in vitro fertilization ( ivf ) involves retrieval of oocytes by super - ovulation which is achieved by exogenously supplementing follicle stimulating hormone ( fsh ) and stimulating the ovaries to produce multiple follicles . several pathways have been postulated for the gonadotropin - induced meiotic maturation ; all of which ultimately function to enhance synthesis of stimulatory factor ( s ) for induction of nuclear and cytoplasmic maturation of oocytes . involvement of steroids such as estradiol and testosterone in the regulation of meiotic maturation in conjunction with gonadotropins , although contemplated , still remains unclear . an earlier study had cited that oocyte priming with estrogen is necessary to develop ca oscillations during maturation . based on the corollary , that oocytes are particularly sensitive to steroids while still at the germinal vesicle stage ; it was speculated that any imbalance in the estrogen - to - androgen ratios in vivo may cause an abnormal ca response thus affecting fertilization . dehydroepiandrosterone ( dhea ) , the most abundant circulating steroid hormone in humans , may be presumed to play an immensely significant role in this context . since dhea is converted to testosterone in ovarian connective tissue ( theca / stroma ) and is subsequently processed by the granulosa cells to form estradiol , it assumes the prohormone status of a predominant endogenous precursor and a metabolic intermediate in ovarian follicular steroidogenesis . dhea may thus be considered a key molecule at the crossroads , maintaining a critical balance between androgen and estrogen production on one hand and affecting oocyte maturation on the other . however , earlier studies with serum / follicular fluid ( ff ) dhea levels in nonpolycystic ovary syndrome ( pcos ) women have provided contradictory results . dhea is reversibly catalyzed by sulfotransferase ( sult2a1 ) to its sulfate ester dhea sulfate ( dhea - s ) . most dhea in blood is found as dhea - s with levels that are about 300 times higher than those of free dhea . whereas dhea levels naturally reach their peak in the early morning hours , dhea - s levels show no diurnal variation . thus from a practical point of view , measurement of dhea - s seems more preferable than dhea since its level remains stable . dhea - s is also the most abundant steroid in follicular fluid of preovulatory follicles being present at micromolar levels . although a few in vitro studies indicate the utilization of dhea - s by granulosa cells via sulfatase for androgen and estrogen production ; no study has as yet been able to obtain a correlation of ff dhea - s levels with fertilization and pregnancy outcome . hence , we attempted to investigate if levels of ff dhea - s correlate with the retrieval of mature oocytes possessing the competence to fertilize , effect early embryonic development , and produce a viable pregnancy in non - pcos women undergoing ivf cycles . a prospective cohort study of women undergoing ivf cycles ( n = 521 ) was carried out at our academically affiliated private infertility clinic from january 2011 to april 2014 . the study protocol was approved by our own hospital ethical committee . no approval from any other ethical committee was required since the procedures did not involve animals or any invasive techniques in humans . all estimations were carried out in ff , which is easily obtained during ovum pick - up ( opu ) , a basic step of ivf treatment ; or in serum samples obtained from blood collected by simple venipuncture . dhea - s levels were estimated in pooled ff by a radio - immuno - assay ( ria ) method using diagnostic kits obtained from beckman coulter ( immunotech im0729 , analytical / functional sensitivity 2.64 g / dl , the intra - assay and interassay coefficient of variations 4.93% and 9.32% , respectively ) . correspondingly , ff e2 levels were also measured using beckman coulter immunotech ria kit ( a21854 , analytical / functional sensitivity 9.58/13.11 pg / ml , the intra - assay and interassay coefficient of variations 14.4% and 14.5% , respectively ) . for assay purposes , only the original follicular aspirate was collected ( even if oocyte was retrieved in the flush ) from all follicles > 16 mm in size and from which oocyte had been retrieved . for each patient per cycle , ff so obtained from all follicles was mixed in equal volume and centrifuged at 3000 g for 15 min to remove blood contaminant / cellular elements and the clear supernatant was used for further assay and analysis . all women were endocrinologically normal , non - pcos and age - matched ( age range 2535 years ) normal responders ( based on their baseline serum amh levels and/or antral follicle count ) . cycles , where intra - cytoplasmic sperm injection had to be performed due to severe male factor , were excluded so as to avoid any bias in fertilization outcome resulting from compromised sperm quality . although ff dhea - s levels were measured in total fertilization failure ( tff ) cycles ( n = 29 ) , these cycles were obviously not included while comparing pregnancy rates . therefore , clinical pregnancy and live birth rates were analyzed in n = 492 cycles . all cycles involved standard stimulation protocol with recombinant fsh ( gonal f , merck - serono , india ) from day 2 and daily administration of antagonist from day 6 until the day of the trigger with injection hcg . transvaginal ultrasound - guided oocyte retrieval under patient sedation was done between 34 and 36 h after hcg administration . conventional insemination was done after oocyte incubation , and morphological assessment of oocyte quality was based on the appearance of coc complex . 