ORIGINAL ARTICLE: ASSISTED REPRODUCTION
Follicular-phase ovarian follicular fluid and plasma cytokine profiling of natural cycle in vitro fertilization patients N. Ellissa Baskind, M.B.Ch.B.,a Nicolas M. Orsi, M.B.Ch.B., Ph.D.,b and Vinay Sharma, F.R.C.O.G., Ph.D.a a Leeds Centre for Reproductive Medicine, Leeds Teaching Hospitals NHS Trust, Seacroft Hospital; and b Women's Health Research Group, Leeds Institute of Molecular Medicine, St. James's University Hospital, Leeds, United Kingdom
Objective: To characterize follicular fluid (FF) and systemic cytokine profiles at various time points during the natural-cycle follicular and periovulatory phases. Design: Observational clinical study across two consecutive cycles. Setting: Hospital-based in vitro fertilization program. Patient(s): Ten women undergoing modified natural-cycle in vitro fertilization (MNC-IVF). Intervention(s): Plasma and follicular fluid (FF) collection. Main Outcome Measure(s): Forty FF cytokine concentrations from individual follicles and plasma from each patient were determined by fluid-phase multiplex immunoassay in two consecutive cycles: 1) tracking cycle—midfollicular or luteal surge; and 2) treatment cycle—periovulatory (at the time of MNC-IVF). Demographic, cycle, and cytokine data were compared with the use of chi-square, paired-scores t test, or Wilcoxon signed ranks tests. Result(s): Fluctuations in various FF cytokines were evident during the follicular phase: Levels of interleukin (IL) 6 and IL-8 were higher in periovulatory samples, and IL-1 receptor antagonist and vascular endothelial growth factor were elevated earlier in the cycle. Luteal surge profiles were similar to those found in periovulatory samples. Conversely, circulatory cytokine concentrations were more stable during the follicular phase. Conclusion(s): These findings present an extensive physiologic reference profile of FF cytokines associated with antral folliculogenesis and highlight the compartmentalization of sysUse your smartphone temic and intraovarian cytokine networks in natural cycles. (Fertil SterilÒ 2014;-:-–-. to scan this QR code Ó2014 by American Society for Reproductive Medicine.) and connect to the Key Words: Natural cycle, in vitro fertilization, follicular fluid, cytokines, correlations Discuss: You can discuss this article with its authors and other ASRM members at http:// fertstertforum.com/baskindne-follicular-fluid-plasma-cytokine-profiling-nc-ivf/
O
ver the past two decades, it has become apparent that contrary to previously held beliefs, the oocyte does not simply passively accept signals from its surrounding granulosa cells (GCs). It is now evident that oocytes, GCs, theca cells (TCs), and stromal interstitial cells engage in an intricate paracrine dialogue that even
involves neighboring follicles, such that a reciprocal cooperation between these different cell types operates at all stages of follicular growth and/or atresia (1, 2). This multifaceted interaction of mediators governs the development of both the oocyte and somatic cells, as well as determining their response to gonadotropins and
Received January 27, 2014; revised April 4, 2014; accepted April 22, 2014. N.E.B. has nothing to disclose. N.M.O. has nothing to disclose. V.S. has nothing to disclose. Reprint requests: Vinay Sharma, F.R.C.O.G., Ph.D., Leeds Centre for Reproductive Medicine, Leeds Teaching Hospitals NHS Trust, Seacroft Hospital, York Road, Leeds LS14 6UH, United Kingdom (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2014 0015-0282/$36.00 Copyright ©2014 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2014.04.032 VOL. - NO. - / - 2014
discussion forum for this article now.*
* Download a free QR code scanner by searching for “QR scanner” in your smartphone’s app store or app marketplace.
