Response of proopiomelanocortin and gonado- or lactotroph systems to in-vitro fertilisation procedures stress

1. Introduction 

Artificial reproductive technologies (ART), especially in-vitro fertilisation (IVF) are associated with emotional and physical stress, and its success may in part depend on differential modes of coping and experiencing anxiety stress and depression [1]. The hypothalamic–pituitary–adrenal (HPA) axis is activated under these conditions. This leads to the release of proopiomelanocortin (POMC) derivatives from the pituitary gland into the cardiovascular compartment. There is only little information on the release of authentic β-endorphin [β-endorphin (1–31)] β-lipotropin as corticotroph POMC derivatives or on the release of melanotroph-type POMC derivatives such as N-acetyl-β-endorphin under these conditions. In addition, methodological inaccuracies such as missing differentiation between β-endorphin, its nine derivatives and β-lipotropin, were responsible for contradictory reports on this topic.

To investigate the corticotroph stress response to IVF we measured ACTH, β-lipotropin (β-LPH) immunoreactive material (IRM), β-endorphin IRM and cortisol in the plasma before and after oocyte retrieval. With a ‘consortium’ of several conventional radioimmunoassays (RIAs) we were well equipped to differentiate between immunoreactive POMC fragments. For determination of authentic β-endorphin [β-endorphin (1–31)] we have developed a highly specific two-site fluid phase immunoprecipitation radioimmunoassay which did not cross-react with any β-endorphin derivative or any other opioid peptide tested (Fig. 1). As an indicator of the melanotroph (“melanotroph cell type”) stress response of the pituitary gland, we also determined Ac-N-β-endorphin IRM (NAC) levels. NAC and authentic β-endorphin have never before been analysed at IVF conditions.

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Fig. 1. Schematic representation of enzymatic processing products of proopiomelanocortin. Signal peptide; N-terminal fragment; γ-MSH; joining peptide (JP); ACTH; β-lipotropin; β-endorphin (END) (1–31); β-END (1–27); β-END (1–26); β-END (1–17); β-END (1–16)+five acetyl-N-β-END derivatives. β-END is usually determined in the plasma using assays which do not differentiate between β-END (1–31) and its derivatives. The principle of these conventional “one-site-radioimmunoassays” is: a polyclonal antibody (AB) is directed against a certain sequence of β-END, which is radioactively labelled. β-END in a sample has the same amino acid sequence and so it competes with the labelled β-END for the antibody binding site. All kinds of β-END derivatives do that, in case they have the same antibody epitope as β-END. Thus, because of cross-reactivities one-site-RIAs are unspecific: not only β-END is determined, just β-END immunoreactive material, which may include various β-END- or even POMC-derivatives (grey arrows). In contrast, a “two-site-RIA” is highly specific for β-END (1–31). The N-terminus of β-END is recognised by a first labelled antibody. The C-terminus of β-END is bound to a second antibody. The second antibody again is bound to a third antibody which is precipitated. Since the radioactive signal is only precipitated when β-END is between these two antibodies like a ‘sandwich’, authentic β-END (1–31) is detected exclusively by this assay (black arrow).

In addition, a possible relationship between hormones of the HPA axis and those of the hypothalamic–pituitary–gonadal (HPG) axis was examined. We also measured prolactin (PRL), luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol (E2) and progesterone (P) in plasma to assess any hormonally induced stress response.

2. Methods 

Twenty-eight patients undergoing their first IVF treatment were investigated (age: mean 29.6±S.D. 3.6 years). The inclusion criteria were: healthy female partners, regular menstrual cycles of 26–34 days and body mass index (BMI) between 20 and 25kg/m2. The exclusion criteria were: acute illness, chronic disorders, organic dysfunction, hormonal imbalance, medication (e.g. psychotropic drugs) and history of pregnancy complications. All patients underwent controlled ovarian stimulation with hMG (Menogon®) starting with 150I.U. (2amp.) on day 4 until day 10 (s.c.). There after the hMG dose was adjusted according to the follicle size. Ovulation induction was performed with human chorionic gonadotropin (hCG, Predalone®), and 10.000I.U. i.m. Buserelin acetate (Suprecur®), an GnRH agonist, was given intranasal three times a day until the ovulation induction. Luteal support was given. 36h after hCG administration oocytes were retrieved by transvaginal ultrasound-directed oocyte recovery. Our investigation was carried out after all patients had signed informed consent forms to donate an additional blood sample for determination of POMC derivatives. Patients received 5–10mg midazolame and 75–150mg ketamine intravenously and oxygen by mask at 3l/min for the anaesthesia during the oocyte recovery procedure. Blood-pressure, heart rate, respiration and O2-saturation were continuously observed.

