Gynecological Endocrinology, 2012; 28(9): 669673 2012 Informa UK, Ltd.

. ISSN 0951-3590 print/ISSN 1473-0766 online DOI: 10.3109/09513590.2012.705386

ART

 HEA supplementation improves follicular microenviroment D in poor responder patients

Paolo Giovanni Artini1, Giovanna Simi1, Maria Ruggiero1, Sara Pinelli1, Olga Maria Di Berardino1, Francesca Papini1, Sara Papini2, Patrizia Monteleone3 & Vito Cela1
1Department of Reproductive Medicine and Child Development, Division of Obstetrics and Gynaecology, University of Pisa,

Pisa, Italy, 2Department of Economics, University of Pisa, Pisa, Italy, and 3S. Francesco Hospital, Barga, Italy

Objective: To analyze the eect of dehydroepiandrosterone (DHEA) supplementation on follicular microenvironment and on in vitro fertilization (IVF) outcomes among poor responder patients. Study design: We enrolled 24 patients diagnosed as poor responders based on ESHRE consensus criteria. One group received 25mg/die three times daily of DHEA supplementation for 3 months previous to IVF cycle, while the other did not receive any treatment. COH was performed with rFSH and hMG, and a GnRH antagonist was administered according to a exible protocol. We evaluated perifollicular vascularization of recruited follicles through power Doppler blood ow analysis and follicles were graded as described by Chui et al. Follicular uids (FF) from F3-F4 follicles were collected, and FF levels of vascular endothelial growth factor (VEGF) and hypoxic inducible factor1 (HIF1) were measured. Results: FF levels of HIF1 were statistically signicant lower in women treated with DHEA (14.76 51.13 vs. 270.03 262.18 pg/ml; p = 0.002). On the contrary, VEGF levels did not dier between the two groups. Concerning COH, in the DHEA-group the mean duration of treatment was signicantly shorter (9.83 1.85 vs. 12.092.81; p = 0.023). Total numbers of oocytes retrieved, fertilized oocytes, good quality embryos, number of transferred embryos and clinical pregnancies tended to be higher in study group, but the results were not signicant. On the other hand, considering the oocytes retrieved in selected F3-F4 follicles, there was a relation between HIF1 levels and oocytes quality. In fact, mature oocytes retrieved in selected follicles were signicantly more numerous in DHEA-group (0.50 0.52 vs. 0.080.29; p = 0.018). Conclusions: The improvement of reproductive parameters after DHEA supplementation in poor responders may be explained through the eect that this prohormone exerts on follicular microenvironment. Keywords: Dehydroepiandrosterone, DHEA, infertility, poor responders, IVF

Introduction
Poor ovarian response (POR) implies a reduced follicular pool and results in a low number of oocytes retrieved at pick up or a low number of developing follicles and low estradiol (E2) levels, despite the high dose of gonadotropins administered during COH [1]. Notwithstanding the advancement in assisted reproductive technologies (ART), POR is still considered one of the most troublesome tasks in reproductive field. Its incidence has