influencing follicular fluid (FF) composition (3, 4). In this manner, a panoply of intrafollicular mediators, including cytokines, chemokines, and protein growth factors (hereafter collectively referred to as cytokines), influence oocyte quality and subsequent embryo viability and support ovulation to the extent that it is increasingly thought that they may affect treatment outcome (5, 6). These agents are produced locally within each follicle in response to the prevailing steroidal milieu and affect the oocyte, both directly and indirectly, through the intermediation of cumulus cells and GCs (7, 8). 1
ORIGINAL ARTICLE: ASSISTED REPRODUCTION Cytokines are small- to medium-size proteins/glycoproteins that act as intercellular mediators and growth factors across a range of immune effector cells, ovarian somatic cells (GCs, TCs, stromal cells), and the oocyte (9, 10). More specifically, intrafollicular cytokines regulate angiogenesis, steroidogenesis, oocyte maturation, leucocyte infiltration and follicle rupture/remodeling during ovulation and, as such, are readily detectable in FF (10–13). Folliculogenesis can be broadly divided into two phases. The first, preantral, phase is gonadotropin independent and characterized by oocyte growth and differentiation, as well as the transition of the primordial follicle to the primary preantral stage. Various cytokines have been implicated in primordial follicle activation and the transition from primary to secondary follicles, including leukemia inhibitory factor (LIF) (14), basic fibroblast growth factor (b-FGF) (15), stem cell factor (SCF) (16), and bone morphogenic protein (BMP) 4 (17). Furthermore, during this phase, oocyte-derived platelet-derived growth factor (PDGF) and b-FGF promote follicle activation and increase GC SCF expression and secretion. In turn, SCF interacts with oocyte-specific receptors to promote oocyte growth (18). Other cytokines play alternative inhibitory or modulatory roles: oocyte-derived stromalderived factor (SDF) 1a is thought to act in an autocrine/paracrine manner to inhibit follicle activation (19). Several factors originating from outside of the immediate oocyte-granulosa follicle unit also regulate the primordial-to-primary follicle transition, such as keratinocyte growth factor (KGF), BMP4, and BMP-7, which are secreted by stromal cells surrounding the primordial follicle (17, 20). Moreover, adjacent follicles produce cytokines that can inhibit follicle activation via intraovarian paracrine mechanisms, such as GC-derived antim€ ullerian hormone (AMH), a member of the transforming growth factor (TGF) b family (21, 22). Other TGF-b family members (e.g., activin), together with oocytederived cytokines, also participate in development beyond the primary follicle (2, 18, 23, 24). Both dominant follicle selection/function and follicular atresia also are regulated by various cytokines. On one hand, dominant follicle E2 production (the plasma concentrations of which are used in monitoring follicular growth and maturity) is influenced by the intraovarian cytokine milieu, and vice versa (25–29). On the other hand, members of the tumor necrosis factor (TNF) family (TNF-a, Fas ligand, and TNF-related apoptosis-inducing ligand [TRAIL]) are recognized inducers of cell surface death receptor signaling and trigger atresia via the extrinsic pathway (30). The second, antral (graafian) or gonadotropindependent, phase of follicle development is principally characterized by follicular growth triggered by a physiologic LH surge that culminates in ovulation (31). The LH surge induces monocyte chemotactic protein (MCP) 1 and interleukin (IL) 8 gene expression by ovarian stromal and granulosa-lutein cells, which results in an influx of monocytes and neutrophils into the preovulatory follicle (13). These cells are then activated and secrete further mediators, thereby promoting tissue degradation through the action of matrix metalloproteinases as well as the vascular changes involved in follicular rupture (32). The presence of a growing volume 2
of FF in antral follicles throughout this phase offers the possibility of assessing its cytokine composition. Earlier studies have measured individual cytokines in FF throughout the menstrual cycle. McClure et al. demonstrated that the endothelial cell chemotactic potential of human FF increases after the LH surge and before ovulation, thus emphasizing the dynamic functional changes in FF composition during the follicular phase (33). This phenomenon is illustrated by IL12, whose concentration is significantly higher in immature follicles than in their preovulatory counterparts. In contrast, FF LIF levels attain their maximum concentration in the preovulatory follicle (34). A number of studies have focused on clarifying the joint impact of disease and various treatment regimens on ovarian follicular development and its associated cytokine profile. However, a comprehensive picture of cytokine profiles in the normal physiology of folliculogenesis is not yet available. The aim of the present study was therefore to characterize the antral FF cytokine milieu in a cohort of normally cycling healthy women: 1) to create a physiologic baseline reference profile; and 2) to better understand how this changes throughout the follicular phase.
