Induction the Proliferation and Differentiation of Primary Testicular Germ Cells From Nonobstructive Azoospermic Patients to Meiotic and Postmeiotic Stages Under In Vitro Conditions.

Maram Azab ¹ Eitan Lunenfeld ² Eliahu Levitas ² Benjamin Fisch ³ Haiem Pinkas ³ Johny Younis ⁴ Shalom Bar-Ami ⁴ Marina Pinchasov ⁵ Raoul Orvieto ⁵ Mahmoud Huleihel ¹

¹The Shraga Segal Dep. of Microbiology, Immunology and Genetics, Ben Gurion University of the Negev
²Fertility and IVF Unit, Dep. OB/GYN, Soroka Medical Center and Faculty of Health Sciences, Ben Gurion University of the Negev
³Infertility and IVF Unit Rabin Medical Center, Beilinson Campus
⁴Reproductive Medicine Unit, Dep. OB/GYN, Poriya Medical Center, Tiberias; Faculty of Medicine in Galilee, Bar-Ilan University
⁵OB/GYN, Barzilai Medical Center

Background: Spermatogenesis includes the proliferation and differentiation of spermatogonial stem cells. Nonobstructive azoospermia (NOA) is 20% of the infertile males.

Aims: To characterize and to evaluate the capability to induce growth, proliferation and differentiation of testicular germ cells from NOA patients in vitro.

Methods: Cells were isolated from testicular biopsies of 26 NOA patients, by enzymatic digestion. Cells from 10 biopsies were cultured in Methylcellulose-Culture-System (3D), from 8 biopsies were cultured in RPMI-Culture-System (2D) and from 3 biopsies were cultured in both systems in the presence of growth factors. Developed colonies were examined after 4-12 weeks for premeiotic markers: OCT4, C-KIT, GFRa-1, CD9, SALL4; meiotic: CREM-1, BOULE, LDH; postmeiotic: PROTAMINE, ACROSIN by PCR and/or immunofluorescence staining (IF).

Results: One or more of the premeiotic markers were detected in 92% of the specimens, meiotic in 50% and postmeiotic in 54% before culture, as examined by PCR and/or IF. Spermatogenic markers were expressed similarly in NOA biopsies with/without sperm. Most patients expressed OCT4, GFRa-1, SALL4 before culture. All patients showed proliferation of premeiotic cells, 5/10 developed meiotic and/or postmeiotic markers in 3D and 4/8 in 2D. Cells isolated from biopsies, where sperm were present and did not express acrosin before culture, expressed acrosin in 2D and 3D. However, cells isolated from biopsies, where no sperm were detected, did not express acrosin in any culture.

Conclusions: This is the first study that shows induction of proliferation and differentiation of testicular germ cells from NOA patients to meiotic and postmeiotic stages in vitro.