Morphometric Analysis of Human Granulosa Cells in ICSI Cycles and Their Correlation with Biochemical Parameters

Abstract

Objective: Granulosa cells are essential somatic cells that play critical roles in oocyte maturation, follicular development, and regulation of the follicular microenvironment through the production of paracrine and autocrine signaling molecules. This study aimed to evaluate the morphometric characteristics of granulosa cells obtained from infertile women undergoing intracytoplasmic sperm injection (ICSI) and to investigate their correlations with serum biochemical parameters, including FSH, LH, TSH, T3, T4, β-hCG, and Ca²⁺. Methods: Fifty infertile women aged 19–42 years who underwent ICSI treatment were included in the study and divided into two groups according to a cutoff age of 35 years. Granulosa cells obtained after the denudation procedure were stained using the Papanicolaou staining method. 30 randomly selected granulosa cells from each patient were analyzed, resulting in a total of 1,500 cells. Cytoplasmic and nuclear areas were measured morphometrically using manual contour tracing and point-counting methods with Stereo Investigator 8 software. Serum FSH, LH, TSH, T3, T4, β-hCG, and Ca²⁺ levels were recorded. Statistical analyses were performed using the Mann–Whitney U test, chi-square test, and Spearman correlation analysis. A P value of <0.05 was considered statistically significant. Results: The mean cytoplasmic and nuclear areas of granulosa cells were 164.34 ± 56.23 µm² and 59.36 ± 14.21 µm², respectively, in the <35-year group, and 181.22 ± 92.86 µm² and 64.46 ± 28.58 µm², respectively, in the ≥35-year group. No statistically significant differences were observed between the groups in cytoplasmic or nuclear areas (P>.05). Similarly, no significant differences were found in serum hormone levels or biochemical pregnancy rates (P>.05). Correlation analysis revealed significant negative correlations between cytoplasmic area and T4 level (r=-0.33; P=.02), nuclear area and T4 level (r=-0.30; P=.03), and age and TSH level (r=-0.30; P=.03). A significant positive correlation was observed between T4 and Ca²⁺ levels (r=0.58; P=.04), whereas FSH and Ca²⁺ levels showed a significant negative correlation (r=-0.49; P=.02). Conclusion: The significant associations between granulosa cell morphometric characteristics and serum T4 and Ca²⁺ levels suggest that these parameters may have potential as non-invasive, low-cost biomarkers for the indirect assessment of oocyte quality in ICSI cycles.

Keywords

• Granulosa cells
• morphometry
• intracytoplasmic sperm injection (ICSI)
• follicular development
• thyroid hormones
• oocyte quality

Ethical Statement

Ethics committee approval was obtained from From the Clinical Research Ethics Committee of Atatürk University Faculty of Medicine (Date: February 23, 2017, Number: 1/37)

ICSI Sikluslarında İnsan Granüloza Hücrelerinin Morfometrik Analizi ve Biyokimyasal Parametreler ile Korelasyonu

Abstract

Amaç: Granüloza hücreleri, oosit maturasyonu, foliküler gelişim ve foliküler mikroçevrenin düzenlenmesinde kritik rol oynayan, parakrin ve otokrin sinyal molekülleri üreten temel somatik hücrelerdir. Bu çalışmada, intrasitoplazmik sperm enjeksiyonu (ICSI) uygulanan infertil kadınlardan elde edilen granüloza hücrelerinin morfometrik özelliklerinin değerlendirilmesi ve bu özelliklerin serum biyokimyasal parametreleri (FSH, LH, TSH, T3, T4, β-hCG, Ca²⁺) ile korelasyonunun araştırılması amaçlandı. Yöntemler: Çalışmaya, ICSI tedavisi uygulanan, 19–42 yaşları arasındaki 50 infertil kadın dahil edildi ve 35 yaş eşik değerine göre iki gruba ayrıldı. Denüdasyon işlemi sonrasında elde edilen granüloza hücreleri Papanicolaou yöntemi ile boyandı. Her hastadan rastgele seçilen 30 granüloza hücresi analiz edilerek toplam 1.500 hücrenin sitoplazma ve çekirdek alanları, Stereo Investigator 8 yazılımı kullanılarak manuel kontur izleme ve nokta sayım yöntemleri ile morfometrik olarak ölçüldü. Serum FSH, LH, TSH, T3, T4, β-hCG ve Ca²⁺ düzeyleri kaydedildi. İstatistiksel analizlerde Mann–Whitney U testi, Ki-kare testi ve Spearman korelasyon analizi kullanıldı. P<,05 değeri istatistiksel olarak anlamlı kabul edildi. Bulgular: <35 yaş grubunda granüloza hücrelerinin ortalama sitoplazma ve çekirdek alanları sırasıyla 164,34±56,23 µm² ve 59,36±14,21 µm², ≥35 yaş grubunda ise 181,22±92,86 µm² ve 64,46±28,58 µm² olarak ölçüldü. Gruplar arasında sitoplazma ve çekirdek alanları açısından istatistiksel olarak anlamlı fark saptanmadı (P>,05). Benzer şekilde, serum hormon düzeyleri ve biyokimyasal gebelik oranları bakımından da gruplar arasında anlamlı farklılık gözlenmedi (P>,05). Korelasyon analizinde, sitoplazma alanı ile T4 düzeyi (r=-0,33; P=,02), çekirdek alanı ile T4 düzeyi (r=-0,30; P=,03) ve yaş ile TSH düzeyi (r=-0,30; P=,03) arasında anlamlı negatif korelasyon; T4 ile Ca²⁺ düzeyi arasında anlamlı pozitif korelasyon (r=0,58; P=,04) ve FSH ile Ca²⁺ düzeyi arasında anlamlı negatif korelasyon (r=-0,49; P=,02) saptandı. Sonuç: Granüloza hücre morfometrik özelliklerinin serum T4 ve Ca²⁺ düzeyleri ile anlamlı ilişki göstermesi, bu parametrelerin ICSI sikluslarında oosit kalitesinin dolaylı olarak değerlendirilmesinde non-invaziv ve düşük maliyetli potansiyel biyobelirteçler olarak kullanılabileceğini düşündürmektedir.

Keywords

• Granüloza hücresi
• morfometri
• intrasitoplazmik sperm enjeksiyonu (ICSI)
• foliküler gelişim
• tiroid hormonları
• oosit kalitesi

Ethical Statement

Etik kurul onayı Atatürk Üniversitesi Tıp Fakültesi Klinik Araştırmalar Etik Kurulu'ndan (Tarih: 23 Şubat 2017, Sayı: 1/37) alınmıştır.

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