MATERIALS AND METHODS Patients and Sample Collection From November 2008 to March 2009, ten women aged 25– 35 years with body mass indexes of 19–30 kg/m2 who required treatment with IVF/intracytoplasmic sperm injection (ICSI) because of unexplained couple infertility or male-factor infertility (MFI) were recruited to the study. These women were invited to undergo ultrasound and endocrine monitoring as well as FF aspiration at a randomly allocated point during the follicular phase in their first cycle (tracking cycle) and during their subsequent modified natural-cycle (MNC) IVF/ ICSI (treatment cycle) at the Assisted Conception Unit (ACU), St. James's University Hospital, Leeds, United Kingdom. Inclusion criteria included no smoking, weekly alcohol intake
Follicular-phase ovarian follicular fluid and plasma cytokine profiling of natural cycle in vitro fertilization patients N. Ellissa Baskind, M.B.Ch.B.,a Nicolas M. Orsi, M.B.Ch.B., Ph.D.,b and Vinay Sharma, F.R.C.O.G., Ph.D.a a Leeds Centre for Reproductive Medicine, Leeds Teaching Hospitals NHS Trust, Seacroft Hospital; and b Women's Health Research Group, Leeds Institute of Molecular Medicine, St. James's University Hospital, Leeds, United Kingdom
Objective: To characterize follicular fluid (FF) and systemic cytokine profiles at various time points during the natural-cycle follicular and periovulatory phases. Design: Observational clinical study across two consecutive cycles. Setting: Hospital-based in vitro fertilization program. Patient(s): Ten women undergoing modified natural-cycle in vitro fertilization (MNC-IVF). Intervention(s): Plasma and follicular fluid (FF) collection. Main Outcome Measure(s): Forty FF cytokine concentrations from individual follicles and plasma from each patient were determined by fluid-phase multiplex immunoassay in two consecutive cycles: 1) tracking cycle—midfollicular or luteal surge; and 2) treatment cycle—periovulatory (at the time of MNC-IVF). Demographic, cycle, and cytokine data were compared with the use of chi-square, paired-scores t test, or Wilcoxon signed ranks tests. Result(s): Fluctuations in various FF cytokines were evident during the follicular phase: Levels of interleukin (IL) 6 and IL-8 were higher in periovulatory samples, and IL-1 receptor antagonist and vascular endothelial growth factor were elevated earlier in the cycle. Luteal surge profiles were similar to those found in periovulatory samples. Conversely, circulatory cytokine concentrations were more stable during the follicular phase. Conclusion(s): These findings present an extensive physiologic reference profile of FF cytokines associated with antral folliculogenesis and highlight the compartmentalization of sysUse your smartphone temic and intraovarian cytokine networks in natural cycles. (Fertil SterilÒ 2014;-:-–-. to scan this QR code Ó2014 by American Society for Reproductive Medicine.) and connect to the Key Words: Natural cycle, in vitro fertilization, follicular fluid, cytokines, correlations Discuss: You can discuss this article with its authors and other ASRM members at http:// fertstertforum.com/baskindne-follicular-fluid-plasma-cytokine-profiling-nc-ivf/
O
ver the past two decades, it has become apparent that contrary to previously held beliefs, the oocyte does not simply passively accept signals from its surrounding granulosa cells (GCs). It is now evident that oocytes, GCs, theca cells (TCs), and stromal interstitial cells engage in an intricate paracrine dialogue that even
involves neighboring follicles, such that a reciprocal cooperation between these different cell types operates at all stages of follicular growth and/or atresia (1, 2). This multifaceted interaction of mediators governs the development of both the oocyte and somatic cells, as well as determining their response to gonadotropins and
Received January 27, 2014; revised April 4, 2014; accepted April 22, 2014. N.E.B. has nothing to disclose. N.M.O. has nothing to disclose. V.S. has nothing to disclose. Reprint requests: Vinay Sharma, F.R.C.O.G., Ph.D., Leeds Centre for Reproductive Medicine, Leeds Teaching Hospitals NHS Trust, Seacroft Hospital, York Road, Leeds LS14 6UH, United Kingdom (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2014 0015-0282/$36.00 Copyright ©2014 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2014.04.032 VOL. - NO. - / - 2014
discussion forum for this article now.*
* Download a free QR code scanner by searching for “QR scanner” in your smartphone’s app store or app marketplace.
