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Hum. Reprod. Advance Access originally published online on June 2, 2007
Human Reproduction 2007 22(8):2249-2253; doi:10.1093/humrep/dem130
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© The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Highly specific and sensitive rise in Days 14–17 pro-{alpha}C inhibin with clinical pregnancy after frozen embryo transfer with ovulatory cycles

S. Tong1,4, L. Rombauts1,2, J. Onwude3, B. Marjono1 and E.M. Wallace1

1 Centre for Women's Health Research, Department of Obstetrics and Gynaecology, Monash Medical Centre, Monash University, 246 Clayton Road, Clayton 3168, Victoria, Australia 2 Monash IVF, Clayton 3168, Victoria, Australia 3 Capio Springfield Hospital, Lawn Lane, Chelmsford CM1 7GU, UK

4 Correspondence address. Tel: +613-9594-5489; Fax: +613-9594-6389; E-mail: stephentong1{at}gmail.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
BACKGROUND: Pro-{alpha}C inhibins are luteal derived analytes peaking in the maternal serum as early as Day 16 after conception. We set out to verify a previous post hoc analysis which suggested that pro-{alpha}C levels measured this early are extremely sensitive in predicting clinical pregnancy success after unstimulated IVF with ovulatory cycles.

METHODS: Prospective observational study of 246 women undergoing frozen embryo transfer with ovulatory cycles. Serum pro-{alpha}C and beta-HCG levels at 14–17 days after conception were measured by enzyme-linked immunosorbent assay and grouped according to whether a clinical pregnancy occurred (demonstrable cardiac activity at ≥6 weeks' gestation).

RESULTS: Of 34 (13.8%) women who achieved a clinical pregnancy, median (25th–75th centile) Days 14–17 pro-{alpha}C levels were 995 pg/ml (758–1463), 6- to 7-fold higher than levels observed in the remainder who did not fall pregnant (112.8 pg/ml (104–121); P < 0.0001). At a fixed 95% specificity, pro-{alpha}C was 100% sensitive in predicting clinical pregnancy. The best specificities achieved at 100% sensitivity were; 94.8% for pro-{alpha}C, 96.7% for beta-HCG and 98.1% when both analytes were combined.

CONCLUSIONS: Clinical pregnancy is always associated with a release of luteal derived pro-{alpha}C 14–17 days after conception. Pro-{alpha}C may play a possible biological role and be a useful clinical biomarker of luteal health.

Key words: pro-{alpha}C/IVF/luteal/inhibin/frozen embryo transfer


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The inhibins are glycoprotein hormones with diverse roles in reproduction (de Krester et al., 2002; Tong et al., 2003). The final {alpha}- and beta-subunits that make up mature dimeric inhibin are products of larger precursor proteins proteolytically cleaved in the cytoplasm. The pro-{alpha}C inhibin assay (Groome et al., 1995Go) detects these precursors but not mature inhibin (Fig. 1), since it utilises an antibody raised against the ‘pro’ region of the precursor {alpha}-subunit, sliced away during processing.


Figure 1
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  		Figure 1: Different inhibin forms, adapted from Groome et al. (1995) (A) and (B) are precursors of (C), mature dimeric inhibin A, where the pro regions of both precursors are proteolytically cleaved and linked by two cysteine bonds. (D) In addition to these forms, confirmed by Groome et al. (1995) to be detected by the pro-{alpha}C assay, other forms with various combinations of the unprocessed subunits may be detected by the assay (e.g. Pro-{alpha}N-{alpha}C/Probeta-betaA: and combinations with Probeta-betaB, the precursor of the inhibin B beta-subunit)

 
During early pregnancy, serum pro-{alpha}C analyte levels are 10-fold that of mature inhibin and display a very different profile across pregnancy (Illingworth et al., 1996Go; Fowler et al., 1998Go). While these facts raise the possibility that pro-{alpha}C analytes are not merely inactive precursors but serve biological roles distinct from dimeric inhibin, they have so far not been shown to be bioactive (Lahiri et al., 2003Go).

Noting that serum pro-{alpha}C inhibin peaks as early as Day 16 after conception (Illingworth et al., 1996Go; Fowler et al., 1998Go), we previously investigated an unselected IVF cohort and observed that serum levels at this gestation can predict the presence of a clinical pregnancy with a sensitivity of 83%, at 95% specificity (Tong et al., 2004Go).