been reported from 9 to 24%, and its estimate prevalence is higher than 50% in patients over 40 years [2,3]. Albeit with limited success, several strategies have been evaluated in these patients, and the use of DHEA prior to IVF cycles have been suggested as an hopeful chance [4]. DHEA and its sulphate ester DHEA-S are the major 5androgens secreted by the conversion of cholesterol primarily by the adrenal cortex. Numerous hypotheses have been made on how DHEA may enhance fertility. First, since DHEA is peripherically converted into E2 and testosterone, it has been supposed that DHEA supplementation may improve the substrate pool for ovarian follicular steroidogenesis [5]. Androgens may act on ovarian follicular development also by increasing the number of FSH receptors expressed in the granulosa cells [6,7], thus stimulating early stages of follicular growth [8,9]. Besides, DHEA seems to increase follicular insulin-like growth factor-I concentration, which can promote the gonadotropin effect by 150%, probably independently of changes in GH secretion [10]. In rat models, DHEA has also shown to create a polycystic environment in the ovaries, with increased levels of active oocytes and decreased atretic effects [8,10]. Plasma concentrations of DHEA remain high during female reproductive life and progressively fall by approximately 2% per year [11,12], leading to the hypothesis that supplementation with DHEA may slow down aging process. Nevertheless, studies have shown the beneficial effect of DHEA administration on vascular function. In fact, DHEA increases vascular endothelial proliferation, migration and vascular tube formation, and nitric oxide synthesis at physiological levels [13]. This effect can be very important in the female reproductive system, considering that ovarian folliculogenesis is accompanied by a very finely regulated angiogenesis [14]. VEGF is a molecule produced by follicular granulose and ovarian thecal cells in response to gonadotropins stimulation [15]. Indeed, VEGF is implied in endothelial sprouting, enhanced vascular permeability, expression of tissue matrix metalloproteinases, and finally in the digestion of matrix, required for the endothelial cells to move [16]. Among its function, VEGF has the role of primary mediator of the formation of a vascular network in the thecal cell layer of the follicle [16,17]. Van Blerkom et al.[18] observed higher VEGF levels in follicles with higher dissolved oxygen contents and with increased blood

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Correspondence: Paolo Giovanni Artini, Department of Reproductive Medicine and Child Development, Division of Obstetrics and Gynaecology, University of Pisa, Pisa, Italy. Tel.: +39.335.349967. E-mail: paolo.artini@med.unipi.it

669

670 P. G. Artini et al. flow observed at Doppler, but they failed to find a specific association between follicular VEGF levels and the presumed extent of perifollicular vascularization. A similar conclusion was obtained by Barroso et al. and Battaglia et al., even if FF levels of VEGF in poor responders patients were found higher than in normo-responders [19,20]. Besides, Kan and associates more recently did not show any difference in FF VEGF concentrations among poor responders with and without high-grade perifollicular vascularity [21]. As a consequence, other angiogenic factors might be involved in the process of cellular reaction to hypoxia, acting synergistically or additionally with VEGF, and actually follicular oxygen content seems not to be predictable only by US. Transcriptional upregulation of VEGF is stimulated by HIF1, during cellular adaptation to hypoxia. HIF1 is a transcription factor sensible to low oxygen tension, which prevents fatal depletion of oxygen and subsequent cell death. As a consequence, FF concentrations of this factor are linked by an inverse correlation the available oxygen, being involved in a certain way in the determination of oocyte developmental competence. In the light of these findings, the purpose of this study was to analyze the effect of DHEA supplementation firstly on follicular microenvironment, evaluating follicular fluid VEGF and HIF1 concentrations and, secondly, on IVF outcomes. hCG injection, transvaginal follicular aspiration was performed for oocyte pick-up. The perifollicular vascularity of recruited follicles in patients ovaries was estimated immediately prior to oocyte retrieval through power Doppler blood flow analysis (GE medical system Logic). Follicles were graded according to the percentage of follicular circumference in which most flow was identified from a single cross-sectional slice as described by Chui et al.[22] The grading system was as follows: <25% follicular circumference in which blood flow was identified (F1), 2650% (F2), 5175% (F3), 76100% (F4). In each patients in both groups we selected two first follicles per ovary with perifollicular vascularity F3 or F4. These two follicles were the first to be drawn at oocyte pick up, and corresponding FF was centifuged and stored at 20C until VEGF and HIF1 assay. VEGF and HIF1 FF levels were measured by ELISA (Endogen Human VEGF ELISA Kit, Pierce Biotechnology, Inc., Rockford; Human/Mouse Total HIF-1 Immunoassay, R&D Systems Europe, Ltd, UK). IVF-embryo transfer (ET) or IVF/ICSI-ET were performed as appropriate, in relation to semen quality. The maturity and potential fertilization of the inseminated oocytes were assessed as well as the quality of the deriving embryos according to the method described by Veeck [23] by a single expert embryologist, 24h and 48h after oocytes insemination. ET was performed on day 2 or 3 under the guidance of abdominal US, using a K-Soft 500 Embryo Transfer Catheter (Cook, Ireland Ltd.). Serum -hCG concentrations were determined 12 days after ET, and if positive (>5 mUI/ml), the test was repeated one week later. Clinical pregnancy was confirmed if a fetal heartbeat was observed by transvaginal US. All patients had luteal phase support with daily vaginal progesterone (Crinone gel 8%, Merck-Serono, Italia) and intramuscolar progesterone every 72 h (Lentogest, AMSA S.r.l., Italia); the therapy was started from the day of the ET and continued until either a serum pregnancy test result was negative or gestational sac was confirmed on US. For patients with a positive pregnancy test, progesterone was continued until the 12th week of pregnancy. The primary outcome measures were HIF1 and VEGF concentrations in FF and the number of mature oocytes among the corresponding retrieved oocytes. Secondary outcome measures were number of retrieved oocytes, mature oocytes, fertilized oocytes, good quality embryos, transferred embryos and clinical pregnancies. Results were expressed as mean SD. Between-group differences were evaluated by means of students t-test. A p value of <0.05 was considered statistically significant.

