. 1999;1:127–136
  3. Seli E, Duleba AJ. Treatment of PCOS with metformin and other insulin-sensitizing agents. Curr Diab Rep. 2004;4:69–75
  4. Musi N, Hirshman MF, Nygren J, Svanfeldt M, Bavenholm P, Rooyackers O, et al. Metformin increases AMP-activated protein kinase activity in skeletal muscle of subjects with type 2 diabetes. Diabetes. 2002;51:2074–2081
  5. Elia E, Sander V, Luchetti CG, Solano ME, Di Girolamo G, Gonzalez C, et al. The mechanisms involved in the action of metformin in regulating ovarian function in hyperandrogenized mice. Mol Hum Reprod. 2006;2:475–481
  6. Issemann I, Green S. Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators. Nature. 1990;347:645–650
  7. Komar CM. Peroxisome proliferator-activated receptors (PPARs) and ovarian function—implications for regulating steroidogenesis, differentiation, and tissue remodeling. Reprod Biol Endocrinol. 2005;3:41–55
  8. Higashiyama H, Billin AN, Okamoto Y, Kinoshita M, Asano S. Expression profiling of peroxisome proliferator-activated receptor-delta (PPAR-delta) in mouse tissues using tissue microarray. Histochem Cell Biol. 2007;127:485–494
  9. Froment P, Fabre S, Dupont J, Pisselet C, Chesneau D, Staels B, et al. Expression and Functional role of peroxisome proliferator-activated receptor-g in ovarian folliculogenesis in the sheep. Biol Reprod. 2003;69:1665–1674
  10. Ibabe A, Grabenbauer M, Baumgart E, Fahimi HD, Cajaraville MP. Expression of peroxisome proliferator-activated receptors in zebrafish (Danio rerio). Histochem Cell Biol. 2002;118:231–239
  11. Crawford MJ, Liversage RA, Varmuza SL. Two isoforms of Xenopus retinoic acid receptor gamma 2 (B) exhibit differential expression and sensitivity to retinoic acid during embryogenesis. Dev Genet. 1995;17:291–302
  12. Mohan M, Malayer JR, Geisert RD, Morgan GL. Expression patterns of retinoid X receptors, retinaldehyde dehydrogenase, and peroxisome proliferator activated receptor gamma in bovine preattachment embryos. Biol Reprod. 2002;66:692–700
  13. Wood JR, Dumesic DA, Abbott DH, Strauss JF. Molecular abnormalities in oocytes from women with polycystic ovary syndrome revealed by microarray analysis. J Clin Endocrinol Metab. 2007;92:705–713
  14. Huang Z, Zhou X, Nicholson AC, Gotto AM, Hajjar DP, Han J. Activation of peroxisome proliferator-activated receptor-alpha in mice induces expression of the hepatic low-density lipoprotein receptor. Br J Pharmacol. 2008;155:596–605
  15. Cui Y, Miyoshi K, Claudio E, Siebenlist UK, Gonzalez FJ, Flaws J. Loss of the peroxisome proliferation-activated receptor gamma (PPARgamma) does not affect mammary development and propensity for tumor formation but leads to reduced fertility. J Biol Chem. 2002;277:17830–17835
  16. Luchetti CG, Solano ME, Sander V, Arcos ML, Gonzalez C, Di Girolamo G, et al. Effects of dehydroepiandrosterone on ovarian cystogenesis and immune function. J Reprod Immunol. 2004;64:59–74
  17. Sander V, Solano ME, Elia E, Luchetti CG, Di Giorlamo G, Gonzalez C, et al. The influence of dehydroepiandrosterone on early pregnancy in mice. Neuroimmunomodulation. 2005;12:285–292
  18. Sander V, Luchetti CG, Solano ME, Elia EM, Di Girolamo G, Gonzalez C, et al. Role of the N, N’dimethylbiguanide metformin in the treatment of female prepuberal BALB/c mice hyperandrogenized with dehydroepiandrosterone. Reproduction. 2006;131:591–602
  19. Solano ME, Elia E, Luchetti CG, Sander V, Di Girolamo G, Gonzalez C, et al. Metformin prevents embryonic resorption induced by hyperandrogenisation with dehydroepiandrosterone in mice. Reprod Fertil Dev. 2006;18:533–544
  20. Belgorosky D, Sander VA, Yorio MP, Faletti AG, Motta AB. Hyperandrogenism alters intraovarian parameters during early folliculogenesis in mice. Reprod Biomed Online. 2010;20:797–807
  21. Dupont J, Chabrolle C, Ramé C, Tosca L, Coyral-Castel S. Role of the peroxisome proliferator-activated receptors, adenosine monophosphate-activated kinase, and adiponectin in the ovary. PPAR Res. 2008;2008:176275
  22. Motta AB, Estévez A, Tognetti T, Gimeno MA, Franchi AM. Dual effect of nitric oxide in functional and regressing rat corpus luteum. Mol Hum Reprod. 2001;7:43–47
  23. Irusta G, Parborell F, Peluffo M, Manna PR, Gonzalez-Calvar SI, Calandra R, et al. Steroidogenic acute regulatory protein in ovarian follicles of gonadotropin-stimulated rats is regulated by a gonadotropin-releasing hormone agonist. Biol Reprod. 2003;68:1577–1583
  24. Solano ME, Sander V, Wald MR, Motta AB. Dehydroepiandrosterone and metformin regulate proliferation of murine T lymphocytes. Clin Exp Immunol. 2008;153:289–296
  25. Parborell F, Abramovich D, Tesone M. Intrabursal administration of the antiangiopoietin 1 antibody produces a delay in rat follicular development associated with an increase in ovarian apoptosis mediated by changes in the expression of BCL2 related genes. Biol Reprod. 2008;78:506–513