Since pro-{alpha}C is luteal derived (Tong et al., 2004Go), we did a post hoc analysis excluding those who underwent hyper-stimulated cycles (where large variations in luteal tissue abundance probably impacted on its performance as a biomarker) and those who underwent anovulatory frozen embryo transfer (ET) protocols (as there would have been no luteal tissue to secrete pro-{alpha}C). Among 118 women whom underwent frozen ET and ovulatory protocols, we found that the sensitivity of pro-{alpha}C in predicting clinical pregnancy rose to 100%, at 95% specificity (Tong et al., 2004Go). This suggests that a very tight biological association exists between a rise in pro-{alpha}C inhibin and evolving clinical pregnancy.

We therefore undertook this study to confirm these findings with the a priori hypothesis that serum pro-{alpha}C levels measured 14–17 days after frozen ET cycles using ovulatory protocols is highly accurate in predicting clinical pregnancy. Importantly, observations made from these women undergoing unstimulated ET with ovulatory cycles may reflect the luteal biology of spontaneous pregnancy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects and samples
We undertook an observational study of 246 women undergoing IVF with a frozen ET using protocols resulting in an ovulatory cycle, and therefore the presence of luteal tissue.

One hundred and twenty-eight of the 246 cases were identified and assayed from a serum bank prospectively collected for the study of potential biomarkers. To increase power, we combined these with 118 cases in the original post hoc analysis previously reported in brief (Tong et al., 2004Go). These two groups, sourced from the same serum bank and collected contemporaneously, were combined after confirming that their performance in predicting outcome was similar.

Blood was collected at Days 12–14 post ET for beta-HCG and pro-{alpha}C inhibin assay. During the study period, embryos were transferred at a time corresponding to 2–3 days after egg collection and fertilisation; the gestation at blood collection therefore corresponds to 14–17 days after conception; or by convention, 4 weeks' gestation.

Baseline patient information was collected, including age, IVF treatment cycle number and the gestational age (in days) when the serum was collected. Women were grouped according to whether they became clinically pregnant, defined by an ultrasound scan at around 6–7 weeks' gestation confirming the presence of one, or more, sacs with a fetal pole and a visible fetal heart beat. The clinically non-pregnant group included both women with a negative pregnancy test and those who had had an initially positive pregnancy test but ultimately no demonstrable viable gestation on ultrasound (i.e. biochemical pregnancies). Approval for the study was granted by the Monash IVF Ethics Committee (Meeting date 8 April 2002).

Treatment protocols
The oocytes used were collected from a previous fresh ET transfer cycle and fertilised in vitro either with or without sperm microinjection. Embryos were maintained in culture for 2–3 days before freezing.

We only included frozen ET regimens that would have resulted in the presence of endogenous luteal tissue. Thus, the two major protocols that we included were a ‘natural cycle’, or a ‘clomiphene citrate’ regimen. The first involves timing the frozen ET with the patients' spontaneous LH surge. The second utilises 25–50 mg clomiphene citrate from Day 2 to Day 6 of the cycle to induce follicle maturation, and timing ET using the spontaneous LH surge or after ovulation induction with HCG. We excluded artificial cycles using oral estradiol valerate and vaginal progesterone as these results in anovulatory cycles.

Assays
Serum beta-HCG (immunometric assay) and progesterone (competitive immunoassay) were assayed at the time of blood collection using an automated system (VitrosECi /OrthoDiagnostics/ Rochester, NY, USA). Excess serum was frozen at –20°C for subsequent measurement of pro-{alpha}C inhibin using a commercially available enzyme-linked immunosorbent assay (Oxford Bionnovation, Oxford). All assays were done according to manufacturer's instructions. The coefficient of variation (CV) was calculated by performing all assays in duplicate (intra-assay CV), and by using the same universal standard between all plates (inter-assay CV). The intra- and inter-assay CV for all assays was <10%. All assays were undertaken blinded to the clinical outcome. The lowest value that the beta-HCG assay reports is <1 mIU/ml, which has been given the value of 1 mIU/ml.

