Morphological and cytochemical determination of cell death by apoptosis Several modes of cell death are now recognized , including necrosis , apoptosis , and autophagy . Oftentimes the distinctions between these various modes may not be apparent , although the precise mode may be physiologically important . Accordingly , it is often desirable to be able to classify the mode of cell death . Apoptosis was originally defined by structural alterations in cells observable by transmitted light and electron microscopy . Today , a wide variety of imaging and cytochemical techniques are available for the investigation of apoptosis . This review will highlight many of these methods , and provide a critique on the advantages and disadvantages associated with them for the specific identification of apoptotic cells in culture and tissues . Introduction The mode of cell death referred to as apoptosis , and now defined by a variety of techniques , including biochemistry , flow cytometry , and imaging was originally defined based solely upon morphological criteria . In 1972 , Kerr et al. -LRB- 1972 -RRB- introduced the neologism `` apoptosis '' to describe a type of cell death which had previously been referred to as `` shrinkage necrosis '' . Recognizing the importance of its kinetic mechanism in controlled cell deletion , either occurring spontaneously or in response to a stimulus , they offered the term apoptosis -LRB- from Greek , meaning `` to fall away from '' , as in leaves from a tree ; thus , the falling away of cells from a tissue -RRB- . They observed apoptotic cells in a wide variety of tissues , including during development and neoplastic transformation . Although they could detect apoptotic cells in many instances by light microscopy , it was their observations by transmission electron microscopy that established the characteristic ultrastructural features now considered the hallmark of apoptosis . These features include -LRB- 1 -RRB- cytoplasmic and nuclear condensation -LRB- pyknosis -RRB- ; -LRB- 2 -RRB- nuclear fragmentation -LRB- karyorrhexis -RRB- ; -LRB- 3 -RRB- normal morphological appearance of cytoplasmic organelles ; and -LRB- 4 -RRB- an intact plasma membrane -LRB- Kerr et al. 1972 ; Wyllie et al. 1980 ; Galluzzi et al. 2007 -RRB- . Frequently , the pyknotic nucleus assumes the appearance of a half-moon or crescent shape , a feature most indicative of an apoptotic cell -LRB- Fig. 1 -RRB- . Following nuclear fragmentation , the cell disaggregates into a number of membrane-bound apoptotic bodies , which are engulfed via phagocytosis by neighboring epithelial cells or macrophages . Fig. 1Transmission electron microscopic image of an apoptotic cell in a human kidney biopsy . Note the pyknotic , shrunken nucleus and the very condensed cytoplasm In more recent years , evaluation of cells and tissues for apoptosis has evolved towards staining for light microscopic and flow cytometric analysis . As the biochemical and cell signaling events involved in the apoptotic cascade have been revealed , new tools for the analysis of apoptosis have emerged . Many of these tools are in the form of antibodies raised against proteins specific for the apoptotic pathway , or against neoepitopes on proteins resulting from action of an activated enzyme . While these new probes may specifically target aspects of the apoptotic pathway , they do not address the ultrastructural changes upon which the term apoptosis was originally defined . This review will focus on some morphological and cytochemical techniques used to demonstrate the presence of apoptotic activity in tissue and cultured cells . For further information on this topic , the reader is referred to the earlier excellent reviews by Willingham -LRB- 1999 -RRB- , Barrett et al. -LRB- 2001 -RRB- and Watanbe et al. -LRB- 2002 -RRB- . However , before we begin to describe these specific detection methods , we need to examine more closely the different classifications of cell death , and then delve into the mechanisms responsible for initiating the apoptotic cascade . Classification of cell death Cell death is now known to be perpetrated through a variety of mechanisms . According to Galluzzi et al. -LRB- 2007 -RRB- , cell death can be classified into four different types , based upon morphological characteristics : apoptosis -LRB- Type 1 -RRB- , autophagy -LRB- Type 2 -RRB- , necrosis -LRB- oncosis , Type 3 -RRB- , and mitotic catastrophe . The morphological changes accompanying apoptosis have been described in detail above . Whereas apoptosis is manifested by volume reduction of the nucleus and cytoplasm -LRB- cell shrinkage -RRB- , necrosis -LRB- the mode of cell death with which apoptosis is most often confused -RRB- is evinced by cytoplasmic swelling , rupture of the plasma membrane , swelling of cytoplasmic organelles -LRB- particularly mitochondria -RRB- , and some condensation of nuclear chromatin -LRB- Galluzzi et al. 2007 -RRB- . Autophagy is distinguished by the accumulation of cytoplasmic vacuoles and membranes , and mitotic catastrophe by multinucleation . Clearly , cellular morphological characteristics must be taken into consideration when determining the mode of cell death . This is of utmost importance when employing the TUNEL assay , since this technique stains both apoptotic and necrotic cells , which as just described display widely different morphological characteristics . This issue will be addressed in detail below . Pathways of apoptosis Cell death through apoptosis is known to occur through two primary pathways , an extrinsic pathway involving death receptors , and an intrinsic pathway via members of the Bcl-2 family -LRB- Adams and Cory 1998 -RRB- . The extrinsic pathway uses the classical death receptors such as Fas -LRB- CD95/APO1 -RRB- , TNF Receptor1 , and TRAIL Receptors . Engagement of these receptors triggers a now well-defined process of recruitment of proteases known as caspases -LRB- cysteine-dependent aspartate-cleaving proteases -RRB- . In the case of Fas , oligomerization by its ligand causes recruitment of the adaptor protein FADD -LRB- Fas-associated via death domain -RRB- through interactions between the death domains -LRB- DD -RRB- found in each protein -LRB- Krueger et al. 2003 -RRB- . FADD then recruits caspase-8 through their common death effector domains -LRB- DED -RRB- . Homodimerization of caspase-8 results in its activation through sequential cleavage at critical aspartate residues . Fully active caspase-8 then cleaves downstream targets such as the Bcl-2 family member Bid to form truncated Bid or tBid -LRB- Li et al. 1998 -RRB- . tBid localizes to the outer mitochondrial membrane where it promotes oligomerization of another Bcl-2 member , Bax , which results in breakdown of the mitochondria and release of cytochrome c and Smac/Diablo -LRB- Verhagen et al. 2000 -RRB- . Cytochrome c engages a complex containing Apaf-1 and caspase-9 that in the presence of ATP forms a large molecular weight complex of active caspase-9 known as the apoptosome -LRB- Cain et al. 2000 -RRB- . The apoptosome can then activate the effector caspases , such as caspase-3 and caspase-6 that ultimately lead to breakdown of cellular components . Among these is cleavage of an inhibitor of caspase-activated DNAse -LRB- ICAD -RRB- that allows the DNAse to degrade DNA . This process is fundamental to various assays of DNA breaks that are described in this review . In parallel with the extrinsic death pathway , the intrinsic pathway ultimately engages the same mitochondria-initiated downstream death pathways . In this case the cascade is initiated by any of a variety of sensors that detect cellular stress . The initiators in this case include a panel of Bcl-2 family members that contain only the BH3 domain -LRB- so-called BH3-only proteins -RRB- -LRB- Strasser 2001 -RRB- . These pro-apoptotic Bcl-2 family members are located throughout the cell somewhat as sentinels that detect cellular perturbations . Upon their activation they induce aggregation and activation of Bax or Bak , which then feeds into the mitochondrial death pathway described above . Activation of the intrinsic death pathway may occur in the form of DNA damage by UV irradiation that activates p53 and then two BH3-only proteins , Puma and Noxa . Glucocorticoids similarly activate Puma and Bim that are critical to induction of cell death by steroids . In fact most chemotherapeutic agents ultimately engage a BH3-only protein in the process of killing cells . As a consequence of these pathways , several death assays measure mitochondrial integrity or membrane potential as described in this review . Additional assays might assess cleavage of caspases or their substrates . Given the localization of many Bcl-2 family members at the mitochondria , considerable interest is now developing over the concept that these proteins may function as well in some normal metabolic pathways . An early and somewhat overlooked study from the Korsmeyer laboratory showed that increased expression of Bcl-2 in T lymphocytes decreased their production of the growth cytokine IL-2 and resulted in somewhat reduced proliferative capacity following stimulation of the T cell antigen receptor -LRB- TCR -RRB- -LRB- Linette et al. 1996 -RRB- . Bcl-2-deficient T cells manifested the opposite phenotype . More recent studies suggest that this may function via regulation of calcium signaling -LRB- White et al. 2005 -RRB- . Other studies suggest that BAD may regulate glucokinase activity in mitochondria and that BAD-deficient β-islet cells are less responsive with insulin secretion to glucose levels -LRB- Wikstrom et al. 2007 -RRB- . In a similar manner , caspase-8 has been shown to be critical to growth of various cell types including T lymphocytes -LRB- Chun et al. 2002 ; Salmena et al. 2003 -RRB- . Thus , analysis of