Prolidase activity in women with polycystic ovarian syndrome undergoing assisted conception

Abstract

Background: Prolidase is a manganese (Mn)-dependent cytosolic exopeptidase that degrades imidodipeptides with C-terminal proline or hydroxyproline. Prolidase recycling from imidodipeptides plays a critical role in collagen resynthesis and extracellular matrix (ECM) remodelling. Following an increase in gonadotropins, ovarian and follicular collagen undergo substantial degradation. Abnormal ovarian ECM composition is associated with polycystic ovary syndrome (PCOS). This study aimed to examine prolidase activity in the serum and follicular fluid (FF) of women undergoing in vitro fertilisation/intracytoplasmic sperm injection (IVF/ICSI) treatment, comparing those with PCOS to those with normal ovarian function.Methods: This prospective study enrolled 50 participants, of whom 44 were included. PCOS diagnosis followed the Rotterdam consensus criteria, with 20 patients constituting the study group. The control group comprised 24 individuals with mild-to-moderate male infertility. Prolidase enzyme activity in serum and FF was measured using the Chinard reagent via spectrophotometric analysis and compared between the groups.Results: Serum and FF prolidase levels were significantly lower in patients with PCOS (p < 0.05). A direct correlation was observed between serum and FF prolidase levels (p < 0.05). Although blastocyst quality scoring (BQS) significantly decreased in PCOS patients, no statistical difference was observed in the clinical pregnancy rate between the groups (p < 0.05) (p > 0.05). A negative correlation existed between serum prolidase levels and total antral follicle (AF) count (p < 0.05). Conversely, both serum and FF prolidase levels positively correlated with BQS (r = 0.574)(p < 0.05) (r = 0.650)(p < 0.05).Conclusions: Patients with PCOS showed lower serum and FF prolidase levels, indicating abnormal degradation of ovarian and follicular collagen, potentially causing anovulation.

PLAIN LANGUAGE SUMMARY

Polycystic ovary syndrome (PCOS), the most prevalent endocrinopathy among reproductive-aged women, affects approximately 3–15% of this demographic. Long-term disorders such as cardiovascular disease, type 2 diabetes mellitus, obesity, and infertility are commonly associated with PCOS, with approximately 70% of affected women experiencing infertility. Although the aetiology of PCOS remains unclear, complex multigenic disorders and environmental factors such as abnormal ovarian extracellular matrix composition, disruption of the inflammatory pathway, and lifestyle factors have been found to be related.

This study addresses the aetiology of PCOS, focusing on the close association between abnormal ovarian extracellular matrix composition and the syndrome, as seen in previous reports. Prolidase is a manganese-dependent cytosolic exopeptidase that degrades imidodipeptides using the C-terminal proline or hydroxyproline. Proline recycling from imidodipeptides by prolidase plays a critical role in the resynthesis of collagen and remodelling of the extracellular matrix. Our aim was to evaluate prolidase activity in the serum and follicular fluid of women diagnosed with PCOS. Our findings revealed a direct correlation between serum and follicular fluid prolidase levels, both of which were diminished in women with PCOS. Furthermore, a negative correlation was observed between serum prolidase levels and total antral follicle count indicating a potential link between prolidase activity and ovarian follicle development. In contrast, both serum and follicular fluid prolidase levels were positively correlated with blastocyst quality. In conclusion, PCOS patients showed lower serum and follicular fluid prolidase levels, indicating abnormal degradation of ovarian and follicular collagen, and potentially causing anovulation. Future studies measuring manganese levels in larger numbers of participants are required.

Keywords:

• Assisted conception
• polycystic ovary syndrome
• prolidase activity

Introduction

Folliculogenesis is a dynamic process that involves oocyte development through the remodelling of the extracellular matrix (ECM) of ovarian tissue with endocrine, paracrine, and autocrine effects. The ECM plays a critical role in various cell functions such as morphology, aggregation, communication, proliferation, survival, and steroidogenesis (Nagyová et al. 2021). Prolidase is a manganese (Mn)-dependent cytosolic exopeptidase that degrades imidodipeptides with C-terminal proline or hydroxyproline. Proline recycling from imidodipeptides by prolidase plays a critical role in the resynthesis of collagen and remodelling of the ECM (Wilk et al. 2021). As members of the matrix metalloproteinases (MMPs) family, prolidases are acute-phase reactants and proteolytic enzymes that play a pivotal role in inflammatory processes and the degradation of ovarian and follicular collagen (Eni-Aganga et al. 2021, Zehravi et al. 2021).