1618 h postinsemination , the metaphase ii ( mii ) status of oocytes was ascertained and fertilization rate assessed . on day 1 post - opu , the presence of one extruded polar body indicated mii status of oocyte whereas the presence of two polar bodies and two pronuclei confirmed fertilization . embryo transfer ( et ) involved transcervical transfer of not more than 2 top / good quality day 5 blastocysts using cooks et catheter . post transfer , the luteal phase was supported by the administration of micronized progesterone 100 mg daily ( susten , sun pharma , india ) until day 14 of et . serum -hcg concentration > 50 miu / ml recorded on day 14 of et was considered as a positive indicator of pregnancy . inter - comparison of various parameters were performed between pregnant ( n = 111 ) and nonpregnant groups ( n = 381 ) excluding the tff cycles . the patients were then classified into low ( < 760 ng / ml ) , medium ( 7601850 ng / ml ) , and high ( > 1850 ng / ml ) ff dhea - s groups on the basis of 25 and 75 percentiles of ff dhea - s concentration . fertilization rate , embryo development to blastocyst stage and live - birth rate were the main outcome measures . statistical analysis was done using graphpad prism v software ( infact , graph pad software is the company ) . chi - square test , student 's t - test , one - way anova , pearson correlation and odds ratio ( or ) were calculated as applicable to obtain statistical significance . all patients included in the study were age matched and did not differ significantly w.r.t age either in pregnant versus nonpregnant ( 30.21  0.27 vs. 30.80  0.17 years ; p = 0.09 ) or in low , medium and high groups ( 31.44  0.28 vs. 31.0  0.2 vs. 30.73  0.3 , p = 0.10 ) . similarly , all women were non - pcos with insignificant differences in their baseline serum amh levels ( 2.83  0.24 vs. 2.61  0.19 ng / ml ; p = 0.12 ) . overall live birth rate achieved was 22.56% ( 111/492 ) . inter - comparison of the basic and embryological parameters between pregnant and nonpregnant groups is depicted in table 1 . significantly higher levels of ff dhea - s ( 1599  77.45 vs. 1372  40.47 ng / ml ; p = 0.01 ) and ff e2 ( 244,500  54,090 vs. 105,500  11,490 pg / ml ; p = 0.016 ) were observed in pregnant as compared to non pregnant group . interestingly , cycles involving tff evinced significantly lower levels of ff dhea - s as compared to remaining cycles in which fertilization occurred ( 686.7  52.76 vs. 1423  36.10 ng / ml ; p < 0.0001 ) . these findings prompted us to further classify the cycles depending upon 25 and 75 percentile value into low , medium , and high ff dhea - s groups . we analyzed the data within these three groups after including as well as excluding the tff cycles . on including the tff cycles , although low ( n = 131 ) , medium ( n = 256 ) , and high ( n = 134 ) groups were similar in terms of age ( 31.44  0.28 vs. 31  0.2 vs. 30.73  0.30 , p = 0.10 ) , day of hcg e2 levels ( 1880  63.45 vs. 2160  72.38 vs. 2320  77.58 , p = 0.32 ) and number of eggs retrieved ( 1112 vs. 2550 vs. 1424 , p = 0.55 ) ; they displayed highly significant differences with respect to number / percent of mii oocytes ( 799 [ 71.85% ] vs. 2115 [ 82.94% ] vs. 1303 [ 91.50% ] , p = 0.0003 ) , number of oocytes fertilized / percent ( 714 [ 64.21% ] vs. 1831 [ 71.80% ] vs. 1123 [ 78.86% ] , p < 0.0001 ) , number of embryos cleaved / percent ( 663 [ 59.62% ] vs. 1706 [ 66.90% ] vs. 1049 [ 73.67% ] , p = 0.0039 ) , and number of blastocysts formed / percent ( 270 [ 24.28% ] vs. 815 [ 31.96% ] vs. 529 [ 37.15% ] , p < 0.0001 ) , respectively . number of tff cycles were significantly higher in the low group compared to medium and high ff dhea - s groups ( 16 [ 12.21% ] vs. 13 [ 5.08% ] vs. 0 [ 0% ] p = 0.0002 ) . inter - comparison of various parameters between pregnant and nonpregnant groups after excluding the tff cycles , there was a highly significant rise in embryo development rates from low ( n = 115 ) to medium ( n = 243 ) to high ( n = 134 ) ff dhea - s groups [ table 2 ] . ff dhea - s levels correlated significantly with percentage of mii oocytes ( pearson r = 0.41 [ 95% confidence interval ( ci ) : 0.340.48 , p < 0.0001 ] ) and fertilization rates ( pearson r = 0.35 { 95% ci : 0.270.42 , p < 0.0001 } ) . or and relative risk ( rr ) of oocytes remaining unfertilized is presented in figure 1 . clinical pregnancy and live birth rates were significantly higher in the high group as compared to both medium and low groups [ figure 2 ] . inter - comparison of data between low , medium and high ff dhea - s groups odds ratio and relative risk in low , medium , high follicular fluid dehydroepiandrosterone sulfate groups clinical pregnancy and live birth rates in low , medium , and high follicular fluid dehydroepiandrosterone sulfate groups our study for the first time evaluates ff dhea - s levels and emphasizes its significance in the process of oocyte maturation and its competence to fertilize . a higher fraction of mii oocytes and subsequently higher fertilization and embryo developmental rates in the high ff dhea - s group over the other two groups obtained in our study justifies our contention . although sulfonated steroids have been