activation of `` death '' molecules will , in the future , need to consider these potential overlapping functions with normal cell metabolism . Detection of apoptosis by electron microscopy It was the early electron microscopic investigations of Kerr et al. -LRB- 1972 -RRB- and Wyllie et al. -LRB- 1980 -RRB- that led to their formulation of the concept of apoptosis as a wide-spread form of cell death in a variety of tissues and physiological situations . Electron microscopy is still considered the `` gold standard '' for the identification of apoptotic cells . However , likely due to the costs associated with performing electron microscopy , the specialized equipment and technical expertise required , and the limited tissue area represented , this technique is no longer routinely used as an apoptosis assay . In many instances , the degree of apoptosis detected , for example following treatment with a specific reagent , is an experimental endpoint . This can easily be accomplished with light microscopy or flow cytometry-based methodologies , but is exceedingly laborious by transmission electron microscopy . However , in instances where quantitation is unimportant , and the mere presence of specifically identifiable apoptotic morphology is the goal , electron microscopy is indispensable . For instance , Abbate et al. -LRB- 2007 -RRB- recently employed this approach as a means to characterize the nature of cell death in the myocardium after an ischemic event . They investigated the ultrastructural appearance of cardiomyocytes by transmission electron microscopy in failing rabbit hearts 16 days following coronary artery ligation . They described cardiomyocytes with severe nuclear derangements , peripheral coarse chromatin condensation , and prevalent mitochondrial and myofibrillar alterations . Although these ultrastructural characteristics are not typical of apoptotic cells , the cells were deemed to be apoptotic based upon an intact cardiomyocyte plasma membrane . These results would suggest that the `` typical '' ultrastructural features ascribed to apoptotic cells may in some instances be cell-type specific . However , further experimentation will be necessary to confirm this interpretation . Scanning electron microscopy has also been used to examine apoptotic cells , with their characteristic rounded-up phenotype well represented by this imaging modality -LRB- Wyllie et al. 1980 ; Germain et al. 2007 -RRB- . Detection of apoptosis by cytochemical optical microscopy A wide variety of probes are commercially available today for the detection of apoptosis -LRB- see Table 1 -RRB- . Many of these are in the form of antibodies raised against proteins specific for the apoptotic pathway , or against neoepitopes on proteins resulting from action of an activated enzyme . These will be discussed later , however , since we will begin with ostensibly the most common cytochemical method for the determination of apoptosis , the TUNEL assay . Table 1Commercial sources for apoptosis reagentsReagentCommercial sourceTUNEL assay kitMBL International , Woburn MAMillipore Corporation , Billerica , MAInvitrogen Corporation , Carlsbad , CAPromega , Madison , WIRoche Applied Science , Indianapolis , INApo stain -LRB- F7-26 -RRB- -LRB- anti-ssDNA -RRB- Axxora , LLC , San Diego , CAAnti-cleaved caspase 3 -LRB- ASP 175 -RRB- Cell Signaling Technology , Inc. , Danvers , MAAnti-cleaved cytokeratin 18Chemicon , Temecula , CAAnnexin V-FITCBD Biosciences Pharmingen , San Diego , CARoche Applied Science , Indianapolis , INInvitrogen Corporation , Carlsbad , CALectins specific for α-d-mannose and β-d-galactoseLectinotest Laboratory , Lviv , UkraineEY laboratories , San Mateo , CACalbiochem , San Diego , CASigma-Aldrich , St Louis , MOAnti-cytochrome c oxidase subunit 1Invitrogen Corporation , Carlsbad , CABD Biosciences Pharmingen , San Diego , CAAnti-apoptosis-inducing factorSanta Cruz Biotechnology , Santa Cruz , CASyntem , Nimes , FranceThis list is not meant to be exhaustive , but to serve as a starting point to search for reagents . Apologies to those companies not listed The TUNEL assay The presence of apoptotic cells has often been inferred from a positive reaction to the TUNEL assay -LSB- terminal deoxynucleotidyl transferase -LRB- TdT -RRB- - mediated dUTP in situ nick end labeling -RSB- . However , the lack of specificity of the TUNEL assay for apoptotic cells has been well documented -LRB- Sloop et al. 1999 ; Jung et al. 2000 ; Walker and Quirke 2001 ; Watanabe et al. 2002 ; Groos et al. 2003 ; Takemura and Fujiwara 2006 -RRB- . TUNEL will detect free 3 ′ - OH terminals of both single - and double-strand DNA breaks , thus potentially labeling both apoptotic and necrotic derived DNA strand breaks . Moreover , the TUNEL assay has also been reported to label mitotic cells -LRB- Ito et al. 2006 -RRB- , cells in the process of gene transcription -LRB- Kockx et al. 1998 -RRB- , cells undergoing DNA repair -LRB- Kanoh et al. 1999 -RRB- , and cells with DNA breaks caused by the microtomy sectioning procedure -LRB- Sloop et al. 1999 -RRB- . The effects of tissue fixation and processing may also alter TUNEL reactivity -LRB- Negoescu et al. 1996 ; Labat-Moleur et al. 1998 ; Tateyama et al. 1998 -RRB- . In a rat model , we found that exposure to asbestos and cigarette smoke resulted in strong TUNEL positivity in bronchiolar epithelial cells from paraffin-embedded sections stained by immunoperoxidase -LRB- Jung et al. 2000 -RRB- . However , when bronchioles from the same animals were examined by transmission electron microscopy , only necrotic epithelial cells were observed , indicating that the TUNEL-positive cells detected by light microscopy were likely necrotic , not apoptotic . The TUNEL-positive nuclei did not display the characteristic shrunken appearance expected for an apoptotic cell , again emphasizing the need to corroborate staining results with morphological observations . Similar conclusions were reached by Negoescu et al. -LRB- 1996 -RRB- and Labat-Moleur et al. -LRB- 1998 -RRB- in their efforts to improve tissue preparation and staining procedures for the TUNEL method . The TUNEL assay maintains its popularity as a marker of apoptotic cells ; it is available in a kit form , is easy to use , and has been available for a number of years , becoming entrenched in the literature . However , given the concerns raised above with its specificity for apoptotic cells , we are of the opinion that other methods should be employed concomitantly with this assay in order to make a determination that the mode of cell death observed is the result of an apoptotic mechanism . Anti-single-stranded DNA antibody -LRB- Apostain -RRB- Frankfurt et al. -LRB- 1994 , 1996 -RRB- and Frankfurt and Krishan -LRB- 2001 -RRB- have pioneered the application of a monoclonal antibody raised against single-stranded DNA -LRB- ssDNA -RRB- as a more specific cytochemical probe for apoptotic cells than the TUNEL assay . They have shown that the combination of a commercially available ssDNA antibody with formamide-induced denaturation of condensed chromatin , results in specific labeling of apoptotic cells in culture and in formalin-fixed , paraffin embedded tissues -LRB- Frankfurt and Krishan 2001 -RRB- . Ito et al. -LRB- 2006 -RRB- expanded upon these observations by adapting the formamide DNA denaturation followed by anti-ssDNA antibody staining technique for the electron microscopic level . They show very convincingly by immunoelectron microscopy that the TUNEL assay results in staining of condensed chromatin in mitotic and necrotic cells in addition to apoptotic cells , whereas the formamide-ssDNA technique stained only the condensed chromatin from apoptotic cells . We have also used this Apostain method for the specific detection of apoptotic pulmonary epithelial -LRB- Buder-Hofmann et al. 2001 -RRB- or rat pleural mesothelial cells -LRB- Shukla et al. 2003 -RRB- after exposure to crocidolite asbestos in culture , and in human lung adenocarcinoma cells -LRB- A-549 -RRB- following treatment with the chemotherapeutic agents paclitaxel and vinorelbine -LRB- Jung et al. 2004 -RRB- . Moreover , we recently combined staining with the ssDNA antibody with a second specific marker of apoptosis in a novel dual staining method on tissue sections , as described below . In our hands , in agreement with studies described above , we find the formamide condensed DNA denaturation/ssDNA technique to be a reliable indicator of apoptotic cells in a variety of cell cultures and tissues . Apoptosis-related neoepitopes Antibodies raised against caspase-cleaved proteins are now commercially available . The enzymatic cleavage of proteins during the apoptotic cascade results in the formation of `` neoepitopes '' on these proteins which can be used as antigens for the production of caspase-mediated apoptotic reagents . Although several such antibodies are currently available , those raised against neoepitopes present on cleaved cytokeratin 18 and caspase 3 have been most frequently used for the detection of apoptosis . Willingham and colleagues -LRB- Barrett et al. 2001 -RRB- demonstrated the utility of using an anti-cleaved cytokeratin 18 antibody for the specific detection of apoptotic cells in culture and in archived paraffin embedded tissues . Specifically stained cells showed the shrunken condensed morphology expected of an apoptotic cell . Naturally , this antibody can only be employed for cells and tissues that express cytokeratin 18 . Likewise , Gown and Willingham -LRB- 2002 -RRB- used a commercially available antibody raised against cleaved caspase 3 to specifically label apoptotic cells in culture and paraffin embedded tissues . Given the ubiquitous presence of caspase 3 in most cells , and the formation of the neoepitope following activation of the apoptotic cascade , this reagent should prove to be a most useful and specific label for cells undergoing caspase-mediated apoptotic cell death . Recently , we combined immunostaining for anti-cleaved caspase 3 with the formamide denaturation/ssDNA method for the detection of two specific apoptosis probes in the same tissue section using confocal scanning laser microscopy -LRB- Taatjes et al. 2007 -RRB- . In dual stained cells in paraffin sections from infarcted mouse ventricle , the anti-cleaved caspase 3 antibody labeled the cytoplasm , whereas the anti-ssDNA antibody stained the cell nucleus . Cells were observed stained singly with either of the two probes , suggesting that cells in different phases of the apoptotic cascade may be identified by their staining properties with these antibodies . We include sections from the thymus of dexamethasone-treated mice as a positive control -LRB- Figs. 2 , 3 -RRB- for the apoptosis assays -LRB- Taatjes et al. 2007 -RRB- , since steroid hormone treatment has been shown by transmission electron microscopy to induce thymic apoptosis -LRB- Wyllie et al. 1980 -RRB- . Fig. 2Paraffin section of thymus from mouse treated with dexamethasone and stained with anti-cleaved caspase 3 antibody -LRB- green -RRB- , and anti-ssDNA antibody -LRB- red -RRB- . Confocal microscopy demonstrates doubly stained thymic cells -LRB- arrowheads -RRB- . Note that the cleaved caspase 3 is localized to the cytoplasm , whereas the ssDNA is localized to the nucleus . Some cells only stained by cleaved caspase 3 antibody are indicated by solid arrows . The nucleus is labeled with DAPI -LRB- blue -RRB- Fig. 3Paraffin section of thymus from mouse treated with dexamethasone and stained with anti-cleaved caspase 3 antibody -LRB- red -RRB- . Confocal microscopy image shows intensely stained cells in the cortex , with neighboring unstained cells . Nuclei have been stained with DAPI -LRB- blue -RRB- . This represents a good positive control for the anti-cleaved caspase 3 antibody technique Annexin V Some markers for apoptotic cells require the use of live cells . For instance , early in the apoptotic cascade of events , phosphatidyl serine residues flip from the internal to external side of the cells ' plasma membrane . Accordingly , annexin V , which binds specifically to phosphatidyl serine residues has been used in this manner as a specific probe for apoptosis -LRB- Koopman et al. 1994 -RRB- . However , as pointed out by Willingham -LRB- 1999 -RRB- , this assay does not seem to label all apoptotic cells , and moreover will stain necrotic cells once the plasma membrane is compromised . Other probes for apoptosis Bilyy et al. -LRB- 2004 , 2005 -RRB- and Bilyy and Stoika -LRB- 2003 -RRB- have championed the examination of plasma membrane glycoconjugates as indicators of apoptosis . They have detected an augmentation in the expression of α-d-mannose - and β-d-galactose-containing glycoproteins in the plasma membrane in response to apoptotic signals . These glycoconjugates can be visualized using a battery of lectins -LRB- carbohydrate-binding proteins of nonimmune origin -RRB- , in conjunction with fluorescence - or peroxidase-conjugated probes for light microscopy . Unlike most of the markers for apoptosis which bind to cytoplasmic or nuclear constituents , these plasma membrane-specific probes offer the means to assess activity occurring at the cell surface during the apoptotic cascade -LRB- similar to annexin V binding to phosphatidylserine -RRB- . Given the enormous diversity in carbohydrate structure , and the role played by carbohydrates in a variety of cell -- cell interactions , it is feasible that they may offer more insight into cellular mechanisms of apoptosis in the future . An early event in the apoptotic cascade is the release of cytochrome c from the outer mitochondrial membrane . Accordingly , the intracellular localization of cytochrome c could serve as an indicator for apoptosis . Jones and colleagues -LRB- Jiang et al. 1999 -RRB- used this approach to show that in a control -LRB- untreated -RRB- osteosarcoma cell line , immunostaining for cytochrome c -LRB- as assessed by confocal microscopy -RRB- was observed in a punctuate cytoplasmic pattern , coincident with mitochondrial staining by MitoTracker Red . When cells were treated with staurosporine to induce apoptosis , the immunolabel for cytochrome c was found to be diffusely present throughout the cytoplasm , consistent with its release from the mitochondria . We have also used immunolabeling for cytochrome c as a marker for mitochondria in intact cells -LRB- Fung et al. 1998 -RRB- , and its further application as a potential marker for apoptotic cells dependent upon its intracellular staining pattern should be further explored . Apoptosis-inducing factor -LRB- AIF -RRB- is a mitochondrial membrane protein involved in caspase-independent cell death -LRB- Daugas et al. 2000 -RRB- . Immunostaining for AIF revealing a translocation from the mitochondrion to the nucleus has been used as a marker for caspase-independent apoptosis -LRB- Daugas et al. 2000 ; Susin et al. 2000 -RRB- . However , Daugas et al. -LRB- 2000 -RRB- have shown that the nuclear translocation of AIF also occurs during a necrotic response , so other methods should be employed in conjunction with AIF immunostaining to differentiate between these two modes of cell death . Indeed , we have used immunostaining of AIF translocation from the cytoplasm -LRB- mitochondrion -RRB- to the nucleus , together with cell morphological assessment by phase contrast microscopy , as an indicator of apoptosis in lung C10 cells in response to asbestos exposure -LRB- Yuan et al. 2004 -RRB- . Detection of apoptosis by video optical microscopy Morphological assessment of cultured cells by video microscopy has been successfully used to follow the cellular changes induced by apoptosis . Cells treated with apoptosis-inducing agents are imaged with a light microscope , typically operating in phase contrast or differential interference contrast mode , and time-lapse recording is made with a ccd camera connected to a video recorder -LRB- Collins et al. 1997 ; Willingham 1999 -RRB- . A major advantage of this type of temporal microscopy is the ability to document cellular morphological changes associated with apoptosis with the time course of the event . Differences in cellular changes observed in necrosis versus apoptosis can be well documented , and the slow course of apoptosis is well suited to time-lapse recording -LRB- Collins et al. 1997 -RRB- . Using phase contrast microscopy in conjunction with video recording has produced dramatic footage of cells exposed to apoptotic agents rounding up and displaying intense surface blebbing -LRB- Collins et al. 1997 -RRB- . Of course , the full apoptotic cascade may require up to 24 h in cultured cells , so movies can also be made by merging files from single ccd camera images and exporting them in a movie-type format . Thorburn and colleagues -LRB- Morgan et al. 2002 -RRB- used a combination of fluorescence and phase contrast microscopy to image apoptosis in live HeLa cells . A Hamamatsu digital camera was used to acquire combination images every 30 min over a 24 h period . The merged image series were exported in Quicktime format , and a representative movie sequence can be seen in the online supplemental material referenced in the manuscript -LRB- Morgan et al. 2002 -RRB- . This type of analysis is particularly useful for determining the temporal sequence of morphological events associated with the introduction of an apoptosis-inducing agent to cell culture media . Detection of apoptosis by laser scanning cytometry Laser scanning cytometry -LRB- LSC -RRB- is a method which combines the efficiency and precision of flow cytometry with the morphological characterization ability of a microscope-based imaging system -LRB- Kamentsky and Kamentsky 1991 ; Kamentsky et al. 1997 ; Taatjes et al. 2001 ; Pozarowski et al. 2006 -RRB- . The ability to examine the morphological appearance of cells following flow cytometry-like analysis overcomes the limitations posed by flow cytometry itself in the analysis of apoptosis ; that is , apoptotic cells appear in the subdiploid peak of a flow cytometric histogram , together with apoptotic bodies and fluorescence debris , and can not be directly visualized . Darzynkiewicz and colleagues have pioneered the use of LSC for analysis of apoptosis , and the reader is referred to their excellent review for further details -LRB- Bedner et al. 1999 -RRB- . In particular , they have used LSC in combination with a variety of apoptosis markers , including annexin-V , markers of mitochondrial transmembrane potential -LRB- rhodamine 123 and DiOC6 -RRB- , nuclear markers -LRB- DAPI , 7-ADD , PI -RRB- , and markers of DNA strand breaks . Importantly , in all of these investigations , they emphasized the combination of fluorescence staining and morphological determination with LSC as being of utmost importance for the identification of apoptotic cells . We have also used LSC for the cytometric and morphological characterization of apoptotic cultured mouse lung epithelial cells -LRB- C10 cell line -RRB- -LRB- Taatjes et al. 2001 -RRB- . Cells were exposed to hydrogen peroxide for 24 h to induce apoptosis , followed by staining with the anti-ssDNA antibody and an Oregon green-conjugated secondary antibody to stain apoptotic cells . All cells were then stained with propidium iodide and analyzed by LSC . Scattergrams from the analysis -LRB- Fig. 4 -RRB- showed that hydrogen peroxide induced an increase in apoptotic cells , evinced by an increase in the population of cells depicted in the subdiploid area . Cells located in this subdiploid portion were then visually confirmed through relocation using the automated stage and microscope of the LSC . These