influencing follicular fluid (FF) composition (3, 4). In this manner, a panoply of intrafollicular mediators, including cytokines, chemokines, and protein growth factors (hereafter collectively referred to as cytokines), influence oocyte quality and subsequent embryo viability and support ovulation to the extent that it is increasingly thought that they may affect treatment outcome (5, 6). These agents are produced locally within each follicle in response to the prevailing steroidal milieu and affect the oocyte, both directly and indirectly, through the intermediation of cumulus cells and GCs (7, 8). 1
ORIGINAL ARTICLE: ASSISTED REPRODUCTION Cytokines are small- to medium-size proteins/glycoproteins that act as intercellular mediators and growth factors across a range of immune effector cells, ovarian somatic cells (GCs, TCs, stromal cells), and the oocyte (9, 10). More specifically, intrafollicular cytokines regulate angiogenesis, steroidogenesis, oocyte maturation, leucocyte infiltration and follicle rupture/remodeling during ovulation and, as such, are readily detectable in FF (10–13). Folliculogenesis can be broadly divided into two phases. The first, preantral, phase is gonadotropin independent and characterized by oocyte growth and differentiation, as well as the transition of the primordial follicle to the primary preantral stage. Various cytokines have been implicated in primordial follicle activation and the transition from primary to secondary follicles, including leukemia inhibitory factor (LIF) (14), basic fibroblast growth factor (b-FGF) (15), stem cell factor (SCF) (16), and bone morphogenic protein (BMP) 4 (17). Furthermore, during this phase, oocyte-derived platelet-derived growth factor (PDGF) and b-FGF promote follicle activation and increase GC SCF expression and secretion. In turn, SCF interacts with oocyte-specific receptors to promote oocyte growth (18). Other cytokines play alternative inhibitory or modulatory roles: oocyte-derived stromalderived factor (SDF) 1a is thought to act in an autocrine/paracrine manner to inhibit follicle activation (19). Several factors originating from outside of the immediate oocyte-granulosa follicle unit also regulate the primordial-to-primary follicle transition, such as keratinocyte growth factor (KGF), BMP4, and BMP-7, which are secreted by stromal cells surrounding the primordial follicle (17, 20). Moreover, adjacent follicles produce cytokines that can inhibit follicle activation via intraovarian paracrine mechanisms, such as GC-derived antim€ ullerian hormone (AMH), a member of the transforming growth factor (TGF) b family (21, 22). Other TGF-b family members (e.g., activin), together with oocytederived cytokines, also participate in development beyond the primary follicle (2, 18, 23, 24). Both dominant follicle selection/function and follicular atresia also are regulated by various cytokines. On one hand, dominant follicle E2 production (the plasma concentrations of which are used in monitoring follicular growth and maturity) is influenced by the intraovarian cytokine milieu, and vice versa (25–29). On the other hand, members of the tumor necrosis factor (TNF) family (TNF-a, Fas ligand, and TNF-related apoptosis-inducing ligand [TRAIL]) are recognized inducers of cell surface death receptor signaling and trigger atresia via the extrinsic pathway (30). The second, antral (graafian) or gonadotropindependent, phase of follicle development is principally characterized by follicular growth triggered by a physiologic LH surge that culminates in ovulation (31). The LH surge induces monocyte chemotactic protein (MCP) 1 and interleukin (IL) 8 gene expression by ovarian stromal and granulosa-lutein cells, which results in an influx of monocytes and neutrophils into the preovulatory follicle (13). These cells are then activated and secrete further mediators, thereby promoting tissue degradation through the action of matrix metalloproteinases as well as the vascular changes involved in follicular rupture (32). The presence of a growing volume 2
of FF in antral follicles throughout this phase offers the possibility of assessing its cytokine composition. Earlier studies have measured individual cytokines in FF throughout the menstrual cycle. McClure et al. demonstrated that the endothelial cell chemotactic potential of human FF increases after the LH surge and before ovulation, thus emphasizing the dynamic functional changes in FF composition during the follicular phase (33). This phenomenon is illustrated by IL12, whose concentration is significantly higher in immature follicles than in their preovulatory counterparts. In contrast, FF LIF levels attain their maximum concentration in the preovulatory follicle (34). A number of studies have focused on clarifying the joint impact of disease and various treatment regimens on ovarian follicular development and its associated cytokine profile. However, a comprehensive picture of cytokine profiles in the normal physiology of folliculogenesis is not yet available. The aim of the present study was therefore to characterize the antral FF cytokine milieu in a cohort of normally cycling healthy women: 1) to create a physiologic baseline reference profile; and 2) to better understand how this changes throughout the follicular phase.
MATERIALS AND METHODS Patients and Sample Collection From November 2008 to March 2009, ten women aged 25– 35 years with body mass indexes of 19–30 kg/m2 who required treatment with IVF/intracytoplasmic sperm injection (ICSI) because of unexplained couple infertility or male-factor infertility (MFI) were recruited to the study. These women were invited to undergo ultrasound and endocrine monitoring as well as FF aspiration at a randomly allocated point during the follicular phase in their first cycle (tracking cycle) and during their subsequent modified natural-cycle (MNC) IVF/ ICSI (treatment cycle) at the Assisted Conception Unit (ACU), St. James's University Hospital, Leeds, United Kingdom. Inclusion criteria included no smoking, weekly alcohol intake