Polycystic ovary syndrome (PCOS), the most prevalent endocrinopathy among reproductive-aged women, affects approximately 3–15% of this demographic. PCOS is associated with long-term disorders such as cardiovascular disease, type 2 diabetes mellitus, obesity, and infertility, with about 70% of affected women experiencing infertility (Hoeger et al. 2021, Fedorcsák et al. 2010). Although the aetiology of PCOS remains unclear, the abnormal ovarian ECM composition and increased pro-inflammatory state that disrupts follicular maturation in ovarian tissue are blamed (Bahçeci et al.2021, Guney et al. 2021, Ambekar et al. 2013).

The selection of antral follicles (AF) for ovulation is based on the intrafollicular microenvironment, where appropriate levels of cytokines, growth factors, proteins, and metabolites should be present in the intrafollicular fluid for the development of high-quality mature oocytes. It has been shown that an average of 480 proteins are found in the follicular fluid (FF), and many of these proteins are involved in ECM remodelling and immune responses (Da Broi et al. 2018). In addition, a significant breakdown of ovarian and follicular collagen through a series of proteolytic enzyme reactions involving plasminogen activators, plasmin, and MMPs has been detected following a gonadotropin surge (Zehravi et al. 2021).

Therefore, this study aimed to examine the activity of prolidase, a member of the MMPs family, in the serum and FF of women who underwent in vitro fertilisation/intracytoplasmic sperm injection (IVF/ICSI) treatment, comparing those with PCOS to those with normal ovarian function.

Methods

Design

The study comprised 50 patients initially, with the final analysis conducted on 44 women, attending the assisted reproduction clinics of Etlik Zübeyde Hanım and Gülhane Training and Research Hospitals. A flowchart of this study is shown in Figure 1. PCOS was diagnosed based on the Rotterdam consensus criteria, and these women constituted the study group (n = 20) (Rotterdam ESHRE/ASRM 2003). Patients diagnosed with mild-to-moderate male factor infertility comprised the control group (n = 24). In the control group, women had regular menstrual cycles (every 21–35 days), Ferriman–Gallwey score < 6, and less than 12 follicles in 2–9 diameter in each ovary.

Figure 1. Strobe study flowchart.

Patients with a body mass index (BMI) > 35 kg/m2, age < 18 or > 35 years, history of smoking, recent infection within the past 14 days, chronic autoimmune diseases, systemic diseases, Cushing’s syndrome, androgen-secreting tumours, congenital adrenal hyperplasia, moderate-to-severe ovarian hyperstimulation syndrome (OHSS), couples with azoospermia and severe oligoasthenospermia, and those who underwent natural or freeze-thawed cycles and more than one embryo transfer were excluded.

Maternal and paternal age and BMI were documented. Basal serum hormone levels on Day 3 (D3), AF count, number of cycles, duration of infertility, number of collected metaphase II (MII) oocytes, number of Grade 1–3 embryos (ESHRE 2011), blastocyst quality scoring (BQS) (Rehman et al. 2007), clinical pregnancy, and serum and FF prolidase levels were measured. The ethics Committee (31.03.2022 2022/36, 29.06.2022 90739940-799) approved the study protocol.

Ovarian stimulation

The Gonadotropin-releasing hormone (GnRH) antagonist protocol was applied for all patients (Lambalk et al. 2017), and human Chorionic Gonadotrophin (hCG), trigger (Ovitrelle®, Merck, Germany) was administered when ≥3 follicles reached a diameter of more than 17–18 mm. FF samples were collected from follicles with a diameter ≥ 16 mm via using a 16-gauge single-lumen aspiration needle, precisely 34–36 hours post-trigger. All collected oocytes were inseminated using ICSI. Embryo grading adhered to the morphological criteria proposed by Gardner and Schoolcraft (ESHRE 2011), with BQS criteria as outlined by Rehman (2007). The same luteal phase support was provided to all patients, involving daily intramuscular progesterone at 100 mg dosage (Progestan®, Koçak Pharma, Turkey) and oral dydrogesterone (Duphaston^®, Abbott, Turkey) administered thrice daily at 10 mg until the twelfth week of gestation. Clinical pregnancy was confirmed through ultrasonography upon detection of the gestational sac.

FF and blood sample collection

FF samples that were not grossly contaminated with blood were selected and centrifuged at 3000 revolutions per minute (rpm) for 10 minutes to remove cellular components. On the day of oocyte retrieval, 5 mL of blood was drawn from the median cubital vein in the morning following an eight hour fast. The blood samples were centrifuged at 3500 rpm for 10 minutes. The serum and FF samples were stored at −80 °C until the day of the assay.