cells were found to be labeled with the anti-ssDNA antibody , and showed the condensed morphological characteristics of apoptotic cells -LRB- Fig. 4 -RRB- . Fig. 4Laser scanning cytometry determination of apoptosis in lung epithelial cells using anti-ssDNA antibody . Upper panel scattergrams showing percentage of ssDNA-positive cells -LRB- green integral vs. orange/red integral -RRB- , the apoptotic fraction -LRB- orange/red integral vs. orange/red maximum pixel -RRB- , and a DNA histogram for untreated C10 cells . Middle panel scattergrams showing percentage of ssDNA-positive cells -LRB- green integral vs. orange/red integral -RRB- , the apoptotic fraction -LRB- orange/red integral vs. orange/red maximum pixel -RRB- , and a DNA histogram for C10 cells treated with 200 μM H2O2 for 24 h. Note the increased number of cells ssDNA-positive , and the enhanced subdiploid DNA fraction in cells treated with H2O2 as compared with sham controls . Bottom panel cell relocation feature of LSC demonstrated for eight cells . Cells within the subdiploid fraction -LRB- elliptical region highlighted on scattergram in middle panel -RRB- were relocated and visually confirmed as apoptotic by morphological appearance and positive staining with the ssDNA antibody . Reprinted with permission from BioTechniques -LRB- Taatjes et al. 2001 -RRB- Detection of apoptosis by atomic force microscopy -LRB- AFM -RRB- The atomic force microscope is a member of the scanning probe microscope family -LRB- Binnig et al. 1986 -RRB- . This instrument utilizes a tactile sensing system , in which a probe -LRB- typically composed of silicon nitride -RRB- is attached to the end of a cantilever and raster scanned in x - and y-directions across a specimen through the activity of a piezoelectric tube to which the cantilever assembly is mounted -LRB- Montigny et al. 2006 -RRB- . A laser beam is directed onto the surface of the cantilever , and the reflected beam is directed up to a four quadrant photodiode , which converts laser movement on the surface into a measurable electrical voltage change . This voltage change is then translated via computer interface into height and amplitude information about the specimen . The complexity of the system belies the simplicity of specimen preparation ; fixed or unfixed cells grown on glass coverslips can be readily imaged by AFM without further processing , or importantly introduction of artifacts . Although AFM initially found its greatest utility in the imaging of isolated molecules and their dynamic interactions -LRB- Hansma and Ho 1994 ; Taatjes et al. 1997 ; Rand et al. 2003 -RRB- , more recently whole cell studies have become more prevalent -LRB- Madl et al. 2006 -RRB- . With this development , and since AFM typically images surface phenomena , it was a natural extension to use this technology to image cells undergoing apoptosis . Hessler et al. -LRB- 2005 -RRB- used AFM to image and measure the cellular volume decrease occurring during apoptosis . They treated the human epidermoid carcinoma cell line KB with staurosporine to induce apoptosis , and determined that after a 3-h exposure , the total cell height decreased 32 % , the total cell volume was decreased by 50 % , and the cell diameter changed by 15 % . Given the temporal experimental capabilities of AFM , it seems as if this would be an ideal modality , in combination with other imaging methods , to investigate the cellular dynamics accompanying the apoptotic cascade , as elegantly shown by Hessler et al. . An example of the importance of delineating apoptosis : cell death in the heart In this brief review , we have attempted to highlight some of the morphological and cytochemical techniques currently in use to classify cell death as an apoptotic response . There are a wide ranging variety of methods to choose from , rendering the choice a matter of the experimental system employed and personal preference . However , the determination of cell death as apoptotic , or through another mode should not be viewed simply as a matter of semantics . For instance , an example from clinical cardiovascular disease will illustrate the importance of the precise determination of the mode of cell death . For several decades the conceptual basis underlying treatment of acute coronary syndromes has been that limitation of myocardial oxygen requirements by diminution of afterload and heart rate coupled with augmentation of myocardial oxygen supply through restoration of myocardial perfusion are the pivotal components -LRB- Maroko et al. 1972 ; Shell and Sobel 1974 ; Sobel and Braunwald 1980 -RRB- . The potential value of `` tissue protective '' interventions including favorable modification of myocardial metabolism has long been recognized as well -LRB- Maroko et al. 1972 -RRB- . Several different types of cell death have been characterized in the heart including coagulation necrosis , the typical manifestation of ischemia sufficient to render myocardium nonviable , contraction band necrosis typical of ischemia followed by reperfusion and presumably mediated in part by calcium overload , autophagy typical of cells insulted by deprivation of nutrients that consequently consume their own components , and apoptosis . The extent to which this final modality , programmed cell death or apoptosis , contributes to overall cardiomyocyte cell death is far from a trivial matter -LRB- Sobel et al. 2007 -RRB- . If apoptosis plays a substantial role as a contributor to total cardiomyocyte cell death and if it can be modified or prevented despite the presence of ischemia , the potential , favorable impact of tissue protective interventions is likely to be considerable . Conversely , if apoptosis plays a small role in overall cardiomyocyte cell death consequent to ischemia , an anti-apoptotic strategy would likely be ineffective . Unfortunately , the implication of apoptosis in cardiomyocyte cell death in studies in laboratory animals has been based virtually exclusively on results with the TUNEL assay -LRB- see above for issues of nonspecificity of this assay -RRB- . As noted by Takemura and Fujiwara -LRB- 2006 -RRB- , there is no unequivocal evidence that apoptosis occurs in zones of infarction . As described above , we have recently sought to quantify apoptosis in myocardium and have developed a novel dual staining method for confocal microscopic identification of apoptotic cells -LRB- Taatjes et al. 2007 -RRB- . Our initial findings indicate that despite massive necrosis induced by ischemia in a transgenic strain of mice deficient in plasminogen activator inhibitor type-1 -LRB- PAI-1 -RRB- hardly any cells were apoptotic as judged from positive reactions to antibodies against both cleaved caspase 3 and ssDNA . Others have observed an absence of apoptosis as judged from electron microscopic criteria -LRB- Ohno et al. 1998 -RRB- despite TUNEL positivity in hearts of animals subject to transitory ischemia followed by reperfusion for several hours . In view of these considerations , it appears important to determine definitively whether the presence of ssDNA coupled with the presence of caspace 3 cleavage product does , indeed , map isomorphically with the cells that exhibit electron microscopic criteria of apoptosis . Furthermore , it will be important to determine the extent to which the positivity of each of these criteria of apoptosis occurs in hearts of experimental animals subjected to ischemia for selected intervals followed by reperfusion for selected intervals as well as in those from animals subjected to myocardial ischemia that is persistent and to define the time course of the evolution of apoptosis , if present , under both conditions . It is , of course , possible that apparently scanty apoptosis at any given time could be of considerable biological importance since apoptotic cells are rapidly destroyed or removed by macrophages . Thus , if a given percentage of cells , though a small one , were exhibiting apoptosis repetitively , the cumulative amount could be substantial . Accordingly , both the temporal and the spatial evolutions of apoptosis associated with myocardial ischemia and infarction require elucidation . This is particularly important because apoptosis in the heart may be secondary to necrosis rather than a primary mode of cell death , a distinction that needs to be clarified because of the disparate therapeutic implications of the two possibilities . Only when armed with information needed to characterize apoptosis in the heart more definitively will it be possible to delineate the potentially beneficial effects of tissue protective interventions designed to retard or prevent apoptotic cell death in cardiomyocytes subjected to ischemia . Concluding remarks Determining the mechanisms of cell death is an area of intense research interest in a wide range of fields including cancer biology , pathology , and toxicology . Certainly , the role of apoptosis as a mode of `` programmed cell death '' during embryonic development has been unequivocally established -LRB- Penaloza et al. 2006 -RRB- . We hope that this brief review has highlighted a variety of imaging modalities suitable for differentiating amongst the different modes of cell death . What should be apparent is that rarely will one method be sufficient for such a determination . Each imaging method has its own strengths with respect to the morphological information it can provide . Such morphological information , combined with biochemical or immunohistochemical assessment can provide a strong indication of the specific mode of cell death observed . The key , in our estimation , is a multiparameter observational approach . Accordingly , the take-home message for any morphological or cytochemical investigation of apoptosis is to employ more than one detection method and run multiple controls .