Blood samples were obtained before induction of anaesthesia (tA) and immediately after oocyte retrieval (tB) in all patients. Blood was centrifuged immediately after withdrawal (1000g/15min at 4°C), plasma was separated and aliquots were saved for determination of ACTH, cortisol, PRL, E2, P, LH and FSH. 10ml plasma was acidified with 1.0M HCl (0.1ml/ml plasma) to block enzymatic degradation of β-endorphin and its derivatives and, in addition, to prepare the samples for acidic extraction. The samples were stored at −20°C until further processing.

3. Results 

Concentrations of βH-endorphin (1–31) and NAC were very low to minimal at all times showing that both authentic β-endorphin and the melanotroph pituitary POMC system did not react to IVF stress under experimental conditions.

Plasma levels of ACTH, β-endorphin IRM and β-LPH IRM were found to be significantly increased immediately after oocyte retrieval (tB) as compared to tA (before induction of anaesthesia) (Table 1, Fig. 2a and b).

Table 1. Statistical significance of differences between steroid, pituitary hormone or POMC derivative concentrations in plasma before induction of anaesthesia (tA) versus immediately after oocyte retrieval (tB) (n=28)

	Parameter	p value
	βH-endorphin (1–31)	1a
	β-endorphin IRM	0.004a
	β-LPH IRM	0.001b
	ACTH	0.001b
	Cortisol	0.512b
	Ac-N-β-endorphin IRM	0.63a
	E2	0.002b
	P	0.026b
	PRL	0.001b
	LH	0.115b
	FSH	0.001b

p values <0.05 are in bold letters.

a Mc Nemar test. b Wilcoxon signed rank test.

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Figs. 2–4 Distribution of plasma concentrations of ACTH (Fig. 2a), β-LPH IRM (Fig. 2b), estradiol (Fig. 3a), progesterone (Fig. 3b), LH (Fig. 4a) and PRL (Fig. 4b) before induction of anaesthesia (tA) and immediately after oocyte retrieval (tB) and the difference between tB and tA; (n=28 patients) described by box and whisker plots. The “*” mark the extreme values, the whiskers the maximum or minimum observation below or above upper or lower fence (1.5 times inter quartile range above the 3rd or below the 1st quartile). The lower and upper line of the box mark the 1st and 3rd quartile, the line within the box marks the median and the “+” marks the arithmetic mean.

The concentrations of E2 and LH decreased significantly from the time before anaesthesia induction (tA) to the time immediately after oocyte retrieval (tB) (Table 1, Figs. 3a and 4a). P showed a significant difference from time (tA) to (tB) (Table 1, Fig. 5b). PRL increased significantly from the time before anaesthesia induction (tA) to the time immediately after oocyte retrieval (tB) (Table 1, Fig. 4b).

Significant correlations between plasma levels of ACTH and β-LPH IRM (Fig. 5a), between ACTH and β-endorphin IRM (p=0.001; rs: 0.746) and between β-endorphin IRM and β-LPH IRM (p=0.001; rs: 0.735) immediately after oocyte retrieval (tB) were observed. The correlation between plasma levels of β-endorphin IRM and β-LPH IRM is compatible with the fact that the β-endorphin IRM RIA is known to detect β-LPH as well.

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Fig. 5. Correlation plot and Spearman's rank correlation coefficients rs and p values, calculated for the interrelationships between β-LPH and ACTH (a) and between progesterone and ACTH (b) in plasma, determined immediately after oocyte retrieval (tB) (n=28).

Significant correlations between POMC derivatives or cortisol and E2, P, PRL, LH or FSH immediately after oocyte retrieval (tB) were not observed, with two exceptions: 1) ACTH and P (Fig. 5b); 2) E2 and β-endorphin IRM (p=0.040; rs: −0.391).

We observed significant negative correlations between P and LH (Fig. 6a) and between PRL and LH (Fig. 6b) at time (tB).

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Fig. 6. Correlation plot and Spearman's rank correlation coefficients rs and p values, calculated for the interrelationships between LH and progesterone (a) and between LH and prolactin (b) in plasma, determined immediately after oocyte retrieval (tB) (n=28).

4. Discussion 

Assisted reproduction is a chronic stress situation. Oocyte retrieval represents an additional acute stress. The pituitary POMC system reflects this acute treatment stress as we conclude from significant increases of ACTH, β-endorphin IRM and β-LPH IRM plasma concentrations after oocyte retrieval. Under these conditions there was a significant correlation between plasma concentrations of ACTH and β-endorphin IRM or between ACTH and β-LPH IRM. This reaction can be called a concerted pattern of a corticotroph stress response.