Measurement of prolidase activity

Prolidase enzyme activity in the serum and FF was measured using the Chinard reagent, based on the spectrophotometric analysis described by Myara (1984). Siemens Advia 1800 chemistry analyser (Siemens Healthcare, Erlangen, Germany) was used for the measurements.

Statistical analysis

Descriptive analyses were presented as mean and standard deviation (mean ± SD) for parametric variables and median (minimum-maximum) for non-parametric variables. Variables between groups were compared using the independent samples t-test or Mann-Whitney U test, based on their distribution. Pearson’s correlation coefficient was used to compare probable associations between variables. Statistical significance was set at p < 0.05. All analyses were performed using Statistical Package for the Social Sciences (SPSS) 20.0 (Armonk, NY, USA).

Results

This prospective study included 44 participants, focusing on prolidase activity as the primary outcome in patients with PCOS. The BQS and clinical pregnancy rate were secondary outcome parameters. No significant differences were noted in maternal or paternal age, BMI, D3 basal serum hormone levels, number of cycles, or duration of infertility between the two groups (p > 0.05) (Table 1). However, the total AF count was significantly higher in the PCOS group than in the control group (p < 0.05) (Table 1).

Table 1. Comparison of demographic characteristics, laboratory data and in vitro fertilisation cycles outcomes.

Parameters	Control Group (n = 24)	Study Group (n = 20)	P value
Maternal age (years)*	29.11 ± 4.00	30.3 ± 4.53	0.493
Paternal age (years)*	32.21 ± 2.44	33.38 ± 2.51	0.540
BMI (kg/m^2)*	26.49 ± 5.2	25.48 ± 5.01	0.760
D3 FSH (mIU/mL)*	6.54 ± 1.48	5.95 ± 1.13	0.175
D3 LH (mIU/mL)*	5.95 ± 2.38	8.33 ± 3.55	0.923
D3 E2 (pg/mL)**	32.7 (12.9–46.7)	44.5(22–125)	0.125
Total AF count**	14 (9–15)	30(19–30)	0.041***
Number of cycle**	2 (1–2)	2(1–4)	0.115
Duration of infertility (month)**	60 (18–144)	45(18–240)	0.646
Retrieved oocyte count*	11.54 ± 5.63	14.06 ± 7.9	0.280
MII oocyte count*	9.46 ± 5.29	11.44 ± 6.47	0.318
Number of Grade 1 embryo**	1 (0–6)	0.5(0–9)	0.965
Number of Grade 2 embryo**	0 (0–2)	1(0–8)	0.071
Number of Grade 3 embryo**	0 (0–3)	0(0–3)	0.29
BQS*	96.60 ± 19.93	23.80 ± 10.16	<0.001***
Clinical pregnancy**	1 (1–2)	2(1–2)	0.126

*Values are mean ± standart deviation, ** Values are median(min-max), ***P-value of less than 0.05 was considered to be statistically significant. Abbreviations: BMI, body mass index; D3, day 3; FSH, follicle-stimulating hormone; LH, luteinizing hormone; E2, oestradiol; AF, antral follicle, MII, metaphase II; BQS, blastocyst quality scoring.

No significant differences were observed in retrieved and MII oocyte counts, number of Grade1, Grade 2, and grade 3 embryos, or rates of clinical pregnancy between the groups (p > 0.05). Nonetheless, the BQS score was significantly lower in the study group than in the control group (p < 0.05) (Table 1).

Serum and FF prolidase levels were significantly lower in the PCOS group than in the control group (p < 0.05) (Table 2). A direct correlation was found between serum and FF prolidase levels (p < 0.05) (Table 3). Conversely, there was a negative correlation between serum prolidase levels and total AF count (r = –0.422) (p < 0.05). In contrast, both serum and FF prolidase levels showed a positive correlation with BQS (r = 0.574) (p < 0.05) (r = 0.650) (p < 0.05) (Table 3).

Table 2. Comparison of serum and FF prolidase level.

Predictors	Control Group (n = 24)	Study Group (n = 20)	P value
Serum prolidase level (U/L)	124.69 ± 18.74	100.17 ± 16.05	=0.005*
Follicle prolidase level (U/L)	74.39 ± 10.69	65.94 ± 8.58	<0.001

* P-value of less than 0.05 was considered to be statistically significant. Independent sample t-test.

Table 3. Correlation analysis of serum and FF prolidase levels.