O O O O O B I O B O O O B I O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B I B I O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B I O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B I O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B I O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B I O B O O O O O B I O O O O O O O O O O O O O O B O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B I O O O O O O O O O O O O O O O O O B O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O

apoptosis
cell death
morphology
microscopy
immunostaining


Predicting the FSH threshold dose in women with WHO Group II anovulatory infertility failing to ovulate or conceive on clomiphene citrate BACKGROUND The objective of this investigation was to establish independent predictors of follicle-stimulating hormone -LRB- FSH -RRB- threshold dose in anovulatory women undergoing ovulation induction with FSH preparations . Introduction Approximately 20 -- 30 % of the women seeking fertility treatment present with anovulation -LRB- Healy et al. , 1994 -RRB- . Clomiphene citrate has been used as the first line treatment for inducing ovulation in anovulatory women . In most women who failed to ovulate or conceive with clomiphene citrate , ovarian stimulation with gonadotrophins leads to ovulation -LRB- Coelingh-Benning et al. , 1998 ; Yarali et al. , 1999 ; Platteau et al. , 2006 ; Balen et al. , 2007 -RRB- . The use of low-dose step-up protocols has been shown to be successful in producing mono-follicular development in more than half of the women undergoing stimulation with gonadotrophins , and thereby minimizing the risk of multiple follicular development and the associated risks of ovarian hyperstimulation syndrome -LRB- OHSS -RRB- and multiple pregnancy -LRB- Homburg and Howles , 1999 -RRB- . A typical low-dose step-up protocol starts with 50 -- 75 IU/day of follicle-stimulating hormone -LRB- FSH -RRB- or menotrophins for ∼ 7 to 14 days , and the dose is then increased by 37.5 -- 50 IU/day if adequate response is not obtained . For some patients , the dose of gonadotrophin needed to provide adequate response is relatively high and the use of low-dose regimens will in these cases lead to stimulations of long duration . A long ovarian stimulation is associated with more monitoring visits and would be expected to result in increased costs and inconvenience for the patients . Selection of the most optimal starting dose of gonadotrophin for each individual patient should minimize these disadvantages , while ensuring adequate response . Imani et al. -LRB- 1998 -RRB- have identified clinical and laboratory parameters such as body mass index -LRB- BMI -RRB- , menstrual cycle history , mean ovarian volume and free androgen index -LRB- FAI -RRB- which can predict ovulation in WHO type II anovulatory women undergoing clomiphene citrate treatment . A similar investigation in anovulatory women undergoing ovulation induction with gonadotrophins suggested BMI , clomiphene citrate resistance , serum insulin-like growth factor-1 -LRB- IGF-1 -RRB- and serum FSH as significant predictors of FSH threshold dose -LRB- Imani et al. , 2002 -RRB- . The clinical application of this latter result is complicated by the fact that the IGF-1 assay is not part of most clinics standard battery for laboratory screening of anovulatory patients . Therefore , further data are needed to identify simple parameters which can predict the FSH threshold dose in women who will undergo low-dose protocols with gonadotrophins . The purpose of the present prospective investigation was to evaluate the influence on the FSH threshold dose of clinical , sonographic and endocrine parameters available at the start of ovarian stimulation as well as type of FSH preparation to be used in a low-dose step-up protocol . The data from this investigation form the basis for constructing a nomogram which provides an indication of the expected FSH threshold dose for the individual patient , facilitating the selection of the most appropriate starting FSH dose for ovarian stimulation . Materials and Methods Study population and design This investigation included 151 women with anovulatory WHO Group II infertility who were included in a randomized , open label , assessor-blind , parallel group , multicentre , multinational , non-inferiority study comparing two different FSH preparations -LRB- highly purified -LRB- HP -RRB- - FSH , BRAVELLE , Ferring Pharmaceuticals A/S , Copenhagen , Denmark and recombinant FSH -LRB- rFSH -RRB- , follitropin alfa , GONAL-F , Serono , Geneva , Switzerland -RRB- with respect to ovulation rate . Patients were recruited in 22 fertility centres -LRB- 12 in Belgium , 7 in Denmark and 3 in the UK -RRB- that on average each included seven patients , ranging from 2 to 20 . A detailed description of the study population , all inclusion and exclusion criteria , all study procedures , as well as the main outcome of the study , is described in detail in a separate publication -LRB- Balen et al. , 2007 -RRB- . Patients were to be 18 -- 39 years of age with BMI 19 -- 35 kg/m2 , have been diagnosed with infertility for ≥ 1 year , have chronic anovulation -LRB- amenorrhea , oligomenorrhea or anovulatory cycles based on progesterone levels in patients with cycle lengths of 21 -- 35 days -RRB- and have failed to ovulate with clomiphene citrate doses of at least 100 mg/day for at least 5 days or failed to conceive after three cycles of ovulation induction with clomiphene citrate . Women with a history of ≥ 12 unsuccessful ovulation induction cycles , persistent ovarian cysts -LRB- ≥ 15 mm -RRB- for > 1 cycle or ovarian endometrioma on ultrasound and any significant systemic disease , endocrine or metabolic abnormalities were excluded . The study was carried out in accordance with the declaration of Helsinki on good clinical practice and ethical committee approval was obtained in all participating centres . All patients underwent screening assessments prior to start of ovarian stimulation . These included evaluation of demographics -LRB- age -RRB- , physical characteristics -LRB- body weight , height , waist and hip circumference -RRB- , obstetric history -LRB- previous pregnancies , previous live births -RRB- , infertility history -LRB- duration of infertility , number of previous ovulation induction cycles with clomiphene citrate and in total , failure to ovulate with clomiphene citrate , failure to conceive with clomiphene citrate -RRB- and menstrual cycle history . Just prior to the first dose of gonadotrophin , patients underwent a transvaginal ultrasound and blood sampling for endocrine evaluation . The sonographic recordings comprised dimensions of right and left ovary , number of follicles -LRB- antral follicles > 2 mm -RRB- in right and left ovary and endometrial thickness . The endocrine evaluation included the following parameters : luteinizing hormone -LRB- LH -RRB- , FSH , estradiol -LRB- E2 -RRB- , prolactin , total testosterone , sex hormone binding globulin -LRB- SHBG -RRB- , FAI , glucose and insulin . Chemiluminescent immunometric assays were used for LH , FSH , prolactin , total testosterone and SHBG , and radioimmunoassays were used for E2 and androstenedione , whereas a hexokinase assay was used for glucose and immulite technology was applied for analysis of insulin . All endocrine analyses were conducted by a central laboratory -LRB- Quest Diagnostics -- Quest Diagnostics Limited , Heston , UK ; Quest Diagnostics Limited , Van Nuys , CA , USA ; Nichols Institute , San Juan Capistrano , CA , USA -RRB- . FAI was calculated as -LRB- total testosterone/SHBG -RRB- × 100 . All patients underwent ovarian stimulation following a low-dose step-up protocol . Stimulation was started 2 -- 5 days after a spontaneous or progesterone-induced menstrual bleed . The starting dose of gonadotrophin was 75 IU daily , which was maintained for 7 days . After the