Although our data are confined to only 28 patients and 5 pregnancies, the concerted pattern of stress response was observed in all women in the present study, without any exception. Furthermore, this study corresponds and extends our previously published findings concerning the POMC response to different stress situations we investigated such as exercise, delivery and orthopaedic surgery [4], [5], [6], [7], [8], [9]. Both in the present as well as in our recent studies, we have examined POMC as multi-hormonal precursor for ACTH, β-LPH and β-endorphins in different stress situations. POMC can also be considered as part of the body's adaptive pituitary adrenal response to acute stress as reflected at IVF oocyte retrieval or delivery [8]. In chronic and especially non-compensated stress situations (e.g. overtraining in physical exercise) this adaptive pituitary adrenal competence is mitigated. A reduced adrenal responsiveness to ACTH as well as a decreased sympathetic activity can be observed in overtraining situations. This non-physiological reaction to chronic stress can only be compensated by an increased pituitary ACTH release [10], (for review see ref. [7]). This fact demonstrates again the significance of POMC for the homoeostasis of physical regulation systems.

The melanotroph pituitary POMC system did not react to IVF stress in contrast to the corticotroph POMC derivatives. This is in accordance with our observations at perioperative conditions [6] and at delivery [8].

In previously published studies, cortisol, ACTH or β-endorphin IRM were determined during IVF treatment. In accordance with our results, the highest POMC derivative and cortisol concentrations were observed immediately after oocyte retrieval [11], [12], [13]. Kerdelhué and co-workers determined ACTH and β-endorphin IRM in the plasma at various time points: before but not immediately after oocyte retrieval and therefore did not find any changes in concentrations of these hormones [14]. Naito and co-workers determined ACTH and cortisol plasma concentrations before induction of anaesthesia for oocyte retrieval, and additionally up to 24h after induction of anaesthesia but not directly after oocyte retrieval [15]. In fact, they observed “negligible” plasma concentrations of ACTH and cortisol, as we did before induction of anaesthesia.

The low concentrations of authentic β-endorphin in comparison with other corticotroph-type POMC derivatives are in accordance with previously published studies applying highly specific assays for authentic β-endorphin under physical stress [4], [5], [16] (for review see ref. [7]) in perioperative stress conditions [6] and at delivery stress [8].

A highly significant rise in PRL plasma concentrations immediately after oocyte retrieval indicates a pronounced lactotroph response to oocyte retrieval stress as reported in the past by Lehtinen and co-workers [11]. Naito and co-workers still found a rise of PRL 0.5h after induction of anaesthesia [15]. Harlow and co-workers also demonstrated an increase in serum PRL in women undergoing stimulated IVF treatment, which interestingly was accompanied by an increase in cortisol and anxiety [17]. Although we found a highly significant increase of POMC derivatives and PRL plasma concentrations after oocyte retrieval, we did not observe any correlation between POMC derivatives or cortisol and PRL. We conclude that oocyte retrieval stress indicates the lactotroph response as previously reported, but the pituitary lactotroph and corticotroph systems react to stress differently. E2 is known to up-regulate the pituitary prolactin release [18], [19]. The correlation between E2 and PRL in plasma is therefore conclusive.

We also observed a significant correlation between P and ACTH plasma concentrations immediately after oocyte retrieval. Information on this relationship is based on studies in various mammals, wherein elevated P plasma levels have been measured after ACTH administration [20], [21], [22], [23], [24], [25].

No molecular endocrinological correlation between E2 and the melanotrophic pituitary system could be observed in this study since we did not see a correlation between NAC and E2. We observed the same phenomenon during caesarean section or after spontaneous delivery [8]. However, a connection between E2 and the corticotroph pituitary systems may exist. It could topically be demonstrated that high E2 causes increases in basal plasma ACTH and corticosterone concentrations [26] and in POMC mRNA of the rat pituitary gland [26], [27]. We also observed a correlation between POMC derivatives on one side and sexual steroids on the other side for β-endorphin IRM and E2 immediately after oocyte retrieval (tB) concordant to observations immediately after spontaneous delivery [8].

Psychosocial and emotional stresses are a direct cause of infertility [28], [29], [30]. Furthermore, women undergoing IVF are subject to an acute physical stress of the surgical procedures. The release of POMC derivatives at follicular puncture reflects this acute stress situation for all women as proved in the present study. But this stress situation should not disturb the later fertilisation rate. Oocyte fertilisation occurs under in vitro conditions and follicle re-implantation is carried out 2 days later at a lower stress condition. In addition, it has to be emphasized that the connection between stress situations and infertility is discussed in the part.

POMC derivatives, such as ACTH, β-endorphin IRM and LPH IRM were released from the pituitary into the blood. We regard the activation of proopiomelanocortin derived peptides as an adaptive response to IVF stress similar to physical exercise, delivery or surgery [4], [5], [6], [8]. The pituitary lactotroph system is also activated, yet integrated in a closed loop control circuit different from the corticotroph pattern. In contrast, the melanotroph POMC system does not react to IVF treatment stress and the reaction of authentic β-endorphin is meagre, shown for the first time in the present study.