predominantly regarded to function as inactive storage reservoirs for steroid hormones ; it may be speculated that the sulfate group of dhea - s plays a role in this maturation process . indeed , in a recent study neunzig and bernhardt , 2014 , noted that dhea - s stringently regulates the first step of steroidogenesis and surmised that the negatively charged sulfate group of dhea - s upregulates the catalytic activity of enzyme cyp11a1 by 75% which binds with higher efficiency to substrate cholesterol to display a 26% increase in pregnenolone formation in presence of dhea - s but not in presence of dhea . earlier , liu and dillon , 2002 had identified a putative membrane receptor for dhea / dhea - s suggesting that they may possess some physiological role . these studies and our own results indicate that the contribution of dhea - s in the process may be more substantial than dhea . therefore , our initial contention that dhea - s may bear a more significant role than just being a reservoir for dhea molecule holds substance . this may probably also be the reason why previous studies obtained contradictory results with dhea . our results demonstrate a concomitant raised ff e2 level along with higher ff dhea - s levels in the pregnant and high groups as against lower levels observed in the non pregnant and medium / low ff dhea - s groups . this result corroborates the relevant role of e2 in the fertilization process and in stipulating endometrial receptivity . at the same time , it may be noted that our results attribute a more significant role for dhea - s than e2 in this process . our findings display a significantly higher fertilization rate and subsequent enhancement in embryo development rates up to the blastocyst stage in the high ff dhea - s group . most importantly , our study denotes that higher fertilization rates correlate more significantly with ff dhea - s ( pearson r = 0.35 , p < 0.0001 ) than with ff e2 ( pearson r = 0.12 , p = 0.03 ) . these results emphasize that dhea - s may act as the primary regulator of estrogen formation . indeed , there are reports and pathways indicating direct utilization of dhea - s as a precursor for estrogen production . it has earlier been documented that an abnormal ca response during fertilization consequently impairs developmental potential of the resulting embryo . dhea - s , owing to its conversion to estriol seems to aid in the generation of ca wave necessary for oocyte maturation . this study , therefore , marks dhea - s as a primary entity in the overall process of oocyte maturation . the exact mechanism by which dhea - s initiates this process still needs to be elucidated . nevertheless , a strong correlation of ff dhea - s levels with the attainment of mii stage oocytes as well as the significantly lower odds / rr of fertilization failure [ figure 1 ] with high ff dhea - s levels obtained in our study validate its involvement in this process . finally , significantly higher embryo development rates presently observed not just at cleavage stage , but also at blastocyst stage leading to significantly higher live birth rates in the high ff dhea - s group establishes ff dhea - s as a robust marker for oocyte competence . ff dhea - s level influences the oocyte maturation process and is predictive of fertilization , embryo development to the blastocyst stage and live birth rates in non - pcos women undergoing conventional ivf cycles .
aim : to investigate if the level of dehydroepiandrosterone sulfate ( dhea - s ) in follicular fluid ( ff ) influences the competence of oocytes to fertilize , develop to the blastocyst stage , and produce a viable pregnancy in conventional in vitro fertilization ( ivf ) cycles.settings and design : prospective study of age - matched , nonpolycystic ovary syndrome ( pcos ) women undergoing antagonist stimulation protocol involving conventional insemination and day 5 blastocyst transfer.materials and methods : ff levels of dhea - s and e2 were measured by a radio - immuno - assay method using diagnostic kits . 
 fertilization rate , embryo development to the blastocyst stage and live birth rate were main outcome measures . 
 cycles were divided into pregnant / nonpregnant groups and also into low / medium / high ff dhea - s groups . 
 statistical analysis was done by graphpad prism v software.results:ff dhea - s levels were significantly higher in pregnant ( n = 111 ) compared to nonpregnant ( n = 381 ) group ( 1599  77.45 vs. 1372  40.47 ng / ml ; p = 0.01 ) . 
 high ( n = 134 ) ff dhea - s group had significantly higher percentage of metaphase ii ( mii ) oocytes ( 91.5 vs. 85.54 vs. 79.44% , p < 0.0001 ) , fertilization rate ( 78.86 vs. 74.16 vs. 71.26% , p < 0.0001 ) , cleavage rate ( 83.67 vs. 69.1 vs. 66.17% , 
 p = 0.0002 ) , blastocyst formation rate ( 37.15 vs. 33.01 vs. 26.95% , p < 0.0001 ) , and live birth rate ( 29.85 vs. 22.22 vs. 14.78% , p = 0.017 ) compared to medium ( n = 243 ) and low ( n = 115 ) ff dhea - s groups , respectively despite comparable number of oocytes retrieved and number of blastocysts transferred . 
 ff dhea - s levels correlated significantly with the attainment of mii oocytes ( pearson r = 0.41 ) and fertilization rates ( pearson r = 0.35).conclusion : ff dhea - s level influences the oocyte maturation process and is predictive of fertilization , embryo development to the blastocyst stage and live birth rates in non - pcos women undergoing conventional ivf cycles .