first 7 days , the gonadotrophin dose was either maintained or increased by 37.5 IU increments according to individual response . Patients were maintained on any specific dose level for at least 7 days . The maximum allowed daily dose was 225 IU and patients were treated with gonadotrophin for a maximum of 6 weeks . Gonadotrophin stimulation was maintained until at least one of the following criteria for human chorionic gonadotrophin -LRB- hCG -RRB- administration was met : one follicle with a diameter of 17 mm or greater , or 2 -- 3 follicles with a diameter of 15 mm or greater . Patients were not given hCG in either of the following situations : no follicular response after 6 weeks of gonadotrophin treatment , ≥ 4 follicles with diameters of ≥ 15 mm or serum E2 levels > 2000 pg/ml . Patients who reached the hCG criteria received a single s.c. or i.m. injection of hCG -LRB- PROFASI , Serono -RRB- at a dose of 5000 IU to trigger ovulation . At least one blood sample was taken during the midluteal phase -LRB- 6 -- 9 days after hCG administration -RRB- and analysed for progesterone by a central laboratory using competitive immunoassay using direct chemiluminometric technology with a sensitivity of 0.48 nmol/l -LRB- Quest Diagnostics Limited , UK -RRB- . Ovulation was defined as a mid-luteal serum progesterone concentration of ≥ 25 nmol/l -LRB- ≥ 7.9 ng/ml -RRB- . The FSH threshold dose was defined as the dose of FSH that was given when the criteria for hCG was reached . The analysis involved the influence of the type of gonadotrophin preparation -LRB- i.e. HP-FSH or rFSH -RRB- as well as of clinical , sonographic and basal endocrine parameters on the FSH threshold dose . All potential predictive variables were investigated : age , BMI , waist -- hip ratio , previous pregnancy , duration of infertility , failure to ovulate on clomiphene citrate , menstrual cycle history -LRB- amenorrhea if the patient had absent menstruation , oligomenorrhea if menstruation intervals were longer than 35 days or cycle length of 21 -- 35 days in which case anovulation had been documented by progesterone levels -RRB- , total number of follicles , mean ovarian volume -LRB- based on length , width and depth of both right and left ovary -RRB- , LH/FSH ratio , androstenedione , total testosterone , SHBG , FAI , prolactin , estradiol , glucose , insulin and insulin/glucose ratio . Statistical analysis The primary end-point of the study was ovulation rate after one cycle of gonadotrophin treatment , and these data have been reported elsewhere -LRB- Balen et al. , 2007 -RRB- . The influence of the selected clinical , sonographic and endocrine parameters as well as type of gonadotrophin on FSH threshold dose were assessed in univariate and multivariate analyses using proportional odds polytomous logistic regression models -LRB- an extension of the model and method for binary response -RRB- . Multivariate analyses were performed including characteristics that had statistically significant influence on the FSH threshold dose in the univariate analysis first by entering all selected characteristics into the polytomous logistic model in a forward selection approach and secondly by entering all selected characteristics into the polytomous logistic model using a backward elimination approach . P-values of < 0.05 were regarded as statistically significant . Score tests were used to evaluate the proportional odds assumption and Wald tests were used to assess the influence of the baseline characteristics on the FSH threshold dose . For the present analysis , all parameters were analysed as continuous variables , except for type of gonadotrophin , previous pregnancy , failure to ovulate on clomiphene citrate and menstrual cycle history -LRB- glucose and insulin were categorical variables as normal/abnormal and insulin/glucose ratio was a continuous variable -RRB- . In addition to the above , the model was evaluated on the subset of patients with mono-follicular development -LRB- one follicle ≥ 17 mm and no follicles of 15 -- 16 mm -RRB- . In this evaluation , the same set of variables was used as selected for the main model according to the above procedure . The predictive performance of the model was assessed by using the ` leave-one-out ' cross-validation procedure . In addition , the residual standard error after a standard multiple regression analysis was evaluated . Finally , ROC curves were produced and AUC under the ROC curves were calculated . For the standard situation where ROC curves are used , the classification is binary , whereas the threshold dose had four possible outcomes -LRB- 87.5 , 112.5 , 150 and 187.5 -RRB- and the prediction model therefore also predicts into four classes . A consensus approach on how to handle multiclass ROC has not been reached -LRB- Landgrebe and Duin , 2007 -RRB- , but when the outcome classes are ranked , ROC curves can be made for each of the possible levels -LRB- Waegeman et al. , 2006 -RRB- . Results The clinical , sonographic and endocrine characteristics of the 151 patients included in the study are shown in Table I . The mean age of the study population was 28.9 years , ranging from 19 to 39 years . The BMI spanned from 18 to 39 kg/m2 , with 54 % of the patients having a normal BMI , 32 % being overweight and 14 % being obese and with the mean BMI being 24.8 kg/m2 . On average , patients had been infertile for ∼ 3 years and had undergone four previous ovulation induction cycles with clomiphene citrate , with 34 % of the patients failing to ovulate on clomiphene citrate . Regarding menstrual cycle history , 56 % of the patients were oligomenorrheic and 15 % amenorrheic . An LH/FSH ratio above 1 was noted for 59 % of the patients . Among the 151 patients who started stimulation with FSH -LRB- HP-FSH 73 , rFSH 78 -RRB- , 132 patients met the hCG criteria -LRB- HP-FSH 65 , rFSH 67 -RRB- and contributed with data to the analysis of FSH threshold dose . There were 19 patients who initiated gonadotrophin therapy but did not contribute to this analysis ; 10 discontinued from the study during ovarian stimulation -LRB- five because of excessive response -RRB- , 2 had spontaneous ovulation and 7 received hCG despite either not meeting or exceeding the hCG criteria . Table I. Clinical , sonographic and endocrine parameters just prior to starting ovarian stimulation with FSH -LRB- Day 1 of stimulation -RRB- . Total -LRB- n = 151 -RRB- Clinical parameters Age -LRB- years -RRB- 28.9 ± 3.7 Body weight -LRB- kg -RRB- 68.6 ± 13.2 BMI -LRB- kg/m2 -RRB- 24.8 ± 4.5 Primary infertility , n -LRB- % -RRB- 97 -LRB- 64 % -RRB- Duration of infertility -LRB- years -RRB- 2.8 ± 1.7 Previous cycles of ovulation induction -LRB- all -RRB- 4.9 ± 2.4 Previous cycles of ovulation induction -LRB- with clomiphene citrate -RRB- 4.1 ± 2.4 Clomiphene citrate non-responders , n -LRB- % -RRB- Failure to ovulate on clomiphene citratea 51 -LRB- 34 % -RRB- Failure to conceive on clomiphene citrateb 100 -LRB- 66 % -RRB- Menstrual cycle history , n -LRB- % -RRB- Amenorrhea 23 -LRB- 15 % -RRB- Oligomenorrhea 84 -LRB- 56 % -RRB- Anovulatory cycles with cycle length 21 -- 35 days 44 -LRB- 29 % -RRB- Sonographic parameters Mean ovarian volume -LRB- cm3 -RRB- 7.1 ± 3.5 Antral follicles > 2 mm 14.8 ± 12.5 Endometrial thickness -LRB- mm -RRB- 4.1 ± 2.2 Endocrine parameters LH -LRB- IU/l -RRB- 7.1 ± 4.6 FSH -LRB- IU/l -RRB- 5.5 ± 2.4 LH/FSH ratio 1.4 ± 0.8 Prolactin -LRB- μg/l -RRB- 16 ± 13 Androstenedione -LRB- nmol/l -RRB- 6.74 ± 3.08 Total testosterone -LRB- nmol/L -RRB- 1.8 ± 0.6 Sex hormone binding globulin -LRB- nmol/l -RRB- 61 ± 40 Free androgen index 4.45 ± 3.47 Estradiol -LRB- pmol/l -RRB- 172 ± 98 Glucose -LRB- mmol/l -RRB- Fasting 5.1 ± 0.6 Non-fasting 5.2 ± 1.5 Insulin -LRB- pmol/l -RRB- Fasting 66.4 ± 43.1 Non-fasting 143.9 ± 192.6 All data are mean ± SD or number of patients -LRB- percentage of patients -RRB- . aOn at least 5 days of 100 mg/day . bAfter