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Materials and methods

This controlled, randomized study enrolled 24 patients, all diagnosed as poor responders based on the Bologna criteria for the definition of poor response, established during ESHRE consensus in 2010 [3]. All selected patients were aged between 31 and 42 years. The study was approved by the Ethical Committee of Pisa University and was performed according to rules of good clinical practice. Informed consent was obtained from each patient. Patients were divided in two groups by randomly choosing sealed, opaque, sequentially numbered envelopes. One group of 12 patients received DHEA supplementation orally, 25mg t.i.d., for 3 months before IVF cycle (study group). The other group of 12 patients did not receive any treatment (control group). All patients received combined oral contraceptives for a month before starting COH, in order to schedule menstrual cycle. COH was carried out in an identical manner in the two groups, with 150450 IU of recombinant FSH (Gonal F pen MerckSerono, Italia) and 150375 IU of highly purified urinary gonadotropine (Meropur fl 75UI, Ferring Italia, hMG) from the day 2 or 3 of the cycle, according to basal FSH levels, age and antral follicle count (AFC). All patients were monitorized with serum E2 and Progesterone assay and transvaginal ultrasonography (US). Daily injections of a GnRH antagonist (Cetrotide 0.25 mg sc, Merck-Serono, Italy) were administered to prevent premature ovulation by using the flexible antagonist protocol, according to a personalized regimen, when the leading follicle reached 14mm in diameter until the day of hCG injection. Measurements were performed with the Elecsys immunoanalyzer (Roche Diagnostics, Manheim, Germany). With this method, intra- and inter-assay coefficients of variation were <3 and 4% for LH, <3 and 6% for FSH, <5 and 10% for E2 and <3 and 5% for progesterone, respectively. Recombinant hCG (Ovitrelle 250 mcg/0.5 ml, MerckSerono, Italy) was administered when a consistent rise in serum E2 concentrations was associated with the presence of two or more follicles >18mm in diameter. After approximately 36h after

Results
The study was conducted between July 2010 and February 2011 at the Department of Reproductive Medicine and Child Development, University of Pisa. There were no differences between treated and non-treated group regarding mean age, BMI, duration of infertility, baseline serum levels of FSH and DHEAS. Androstenedione levels resulted lower in controls, but all values were in the range accepted as normal by our laboratory (see Table I). All selected follicles showed a homogeneous vascularity grading, either at least F3-F4 upon power Doppler evaluation.