  26. Gasic S, Bodenburg Y, Nagamani M, Green A, Urban RJ. Troglitazone inhibits progesterone production in porcine granulosa cells. Endocrinology. 1998;139:4962–4966
  27. Komar CM, Braissant O, Wahli W, Curry TE. Expression and localization of PPARs in the rat ovary during follicular development and the periovulatory period. Endocrinology. 2001;142:4831–4838
  28. San-Millán JL, Escobar-Morreale HF. The role of genetic variation in peroxisome proliferator-activated receptors in the polycystic ovary syndrome (PCOS): an original case-control study followed by systematic review and meta-analysis of existing evidence. Clin Endocrinol (Oxf). 2010;72:383–392
  29. Day C. Thiazolidinediones: a new class of antidiabetic drugs. Diabet Med. 1999;16:179–192
  30. Iuorno MJ, Nestler JE. Insulin-lowering drugs in polycystic ovary syndrome. Obstet Gynecol Clin North Am. 2001;28:153–164
  31. Girard J. Mechanisms of action of thiazolidinediones. Diabetes Metab. 2001;27(2 Pt 2):271–278
  32. Seli E, Duleba AJ. Treatment of PCOS with metformin and other insulin-sensitizing agents. Curr Diab Rep. 2004;4:69–75
  33. Minge CE, Ryan NK, Van Der Hoek KH, Robker RL, Norman RJ. Troglitazone regulates peroxisome proliferator-activated receptors and inducible nitric oxide synthase in murine ovarian macrophages. Biol Reprod. 2006;74:153–160
  34. Brannian JD, Eyster KM, Weber M, Diggins M. Pioglitazone administration alters ovarian gene expression in aging obese lethal yellow mice. Reprod Biol Endocrinol. 2008;18:6–10
  35. Sander VA, Facorro GB, Piehl L, Rubín de Celis E, Motta AB. Effect of DHEA and metformin on corpus luteum in mice. Reproduction. 2009;138:571–579
  36. Simpson ER, McCarthy JL, Peterson JA. Evidence that the cycloheximide-sensitive site of adrenocorticotropic hormone action is in the mitochondrion. Changes in pregnenolone formation, cholesterol content, and the electron paramagnetic resonance spectra of cytochrome P-450. J Biol Chem. 1978;253:3135–3139
  37. Stocco DM, Clark BJ. Role of the steroidogenic acute regulatory protein (StAR) in steroidogenesis. Biochem Pharmacol. 1996;51:197–205
  38. Stocco DM, Clark BJ. Regulation of the acute production of steroids in steroidogenic cells. Endocr Rev. 1996;17:221–244
  39. Stocco DM. The steroidogenic acute regulatory (StAR) protein two years later. An update. Endocrine. 1997;6:99–109
  40. Devoto L, Kohen P, Vega M, Castro O, González RR, Retamales I, et al. Control of human luteal steroidogenesis. Mol Cell Endocrinol. 2002;186:137–141
  41. Yazawa T, Inaoka Y, Okada R, Mizutani T, Yamazaki Y, Usami Y, et al. PPAR-gamma coactivator-1alpha regulates progesterone production in ovarian granulosa cells with SF-1 and LRH-1. Mol Endocrinol. 2010;24:485–496
  42. Li MY, Yuan H, Ma LT, Kong AW, Hsin MK, Yip J, et al. Roles of PPAR{alpha} and PPAR{gamma} in the development of non-small cell lung cancer. Am J Respir Cell Mol Biol. 2010;[Epub ahead of print]
  43. Yan KH, Yao CJ, Chang HY, Lai GM, Cheng AL, Chuang SE. The synergistic anticancer effect of troglitazone combined with aspirin causes cell cycle arrest and apoptosis in human lung cancer cells. Mol Carcinog. 2010;49:235–246
  44. Shim J, Kim BH, Kim YI, Kim KY, Hwangbo Y, Jang JY, et al. The peroxisome proliferator-activated receptor gamma ligands, pioglitazone and 15-deoxy-Delta(12,14)-prostaglandin J(2), have antineoplastic effects against hepatitis B virus-associated hepatocellular carcinoma cells. Int J Oncol. 2010;36:223–231
  45. Park SH. Current status of liver disease in Korea: nonalcoholic fatty liver disease. Korean J Hepatol. 2009;15:34–39
  46. Milkevitch M, Beardsley NJ, Delikatny EJ. Phenylbutyrate induces apoptosis and lipid accumulations via a peroxisome proliferator-activated receptor gamma-dependent pathway. NMR Biomed. 2010;23:473–479
  47. Bruedigam C, Eijken M, Koedam M, van de Peppel J, Drabek K, Chiba H, et al. A new concept underlying stem cell lineage skewing that explains the detrimental effects of thiazolidinediones on bone. Stem Cells. 2010;28:916–927
  48. Chen Q, Sun X, Chen J, Cheng L, Wang J, Wang Y, et al. Direct rosiglitazone action on steroidogenesis and proinflammatory factor production in human granulosa-lutein cells. Reprod Biol Endocrinol. 2009;7:147

 M.F. has nothing to disclose. E.M.E. has nothing to disclose. F.P. has nothing to disclose. N.C.C. has nothing to disclose. M.T. has nothing to disclose. A.B.M. has nothing to disclose.

 Supported by Agencia Nacional de Promoción Científica y Tecnológica (grants PICTR 32529/05 and PICT 949/06) and the PIP 185 from Consejo Nacional de Investigación Científica y Técnica.

PII: S0015-0282(10)02209-0

doi:10.1016/j.fertnstert.2010.07.1083

Fertility and Sterility
Volume 95, Issue 1 , Pages 333-337, January 2011

Article Tools

* Image Usage & Resolution