three cycles . Table II provides the outcome of the univariate and multivariate analysis for the predictors of FSH threshold dose . Eight parameters were statistically significant predictors of FSH threshold dose -LRB- P < 0.05 -RRB- : age , BMI , failure to ovulate with clomiphene citrate , menstrual cycle history -LRB- amenorrhea , oligomenorrhea or anovulatory cycles with cycle length 21 -- 35 days -RRB- , mean ovarian volume , LH/FSH ratio , testosterone and FAI . The type of gonadotrophin preparation , HP-FSH or rFSH , did not predict FSH threshold dose . All eight significant parameters were included in a multivariate analysis which showed that only three of the parameters were independently statistically significant -LRB- P-values < 0.001 -RRB- predictors of FSH threshold dose : BMI , mean ovarian volume and menstrual cycle history -LRB- Table III -RRB- . The odds for needing > 75 IU FSH were 1.18 -LSB- 95 % confidence interval -LRB- CI -RRB- : 1.07 -- 1.29 -RSB- for BMI and 1.22 -LSB- 95 % CI : 1.09 -- 1.37 -RSB- for mean ovarian volume ; thus the higher the BMI and ovarian volume prior to start of stimulation , the higher the FSH threshold dose . Regarding the menstrual cycle history , the odds for needing a FSH dose > 75 IU were 11.9 -LSB- 95 % CI : 3.35 -- 42.0 -RSB- and 2.57 -LSB- 95 % CI : 0.97 -- 6.79 -RSB- for amenorrhea and oligomenorrhea , respectively , compared with anovulatory cycles with a length of 21 -- 35 days . The observed data for these three independent predictors are presented in Table III . Table II . Statistically significant predictors of FSH threshold dose -LRB- univariate and multivariate analysis -RRB- . Univariate analysis Multivariate analysis OR -LRB- 95 % CI -RRB- P-valuea OR -LRB- 95 % CI -RRB- P-valuea Age -LRB- years -RRB- 0.91 -LRB- 0.83 -- 1.00 -RRB- 0.040 -- -- BMI -LRB- kg/m2 -RRB- 1.10 -LRB- 1.02 -- 1.19 -RRB- 0.010 1.17 -LRB- 1.07 -- 1.29 -RRB- < 0.001 Failure to ovulate on clomiphene citrate : yes versus no 2.27 -LRB- 1.14 -- 4.52 -RRB- 0.020 -- -- Menstrual cycle history < 0.001 < 0.001 Amenorrhea versus cycle length 21 -- 35 days 8.33 -LRB- 2.81 -- 24.7 -RRB- 11.88 -LRB- 3.35 -- 42.1 -RRB- Oligomenorrhea versus cycle length 21 -- 35 days 3.48 -LRB- 1.51 -- 8.03 -RRB- 2.57 -LRB- 0.97 -- 6.79 -RRB- Mean ovarian volume -LRB- cm3 -RRB- 1.18 -LRB- 1.06 -- 1.31 -RRB- 0.002 1.22 -LRB- 1.08 -- 1.37 -RRB- < 0.001 Free androgen index -LRB- nmol/l -RRB- 1.25 -LRB- 1.13 -- 1.38 -RRB- < 0.001 -- -- Total testosterone -LRB- nmol/l -RRB- 2.14 -LRB- 1.14 -- 4.03 -RRB- 0.018 -- -- LH/FSH ratio -LRB- IU/l -RRB- 1.59 -LRB- 1.03 -- 2.45 -RRB- 0.038 -- -- aWald test for effect of factor/covariate . Table III . BMI , mean ovarian volume and menstrual cycle history by observed FSH threshold dose . BMI -LRB- kg/m2 -RRB- Mean ovarian volume -LRB- cm3 -RRB- Menstrual cycle history -LRB- % -RRB- Amenorrhea / oligomenorrhea / cycle length 21 -- 35 days 75 IU -LRB- n = 67 -RRB- 23.8 ± 4.2 6.3 ± 2.9 8 % / 48 % / 45 % 112.5 IU -LRB- n = 46 -RRB- 25.5 ± 4.8 7.3 ± 3.4 22 % / 63 % / 15 % 150 IU -LRB- n = 15 -RRB- 25.8 ± 4.0 9.5 ± 4.8 33 % / 47 % / 20 % 187.5 IU -LRB- n = 4 -RRB- 28.1 ± 4.6 10.2 ± 2.3 25 % / 75 % / 0 % All data are mean ± SD or percentage of patients . The model predicted the FSH threshold dose to be the same as the dose observed in the study for 59 % of the patients . The AUC under the ROC curve was 0.78 for 75 IU -LRB- Fig. 1a -RRB- and 0.79 for 112.5 IU -LRB- Fig. 1b -RRB- . In a multiple regression analysis with the same three variables as in the logistic model , the FSH threshold dose was estimated on a continuous scale . From this estimation , the residual mean square error was 25 IU and the R2 = 25 % . Figure 1 : ROC curves for threshold dose -LRB- a -RRB- 75 IU versus < 75 -LRB- AUC = 0.78 -RRB- and -LRB- b -RRB- ≤ 112.5 IU versus > 112.5 IU -LRB- AUC = 0.79 -RRB- . A simple nomogram can be constructed based on the two clinical and one sonographic parameters that significantly predict the FSH threshold dose . Figure 2 displays three nomograms -- one for each type of menstrual cycle history -- where the shaded areas indicate the predicted FSH threshold dose , based on the BMI displayed on the x-axis and mean ovarian volume on the y-axis . For example , the expected threshold FSH dose for a patient with amenorrhea -LRB- Fig. 2a -RRB- , mean ovarian volume of 10.5 cm3 and a BMI of 30 kg/m2 will be 150 IU . In the case that the patient presents with oligomenorrhea -LRB- Fig. 2b -RRB- , but similar mean ovarian volume and BMI , the expected threshold FSH dose would be 112.5 IU . As another example , a threshold FSH dose of 75 IU will be expected for a patient with anovulatory cycles of 21 -- 35 days , mean ovarian volume of 8.5 cm3 and BMI 27 kg/m2 , whereas it will be 112.5 IU for a patient with oligomenorrhea and similar mean ovarian volume and BMI . Figure 2 : Nomograms for prediction of individual FSH threshold dose in anovulatory patients undergoing ovulation induction with FSH preparations , according to menstrual cycle history , BMI and mean ovarian volume . -LRB- a -RRB- Amenorrhea , -LRB- b -RRB- oligomenorrhea and -LRB- c -RRB- anovulatory cycles with cycle length 21 -- 35 days . The accuracy of the model was 60 % when applied to the subset of patients with mono-follicular development -LRB- 80 patients -RRB- , and thus similar to that achieved for the overall study population . Discussion The main finding in the present study was identification of the three variables : menstrual cycle history , BMI and mean ovarian volume , as independent predictors of the FSH threshold dose during FSH treatment of anovulatory infertility . For clinical use , such data could be implemented and used as screening predictors to determine the individual FSH threshold dose of any patient before start of gonadotrophin treatment , in order to select the most appropriate starting dose . For this purpose , we have used the three independent predictors to develop the FSH dose nomograms presented in Fig. 2 . The proposed model had an accuracy of 59 -- 60 % , which is well above the 25 % that would be expected by random allocation to one of the four dose levels evaluated in this study . Our findings are partly in line with those obtained by Imani et al. -LRB- 2002 -RRB- who studied 90 anovulatory women , also treated with either urinary or rFSH , and showed that BMI , clomiphene resistance , free IGF-1 and basal FSH were independent predictors of the FSH threshold dose . Imani et al. -LRB- 2002 -RRB- made an equation showing that the predicted dose was : -LSB- 4 BMI -LRB- in kg/m2 -RRB- -RSB- + -LSB- 32 clomiphene citrate resistance -LRB- yes = 1 or no = 0 -RSB- + -LSB- 7 initial free IGF-I -LRB- in ng/ml -RRB- -RSB- + -LSB- 6 initial serum FSH level -LRB- in IU/l -RRB- -RSB- − 51 . Similar to our study , it was found that menstrual bleeding interval as well as clomiphene resistance were significant predictors in the univariate analysis , but among the possible clinical predictors only clomiphene resistance and BMI remained significant in the multivariable analysis . The population studied by Imani et al. -LRB- 2002 -RRB- seems very similar to the present study , even though the latter included patients if they failed to conceive after three clomiphene cycles , whereas the study by Imany et al. -LRB- 2002 -RRB- only included women with six failed ovulatory clomiphene citrate cycles , but it seems unlikely that this would explain the difference in predictive factors . A more plausible explanation could be that the study by Imani et al. -LRB- 2002 -RRB- included , in the forward step-wise multivariate analysis , some endocrine factors , such as free-IGF-I and its binding proteins , that may alter the predictors that remained in the final model during the analysis . It is worth briefly discussing a statistical methodological difference between the present model and that suggested by Imani et al. -LRB- 