Gynecological Endocrinology

DHEA for poor responders671

Table I. Baseline data [mean SD]. Study group Control group Normal range AGE of female 36.58 3.32 37.00 4.61 partner (y) AGE of male 38.1 4.8 38.3 7.3 partner (y) 21.7 3.1 21.6 3.1 Body mass index (kg/m2) Duration of 4.6 3.5 5.0 4.2 infertility (y) BASAL FSH 5.78 1.45 6.68 1.79 2.012 (mU/ml) BASAL DHEAS 4.12 2.12 2.63 2.22 1.23.6 (g/ml) BASAL 2.54 0.01 1.98 0.56 0.23.1 Androstenedione (ng/ml) Gynecol Endocrinol Downloaded from informahealthcare.com by 189.167.75.245 on 11/20/12 For personal use only. p value 0.792 0.938 0.853 0.619 0.175 0.093 0.001 Figure 1. Comparison of follicular fluid HIF-1 levels in study group and control group (pg/ml). Table II. Study and control outcomes. Unless otherwise indicated, data are expressed in means (SD). E2, Estradiol; P, Progesterone. DHEA group Control group (n = 12) (n = 12) 648.00 303.30 1154.50 860.14 1770.25 1125.96 1535.56 429.13 9.83 1.85 12.09 2.81 241.67 101.32 187.50 75 2844.17 1412.98 1881.25 1244.95 0.08 0.29 748.29 503.02 270.03 262.18 2.67 2.50 0.92 1.31 1.25 1.14 1.00 1.21 1.25 1.14 0.17 0.39 p Value 0.056 0.488 0.023 0.444 0.54 0.859 0.555 0.02 0.997 0.002 0.39 0.853 0.255 0.754 0.45 0.62

FF levels of HIF1 were statistically significant lower in women treated with DHEA (14.76 51.13 vs. 270.03 262.18 pg/ml; p = 0.002) (Figure 1). VEGF levels did not differ between the two groups (749.08 474.31 vs. 748.29 503.02 pg/ml; p = 0.997). Besides, gonadotropin requirements during COH were slightly lower in DHEAtreated group, although it was not statistically significant. However, in the DHEA-group the mean duration of stimulation was significantly shorter (9.83 1.85 vs. 12.092.81; p = 0.023). Total numbers of oocytes retrieved, fertilized oocytes, good quality (I-II) embryos, transferred embryos and clinical pregnancies per cycle tended to be higher in study group, but the results (Table II) were not significant. On the other hand, considering oocytes retrieved in selected F3-F4 follicles, there was a correlation between HIF1 levels and oocytes quality. In fact, mature oocytes retrieved in selected follicles were significantly higher in DHEA-group (0.50 0.52 vs. 0.080.29; p = 0.02). Only one patient did not complete the cycle and was suspended because of insufficient response to COH, and she belonged to control group. The supplementation was well tolerated by all patients. Some women reported increased sebum production and a few developed transitional hirsutism, but no patient dropped out of the study because of side effects attributed to the treatment.

Discussion
Oocyte quality and embryo development have been hypothesized to be depending on the intrafollicular oxygen levels, that determine the adequate formation and stabilization of meiotic spindle. An adequate perifollicular vascularity is crucial in the establishment of oocyte developmental potential, but studies in literature demonstrate that perifollicular blood flow is not strictly related with intrafollicular oxygen content [20,21]. In the light of this fact, in this study we selected follicles with high-grade vascularity, focusing our attention only on the analysis of follicular fluids concentration of two key molecules in the process of follicular oxygenation, VEGF and HIF1. In our study, VEGF FF levels were comparable between the two groups. Nevertheless, data in literature did not observe any specific association between VEGF FF concentrations and presumed extent of perifollicular angiogenesis [1921]. Thus, this result should not be read as a lack of effect of the treatment. On the contrary, HIF1 concentrations have been found significantly lower in FF of patients treated with DHEA if compared to the control group. Even if more studies are needed to elucidate
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E2 on hCG day (pg/ml) P on hCG day (pg/ml) Duration of stimulation (days) Initial dose rFSH (UI) 272.92 104.15 Initial dose hMG (UI) 168.75 79.15 Total dose of rFSH (UI) 2756.25 1073.66 Total dose of hMG (UI) 1637.50 787.15 No. of mature oocytes 0.50 0.52 retrieved in selected follicles (F3-F4) Follicular fluid VEGF 749.08 474.31 levels (pg/ml) Follicular fluid HIF-1 14.76 51.13 levels (pg/ml) No. of oocytes retrieved 3.58 2.84 No. of mature oocytes 0.83 0.94 retrieved No. of oocytes fertilized 1.75 1.06 Good quality embryos 1.17 1.47 (I-II) No. of embryos transferred 1.58 1.08 Clinical pregnancies per 0.25 0.45 cycle