Statistical analyses
All comparisons were undertaken using chi-square for categorical data; and parametric, or non-parametric tests depending on whether or not the data was normally distributed. [Levels of all analytes were not normally distributed. Therefore, data for these variables were expressed as median (25th–75th centiles).]

The cut-off levels for assessing the performance of pro-{alpha}C inhibin and beta-HCG in predicting viable pregnancy were determined by non-parametric receiver operating characteristic analysis. Statistical tests were done using state 8.1 (Stata Corporation, NJ, USA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The mean (SD) age of all women in the study was 34.6 (4.9) years and the median IVF treatment cycle number was four. The median gestation of blood collection was day 16 (range 14–17). None of these baseline variables differed between the non-pregnant and clinical pregnancy groups (Table 1). Pro-{alpha}C inhibin levels did not vary across Days 14–17.


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  		Table 1: Baseline characteristics of 246 women undergoing frozen embryo transfer with ovulatory cycles

 
Of the 246 frozen ET cycles, there were 34 (13.8%) pregnancies detected at early pregnancy ultrasound. All were singleton pregnancies, except one set of twins. Fig. 2 depicts the spread of Days 14–17 maternal serum pro-{alpha}C inhibin levels in the non-pregnant and clinically pregnant groups. The median (25th–75th centile) pro-{alpha}C inhibin levels in the non-pregnant group were 112.8 pg/ml (103.5–121.4), significantly lower than the clinically pregnant group (995 pg/ml (757.6–1463); P < 0.0001). Median (25th–75th centile) beta-HCG levels in the non-pregnant group were 1(1–1) mIU/ml, also significantly lower than the clinically pregnant group (265 mIU/ml (161–423); P < 0.0001).


Figure 2
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  		Figure 2: Days 14–17 maternal serum pro-{alpha}C levels in women undergoing frozen embryo transfer with ovulatory cycles. Dotted line at the 403-pg/ml cut-off which provides 100% sensitivity at 95% specificity in predicting clinical pregnancy (see text). ({blacksquare}) Not clinically pregnant, ({blacktriangleup}) clinically pregnant

 
At 95% specificity, a pro-{alpha}C level of 403 pg/ml at 14–17 days after ET afforded 100% sensitivity in the prediction of a successful clinical pregnancy. At the same specificity, a beta-HCG cut-off of 23 mIU/ml also yielded 100% sensitivity. At a fixed 100% sensitivity, the best specificities achieved were 94.8% for pro-{alpha}C inhibin, 96.7% for beta-HCG and 98.1% when both analytes were combined (Table 2).


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  		Table 2: Performance of beta-HCG and Pro – {alpha}C in detecting viable pregnancies; results of receiver operating characteristic analysis

 
Of 14 biochemical pregnancies (beta-HCG > 5, but did not become clinically pregnant), five had pro-{alpha}C levels under 403 pg/ml, meaning that they would have been identified as non-viable.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have shown that by the end of the first week of implantation there is increased production of one or more of the various inhibin forms detectable by the pro-{alpha}C inhibin assay (Fig. 1), that are secreted from luteal tissue (Illingworth et al., 1996Go; Tong et al., 2004Go). This rise in pro-{alpha}C in the maternal serum appears highly sensitive and specific to a subsequent clinical pregnancy. Although an IVF cohort was studied in order to obtain bloods so early in gestation, it is important to note that our study is likely to reflect the luteal biology of spontaneous pregnancies since we specifically excluded hyper-stimulated (fresh ET) cycles, and those who had anovulatory cycles.

Although pro-{alpha}C has a very strong association with clinical pregnancy in non-hyper-stimulated ovulatory cycles and indeed improves on the performance of the current marker beta-HCG, the relatively small gains mean that it is unlikely to be adopted as a clinical biomarker in the exact setting we have studied. However, as a product highly specific to both the corpus luteum and pregnancy success, it merits further investigation as a possible luteal biomarker in other situations. For instance, there has been considerable effort in finding a test in very early pregnancy which can tease out who will be among the 30% undergoing IVF fated to have a ‘biochemical’ pregnancy. Various studies exploring various cut-offs in beta-HCG levels at Days 12–16 have developed models that identify biochemical pregnancies with around 75–85% sensitivity and 79–83% specificity (Qasim et al., 1996Go; Bjercke et al., 1999Go; Sugantha et al., 2000Go; Poikkeus et al., 2002Go). Being of a different tissue source, pro-{alpha}C may potentially add to performance of the model. Furthermore, as a possible marker reflecting luteal health, pro-{alpha}C may be a novel tool to investigate luteal phase defect, still quoted recently by some authorities to be involved in 10–28% of recurrent miscarriage (Potdar and Konje, 2005Go). Finally, it might have a role as a non-invasive marker to discriminate the likelihood of a pregnancy being intra-uterine or ectopic during very early pregnancy, an area of active research (Gevaert et al., 2006Go).