2002 -RRB- . The polytomous logistic regression model used on the present data set predicts FSH threshold doses that correspond to actual dose levels applied in the clinical trial , in contrast to a multiple regression where the predicted dose is on the continuous scale , as the equation published by Imani et al. -LRB- 2002 -RRB- . In our model , the residual mean square error was 25 IU and therefore the individual prediction is at least as good as in the model suggested by Imani et al. -LRB- 2002 -RRB- where the residual mean square error was 35 IU . The R2 value of 25 % in the present model is explained by the relatively few patients in the two highest dose levels in our trial compared with a more even distribution of patients in the trial conducted by Imani et al. -LRB- 2002 -RRB- yielding an R2 of 39 % in their model . Our finding that the menstrual status was important is in line with expectations . Patients with amenorrhea may need more FSH to reach the threshold than a patient with oligomenorrhea , who may also need more FSH compared with patients with anovulatory cycles , indicating that the severity of the ovarian dysfunction may well be reflected in the degree of menstrual cycle disturbance . The relative resistance to FSH in amenorrheic patients is supported by the finding that these patients also have a relative resistance to endogenously released FSH during clomiphene citrate treatment -LRB- Imani et al. , 1998 -RRB- , as patients with clomiphene resistance do release a similar amount of endogenous FSH as the clomiphene sensitive women -LRB- Polson et al. , 1989 -RRB- . This supports the concept that it is indeed the threshold of the ovarian sensitivity that is altered . A high BMI was associated with a higher FSH threshold dose ; an observation that is supported by the findings that the total dose of gonadotrophins needed to induce ovulation is increased in parallel with body weight -LRB- Hamilton-Fairly et al. , 1992 ; Balen et al. , 2006 -RRB- . The cause of the association with BMI may be that the endocrine disturbance is related to the degree of body fat and hyperinsulinaemia , but it may also be due to a lower bioavailability of exogenous gonadotrophins in obese women -LRB- Chan et al. , 2003 ; Steinkampf et al. , 2003 -RRB- . The type of gonadotrophin used for stimulation was not a significant predictor of FSH threshold dose , and thus of limited relevance compared with the patient 's clinical characteristics . In theory , the threshold is defined as the lowest dose that induces growth of the single most sensitive follicle . In this clinical trial , a dual criterion for hCG administration was used , covering not only the development of a single dominant follicle , defined as one follicle of 17 mm or greater , but also the presence of 2 -- 3 follicles of 15 mm or greater . Therefore , our threshold is based on the stimulation in those 132 -LRB- 87 % -RRB- patients who reached either of these criteria , including those patients with up to 3 follicles ≥ 15 mm . The threshold dose in the study and subsequently the model could also be termed the ` response dose ' and can be viewed as a surrogate for the real threshold . It should be considered that serum FSH may accumulate over time so the theoretically ideal threshold should be defined as the dose that caused growth of a single dominant follicle to around 9 -- 10 mm , where dominance is normally established . The exact timing of dominance was not determined in the present study , but it is important to note that the model for threshold dose predictions also applied in the subset of patients with mono-follicular development defined as only one follicle ≥ 17 and no follicles of 15 -- 16 mm . The average number of follicles ≥ 15 mm was 1.5 -LRB- Balen et al. , 2007 -RRB- indicating that some patients did indeed receive FSH doses slightly above the threshold for single dominant follicle development . However , the analysis restricted to patients with mono-follicular development showed that the accuracy of the model was the same for this subset of patients as for the overall study population . On the basis of the intention to treat population in the present study , 92 % of the patients received hCG and the ongoing pregnancy rate was 19 % , of which 79 % accounted for singletons and 21 % for multiple pregnancies -LRB- Balen et al. , 2007 -RRB- . These figures are much in line with literature data on 7 day step-up protocols -LRB- Homburg and Howles , 1999 -RRB- , but it could be argued that the multiple pregnancy rate is somewhat high , compared with a number of recent studies using starting doses of FSH < 75 IU/day -LRB- Balasch et al. , 2000 ; Calaf Alsina et al. , 2003 -RRB- . Indeed , multiple pregnancy rates as low as 6 % have been reported in a large clinical trial using starting doses of 50 IU/day , even though up to 3 follicles > 16 mm before hCG administration was accepted -LRB- Calaf Alsina et al. , 2003 -RRB- . An alternative approach is to restrict hCG administration to those patients who have only one or two mature follicles , but this would increase the cancellation rates . As published -LRB- Balen et al. , 2007 -RRB- , 3.3 % -LRB- 5/151 -RRB- of the patients had their cycle cancelled before hCG , due to development of multiple follicles . Identification of these patients with a very low threshold would have necessitated a starting dose of < 75 IU/l . Ovarian volume was another significant parameter predicting the FSH threshold dose . The larger the ovaries , the more FSH was needed to obtain adequate ovarian response . This finding is consistent with earlier clinical studies of ovulation induction with gonadotrophins , where it has been shown that patients with larger ovaries need longer duration of stimulation and higher doses of gonadotrophins -LRB- Lass et al. , 2002 -RRB- . Van den Meer et al. -LRB- 1994 -RRB- studied three consecutive cycles in 16 patients during gradually increased doses of intravenously infused urinary FSH and elegantly documented how the inter-individual variability in FSH threshold dose was far greater than the variability from cycle to cycle . The key problem is therefore to define the appropriate dose for any individual patient and the present study provides a suggestion for nomograms that may be used clinically . A major concern , if patients were dosed according to the nomograms , would be that 13 % would receive a higher starting dose that the observed threshold dose . The implications of an excessive starting dose are likely to be the same as when step-down protocols are used , i.e. risk of multiple follicular development . Indeed , van Santbrink et al. -LRB- 2002 -RRB- have used the prediction model developed by the same group of investigators -LRB- Imani et al. , 2002 -RRB- , and retrospectively evaluated the doses used in a step-down regimen . In that study , even though the average excessive dose was 28.5 IU/day above the threshold , the step-down approach resulted in an overall multiple pregnancy rate of 17 % and not a single case of OHSS -LRB- van Santbrink et al. , 2002 -RRB- . The expected main advantage of dosing according to the nomogram would be a shortening of the stimulation phase . It is important to note that the suggested dosage nomograms could be easily used clinically because they only include simple characteristics that are routinely recorded in all anovulatory patients . Before using this model in clinical practice , a randomized controlled trial should be conducted to compare the presently used low-dose step-up protocol with a flexible protocol using this model . Funding The study was sponsored by Ferring Pharmaceuticals A/S , Copenhagen , Denmark . Funding to pay the Open Access publication charges for this article was provided by Ferring Pharmaceuticals A/S , Copenhagen , Denmark .

O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O 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