Materials and methods

This was an observational descriptive study conducted on human fetal testicular tissue samples collected from therapeutically aborted fetuses between 2017 and 2018. The study was carried out in the Department of Anatomy in collaboration with the Department of Obstetrics and Gynecology, All India Institute of Medical Sciences, Bhubaneswar. A total of 10 fetal testicular tissue samples representing different gestational periods (weeks) of the first, second, and third trimesters were included in the study.

The gestational age of each fetus was determined based on maternal history, ultrasonographic findings, and fetal biometric parameters. The collected samples were categorized according to trimester to evaluate developmental changes in testicular morphology, proliferative activity, and ultrastructural characteristics.

Inclusion and exclusion criteria

Fetal testicular tissue samples obtained from therapeutically terminated pregnancies were included in the study after obtaining written informed consent from the legal guardians. Fetuses were included if the fetus had a confirmed male phenotype, testicular tissue was grossly identifiable and adequate for histological and ultrastructural evaluation, and the fetus had no evidence of congenital anomalies affecting the reproductive system. Samples were excluded if external congenital abnormalities were present; there was evidence of cryptorchidism, testicular agenesis, or gross developmental abnormalities of the testes; the tissue sample was inadequate or poorly preserved for immunohistochemistry or electron microscopy analysis; and there was evidence of maceration or autolysis of fetal tissue.

Ethical considerations

The study was approved by the Institutional Ethics Committee and Institutional Review Board of All India Institute of Medical Sciences, Bhubaneswar (Approval No. T/IM-NF/Anatomy/18/84). Written informed consent was obtained from the parents/legal guardians prior to sample collection. All procedures were performed according to institutional ethical guidelines and the principles of the Declaration of Helsinki.

Sample size

The sample size was determined based on the availability of fetal testicular specimens during the study period and the feasibility of detailed histological, immunohistochemical, and ultrastructural analysis. As this was an exploratory morphological study involving human fetal tissue, a purposive sampling method was adopted. A total of 10 samples distributed across the first, second, and third trimesters were analyzed to identify developmental variations in testicular architecture and cellular proliferation.

Study parameters

The following parameters were evaluated: (1) histomorphological assessment (general architecture of fetal testicular tissue, arrangement and morphology of seminiferous cords/tubules, morphological characteristics of Sertoli cells and germ cells, and presence and appearance of interstitial Leydig cells); (2) immunohistochemical assessment (expression of Ki-67 as a marker of cellular proliferation, percentage of Ki-67-positive cells among testicular cell populations, and comparison of proliferative activity between different gestational periods); and (3) ultrastructural assessment (surface morphology of testicular components using scanning electron microscopy (SEM) and cellular ultrastructure and organelle characteristics using transmission electron microscopy (TEM).

Immunohistochemistry

Testicular tissue samples were fixed in 10% neutral buffered formalin, routinely processed, and embedded in paraffin wax. Sections of approximately 3 µm thickness were prepared and subjected to heat-induced antigen retrieval using Danish Antibody Company (DAKO) Target Retrieval Solution.

Sections were incubated with Ki-67 primary antibody, followed by detection using a labeled avidin-biotin immunoperoxidase technique on a DAKO Auto Stainer. Diaminobenzidine (DAB) was used as the chromogen, and hematoxylin was used for counterstaining. Ki-67 immunoreactivity was evaluated semi-quantitatively according to the percentage of positive cells and graded as 0: no positive cells; 1+: <10% positive cells; 2+: 10%-25% positive cells; 3+: 26%-50% positive cells; 4+: >50% positive cells.

Scanning electron microscopy

Small pieces of fetal testicular tissue were fixed in phosphate-buffered fixative followed by postfixation with osmium tetroxide. Samples were dehydrated through ascending grades of acetone and subjected to critical point drying using liquid carbon dioxide. The dried specimens were mounted on aluminum stubs, sputter-coated with gold, and examined under a scanning electron microscope for surface ultrastructural evaluation.

Transmission electron microscopy

For ultrastructural evaluation, tissue fragments measuring approximately 2 × 2 mm were fixed in Karnovsky’s fixative followed by postfixation in osmium tetroxide. The samples were dehydrated through graded alcohol concentrations and embedded in epoxy resin. Ultrathin sections (50-70 nm) were prepared using an ultramicrotome, stained with uranyl acetate and lead citrate, and examined under a transmission electron microscope.

Statistical analysis

As the study was exploratory in nature with a limited sample size, formal inferential statistical analysis was not performed. Observations were described qualitatively and semi-quantitatively.