The biology of circulating inhibin precursor forms detected by the pro-{alpha}C assay has received little interest compared to dimeric inhibin since they are widely regarded to lack biological activity (Tong et al, 2004; Lahiri et al., 2003Go). This premise is strengthened by the fact that pregnancies can occur in anovulatory IVF cycles where exogenous progesterone alone seems sufficient as luteal support. However, there is now a significant body of evidence to raise the possibility that certain pro-{alpha}C molecular weight forms may be biologically active and play a role in the establishment of pregnancy.

First, there appears to be a highly specific and early surge of pro-{alpha}C from the corpus luteum, specifically timed to peak as early as at Day 16 after conception (Illingworth et al., 1996Go; Fowler et al., 1998Go), a rapid maternal response to pregnancy. We have now shown that this occurs consistently with all clinical pregnancies (100% sensitivity). Such a highly predictable rise would be in keeping with some sort of biological role.

Secondly, several lines of evidence would suggest that the various {alpha}-subunit forms detected by the pro-{alpha}C assays may be more than inactive precursors of mature dimeric inhibin (Thirunavukarasu et al., 2001Go). The serum profile of pro-{alpha}C across pregnancy (Illingworth et al., 1996Go; Fowler et al., 1998Go) and likely tissue source (Lahiri et al., 2003Go; Thirunavukarasu et al., 2001Go) are very different to inhibin A. Also, two studies have shown that it is possible to artificially induce differential secretion of pro-{alpha}C and inhibin A. Lahiri et al. (2003) looked at the response of several analytes to mifepristone given to a cohort undergoing medical termination of pregnancy. In response to this progesterone antagonist, an early acute decline of serum pro-{alpha}C occurred, seen much earlier than the subsequent fall in inhibin A, beta-HCG and progesterone. In a non-pregnant cohort, the administration of GnRH to down-regulate the pituitary caused a decrease in inhibin A secretion, but pro-{alpha}C levels remained unchanged (Casper et al., 2001Go).

Thirdly, indirect evidence exists suggesting that {alpha}-subunit forms may indeed have biological activity. In the study of pregnant women given mifepristone, Lahiri et al. (2003) concluded that their findings implicate pro-{alpha}C analytes in having a role in ovarian steroidogenesis. In vitro studies on marmoset luteal cells found that antibodies directed at the aminoterminal sequence of the {alpha}-subunit decreased progesterone output, again suggesting a local ovarian paracrine role (Webley et al., 1994Go). It has also been shown that active immunisation of ewes against the aminoterminal sequence on the {alpha}-subunit can decrease the fertility of ewes, possibly by inhibiting a role in ovulation (Findlay et al., 1994Go). Thus, animal and human studies point to a possible role in ovarian steroidal/luteal physiology.

We have demonstrated that clinical pregnancy is always associated with an early rise of luteal derived pro-{alpha}C. Further studies may be worthwhile to determine whether any of the inhibin forms detectable by the pro-{alpha}C assay have potential use as a clinical biomarker of luteal health or early ectopic pregnancies, or a biological role in aiding the establishment of early pregnancy. If a biological role in early pregnancy is subsequently demonstrated, then the possibility exists of exploiting this therapeutically as an adjunct to fertility treatment.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Bjercke S, Tanbo T, Dale PO, Morkrid L, Abyholm T. Human chorionic gonadotrophin concentrations in early pregnancy after in-vitro fertilization. Human Reprod (1999) 14:1642–1646.[Abstract/Free Full Text]