Predictors		Follicle prolidase	Total AF count	BQS
Serum prolidase (U/L)	p	0.001*	0.007*	<0.001*
	r	0.491	−0.422	0.574
Follicle prolidase (U/L)	p	–	0.201	<0.001*
	r	–	−0.214	0.650

* P-value of less than 0.05 was considered to be statistically significant. Pearson’s correlation test. Abbreviations: AF, antral follicle; BQS, blastocyst quality scoring.

Discussion

To our knowledge, this is the first study to investigate prolidase activity in the serum and FF of patients with PCOS who underwent IVF/ICSI treatment. In the present study, serum and FF prolidase levels were found to be directly correlated, and both were decreased in PCOS patients. Notably, a negative correlation was observed between serum prolidase levels and total AF count, whereas BQS was positively correlated with both serum and FF prolidase levels.

Contrary to findings by Hilali (2013), who reported higher serum prolidase activity in a small sample of patients with PCOS, our study revealed decreased serum prolidase levels in PCOS patients. Additionally, unlike Hilali (2013), we measured FF prolidase levels, which seem to offer a more accurate assessment than serum analysis. To our knowledge, no prior studies have been conducted to evaluate the FF prolidase levels in patients with PCOS.

Prolidase is a member of the MMPs family. Following the gonadotropin surge, a number of proteolytic enzymes, such as plasminogen activators, plasmin, and MMPs, cause a significant break down of follicular and ovarian collagen. It seems that the imbalance in the concentration of MMPs might get involved in the pathogenesis of PCOS (Zehravi et al. 2021).

Furthermore, Mn is a prolidase cofactor and is located at the active site of the enzyme. Chakraborty (2013) reported significant lower serum Mn levels PCOS patients, leading us to hypothesise that the Mn deficiency may lead to decreased serum and FF prolidase levels in these patients. However, previous studies have shown that low-grade chronic inflammation may be responsible for the development of PCOS (Rudnicka et al. 2021, Awonuga et al. 2023). Prolidase has a pivotal role in the immune response through its regulatory effects on the expression of the interferon α/β receptors and nuclear factor κβ. Ergin Tuncay et al. (2022) revealed lower prolidase activity in patients with inflammatory conditions, including coronavirus disease of 2019 (COVID-19), despite normal Mn levels.

Our study revealed a negative correlation between serum prolidase levels and total AF count, suggesting that the observed decrease in serum prolidase levels in PCOS patients may contribute to increased AF count. In contrast, Syabakhash Raghdaa et al. (2020) reported a positive correlation between the number of AF and serum prolidase activity. Additionally, it was found that the BQS was significantly lower in the PCOS group and demonstrated a positive correlation with both serum and FF prolidase levels. In agreement with the present study, Liu (2021) reported that patients with PCOS had fewer high-quality embryos with increased oxidative stress parameters in both serum and FF samples. However, clinical pregnancy rates were similar in both groups, most likely due to the exclusion of frozen-thawed cycles in our present study.

A limitation of this study was the small sample size, which was not sufficient to analyse PCOS subgroups. Furthermore, the results of this study can be strengthened by measuring the Mn levels.

In conclusion, prolidase catalyses the dipeptides that are primarily produced during collagen biosynthesis. Therefore, a better understanding of prolidase enzyme activity in the serum and FF will help identify the pathophysiology of PCOS, offering possible therapeutic applications. In this study, patients with PCOS showed lower serum and FF prolidase levels, indicating abnormal degradation of ovarian and follicular collagen, leading to anovulation. Future studies measuring manganese levels in larger numbers of participants are required to confirm our results.

Authors’ contribution

K. Erdogan: Conception and design, analysis and interpretation of data, drafting the work, writing the manuscript, approval of the final version to be published.

E.U. Ozen: Conception and interpretation of data and drafting the work, approval of the final version to be published.

I. Kahyaoglu: Conception and design, analysis of data, approval of the final version to be published.

S. Neselıoglu: Analysis of data and writing the manuscript, approval of the final version to be published.

O. Erel: Drafting the work and critical interpretation of content, approval of the final version to be published.

S. Akar: Writing the manuscript, approval of the final version to be published.

O. Ozdemir: Data analysis and collection, approval of the final version to be published.

C.M. Ercan: Data analysis and collection, approval of the final version to be published.

Y.E. Ustun: Drafting the work, writing the manuscript and critical interpretation of content, approval of the final version to be published.

Data availability statement

This research data is available upon request from the corresponding author.

Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.