the correlation between perifollicular blood flow and intrafollicular homeostasis, as a consequence of the significant reduction of HIF1 in follicular fluid we can suppose that the quality of follicular environment may be improved by supplementation with DHEA 75mg orally daily for 3 months before COH, as HIF1 represents a key molecule in the reaction of cells to hypoxia. For what concerns the actions of DHEA supplementation on poor responder patients, Casson and associates firstly speculated on a possible improvement of ovarian function following exogenous administration of DHEA in a case series involving women with a history of POR [10,24]. Few years later, Barad and Gleicher reported an increase oocyte yeld, a higher fertilization rate and higher embryo grade in women pre- and post-treatment [25,26]. The following year, the same authors published a study on patients with premature ovarian aging, who received DHEA for at least 1 month, and they reported a reduction in aneuploidy,

672 P. G. Artini et al. a lower cancellation rate and a better clinical pregnancy rate (PR), compared to control group [27]. In a subsequent observational study, they also demonstrated a significantly higher clinical PR [28] and a lower miscarriage rate in DHEAtreated patients, compared to the rate in the National US IVF database [29]. More recently, Mamas and Mamas published a case series in patients with premature ovarian failure. DHEA supplementation led to regular periods and to a decrease in serum FSH concentrations, with one spontaneous pregnancy during the treatment [30]. In the same year, Sonmezer and colleagues compared IVF outcomes in patients treated with DHEA with the parameters of the previous, failed, cycle of stimulation [31]. They described a significant improvement in number of follicles recruited, oocytes retrieved, MII oocytes and day-3 high-quality embryos, as well as a statistically significant improvement in cPR. The first randomized prospective, controlled study regarding supplementation with DHEA before IVF in poor responders was conducted by Wiser and associates [32], who showed a significantly higher cumulative live birth rate among the study group. Our finding of an improved response to COH in poor responders after DHEA supplementation is in line with the cited data [2529,32,33]. The lack of significance of some results may ensue in part from the limited number of patients enrolled and partly from the wide range of factors influencing the mechanisms of implantation and subsequent pregnancy establishment. As far as we are aware, this is the first time that HIF1 levels are analyzed in FF of patients undergoing IVF. The improvement of biological reproductive parameters after DHEA supplementation may be explained through the effect that this pro-hormone exerts on follicular microenvironment. Women undergoing DHEA treatment might deal with possible virilizing side effects, that appear minimal with the therapeutic dose of 75 mg/day [34]. Notwithstanding, no patients left our study as a consequence of side effects. It is noteworthy that the observation on improving oocyte microenvironment could be useful to provide an augmented chance of success of IVF cycles. Even though couples should be informed of the fact that DHEA is used an experimental treatment option, it should be considered in the light of the available data as a potential resort for poor responder patients, first of all because of the limited number of options in this group of patients, and furthermore by virtue of the high compliance to its use and the low incidence of side effects. Declaration of Interest: The authors report no declarations of interest.

References
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Conclusions
More studies are needed to elucidate the correlation between perifollicular vascularization and paracrine/autocrine factors involved in intrafollicular homeostasis. In the same way, how changes in FF may alter the developmental capacity of the oocyte and embryos is still to be clarified. Although, the fact that levels of HIF1 are significantly lower in FF of patients treated with DHEA authorize to conjecture that the improvement of reproductive parameters may be a consequence of the effect that this pro-hormone exerts on follicular microenvironment, as this molecule represents a marker of cellular adaptation to hypoxia. Our study group is limited. Nevertheless, despite the small size, the differences between the groups are well marked. Hence, it seems obvious that DHEA represents a useful option for the treatment of a large number of women who are among the foremost challenging dilemma for IVF clinicians. In addition to the possible benefits for fertility outcome, DHEA opens the possibility of choosing a milder and more cost-effective hormonal protocol. Without supplementation with DHEA, specialists would be forced to use heavy hormonal doses, without any certainty of optimal response.