Casper FW, Seufert RJ, Schaffrath M, Pollow K. Concentrations of inhibins and activin in women undergoing stimulation with recombinant follicle-stimulating hormone for in vitro fertilization treatment. Fertil Steril (2001) 75:32–37.[CrossRef][Web of Science][Medline]

de Kretser DM, Hedger MP, Loveland KL, Phillips DJ. Inhibins, activins and follistatin in reproduction. Hum Reprod Update (2002) 8:529–541.[Abstract/Free Full Text]

Findlay JK, Russell DL, Doughton B, Tsonis CG, Borchers C, Forage RG. Effect of active immunisation against the amino-terminal peptide (alpha N) of the alpha 43 kDa subunit of inhibin (alpha 43) on fertility of ewes. Reprod Fertil Dev (1994) 6:265–267.[CrossRef][Medline]

Fowler PA, Evans LW, Groome NP, Templeton A, Knight PG. A longitudinal study of maternal serum inhibin-A, inhibin-B, activin-A, activin-AB, pro-alphaC and follistatin during pregnancy. Hum Reprod (1998) 13:3530–3536.[Abstract/Free Full Text]

Gevaert O, De Smet F, Kirk E, Van Calster B, Bourne T, Van Huffel S, Moreau Y, Timmerman D, De Moor B, Condous G. Predicting the outcome of pregnancies of unknown location: Bayesian networks with expert prior information compared to logistic regression. Hum Reprod (2006) 21:1824–1831.[Abstract/Free Full Text]

Groome NP, Illingworth PJ, O'Brien M, Priddle J, Weaver K, McNeilly AS. Quantification of inhibin pro-alpha C-containing forms in human serum by a new ultrasensitive two-site enzyme-linked immunosorbent assay. J Clin Endocrinol Metabol (1995) 80:2926–2932.[Abstract/Free Full Text]

Illingworth PJ, Groome NP, Duncan WC, Grant V, Tovanabutra S, Baird DT, McNeilly AS. Measurement of circulating inhibin forms during the establishment of pregnancy. J Clin Endocrinol Metab (1996) 81:1471–1475.[Abstract]

Lahiri S, Anobile CJ, Stewart P, Ledger WL. Changes in circulating concentrations of inhibin A and pro-alpha C during first trimester medical termination of pregnancy. Hum Reprod (2003) 18:744–748.[Abstract/Free Full Text]

Poikkeus P, Hiilesmaa V, Tiitinen A. Serum beta-HCG 12 days after embryo transfer in predicting pregnancy outcome. Human Reprod (2002) 17:1901–1905.[Abstract/Free Full Text]

Potdar N, Konje JC. The endocrinological basis of recurrent miscarriage. Curr Opin Obstet Gynecol (2005) 17:424–428.[Web of Science][Medline]

Qasim SM, Callan C, Choe JK. The predictive value of an initial serum beta human chorionic gonadotrophin concentration for pregnancy outcome following in vitro fertilization. J Assist Reprod Genet (1996) 13:705–708.[CrossRef][Web of Science][Medline]

Sugantha SE, Webster S, Sundar E, Lenton EA. Predictive value of plasma human chorionic gonadotrophin following assisted conception treatment. Hum Reprod (2000) 15:469–473.[Abstract/Free Full Text]

Thirunavukarasu P, Stephenson T, Forray J, Stanton PG, Groome NP, Wallace EM, Robertson DM. Changes in molecular weight forms of inhibin A and Pro-{alpha}C in maternal serum during human pregnancy. J Clin Endocrinol Metab (2001) 86:5794–5804.[Abstract/Free Full Text]

Tong S, Wallace EM Burger HG. Inhibins and activins: clinical advances in reproductive medicine. Clin Endocrinol (2003) 58:115–127.[CrossRef][Medline]

Tong S, Rombauts L, Mulder A, Marjono B, Onwude JL, Wallace EM. Increased day 15–17 serum pro-{alpha}C inhibin levels specific to successful pregnancy. J Clin Endocrinol Metab (2004) 89:4464–4468.[Abstract/Free Full Text]

Webley GE, Marsden PL, Knight PG. Differential control of immunoreactive {alpha}-inhibin and progesterone production by marmoset luteal cells in vitro: evidence for a paracrine action of {alpha}-inhibin on basal and gonadotrophin-stimulated progesterone production. Biol Reprod (1994) 50:1394–1314.[Abstract]

Submitted on February 13, 2007; resubmitted on April 16, 2007; accepted on April 19, 2007.


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