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19. Barroso G, Barrionuevo M, Rao P, Graham L, Danforth D, Huey S, Abuhamad A, Oehninger S. Vascular endothelial growth factor, nitric oxide, and leptin follicular fluid levels correlate negatively with embryo quality in IVF patients. Fertil Steril 1999;72:10241026. 20. Battaglia C, Genazzani AD, Regnani G, Primavera MR, Petraglia F, Volpe A. Perifollicular Doppler flow and follicular fluid vascular endothelial growth factor concentrations in poor responders. Fertil Steril 2000;74:809812. 21. Kan A, Ng EH, Yeung WS, Ho PC. Perifollicular vascularity in poor ovarian responders during IVF. Hum Reprod 2006;21:15391544. 22. Chui DK, Pugh ND, Walker SM, Gregory L, Shaw RW. Follicular vascularitythe predictive value of transvaginal power Doppler ultrasonography in an in-vitro fertilization programme: a preliminary study. Hum Reprod 1997;12:191196. 23. Veeck LL. The morphological estimation of mature oocytes and their preparation for insemination. In vitro fertilization-Norfolk. Baltimore: Williams and Wilkins. 1986; In: Jones HW Jr, Jones GS, Hodgen GD, Rosenwaks Z, editors. 24. Casson PR, Lindsay MS, Pisarska MD, Carson SA, Buster JE. Dehydroepiandrosterone supplementation augments ovarian stimulation in poor responders: a case series. Hum Reprod 2000;15:21292132. 25. Barad DH, Gleicher N. Increased oocyte production after treatment with dehydroepiandrosterone. Fertil Steril 2005;84:756. 26. Barad D, Gleicher N. Effect of dehydroepiandrosterone on oocyte and embryo yields, embryo grade and cell number in IVF. Hum Reprod 2006;21:28452849. 27. Gleicher N, Weghofer A, Barad D. Increased euploid embryos after supplementation with dehydroepiandrosterone (DHEA) in women with premature ovarian aging. Fertility and sterility 2007;88:S232. 28. Barad D, Brill H, Gleicher N. Update on the use of dehydroepiandrosterone supplementation among women with diminished ovarian function. J Assist Reprod Genet 2007;24:629634. 29. Gleicher N, Ryan E, Weghofer A, Blanco-Mejia S, Barad DH. Miscarriage rates after dehydroepiandrosterone (DHEA) supplementation in women with diminished ovarian reserve: a case control study. Reprod Biol Endocrinol 2009;7:108. 30. Mamas L, Mamas E. Premature ovarian failure and dehydroepiandrosterone. Fertil Steril 2009;91:644646. 31. Snmezer M, Ozmen B, Cil AP, Ozkavuku S, Tasi T, Olmus H, Atabekoglu CS. Dehydroepiandrosterone supplementation improves ovarian response and cycle outcome in poor responders. Reprod Biomed Online 2009;19:508513. 32. Wiser A, Gonen O, Ghetler Y, Shavit T, Berkovitz A, Shulman A. Addition of dehydroepiandrosterone (DHEA) for poorresponder patients before and during IVF treatment improves the pregnancy rate: a randomized prospective study. Hum Reprod 2010;25:24962500. 33. Gleicher N, Weghofer A, Barad DH. Dehydroepiandrosterone (DHEA) reduces embryo aneuploidy: direct evidence from preimplantation genetic screening (PGS). Reprod Biol Endocrinol 2010;8:140. 34. Kroboth P, Salek F, Pittenger A, Fabian T, Frye R. DHEA and DHEA-S: a review. The Journal of Clinical Pharmacology 1999;39:327348.

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