Telomere Length in Human Spermatogenic Cells as a New Potential Predictor of Clinical Outcomes in ART Treatment with Intracytoplasmic Injection of Testicular Spermatozoa

Abstract

Predicting the clinical outcomes of intracytoplasmic sperm injection (ICSI) cycles that use the testicular spermatozoa of azoospermic patients presents a challenge. Thus, the development of additional approaches to assessing the competence of a testicular-sperm-derived embryo without causing damage to gametes or the embryo is necessary. One of the key parameters in determining such developmental competence is telomere length (TL). We aimed to analyze TLs in spermatogenic cells from the testicular biopsy samples of azoospermic patients and determine how this parameter influences embryo competence for pre- and post-implantation development. Using Q-FISH, we studied the TL of the chromosomes in spermatogonia and spermatocytes I from the TESE biopsy samples of 30 azoospermic patients. An increase in TL was detected during the differentiation from spermatogonia to spermatocytes I. The patients' testicular spermatozoa were used in 37 ICSI cycles that resulted in 22 embryo transfers. Nine pregnancies resulted, of which, one was ectopic and eight ended in birth. The analysis of embryological outcomes revealed a dependence between embryo competence for development to the blastocyst stage and the TL in spermatogenic cells. The TLs in spermatogonia and spermatocytes I in the testicular biopsy samples were found to be higher in patients whose testicular sperm ICSI cycles resulted in a birth. Therefore, the length of telomeres in spermatogenic cells can be considered as a potential prognostic criterion in assessing the competence of testicular-sperm-derived embryos for pre- and post-implantation development. The results of this study provide the basis for the development of a laboratory test for the prediction of testicular sperm ICSI cycle outcomes.

Keywords: ICSI; Q-FISH; TESE; azoospermia; human spermatogenesis; spermatocytes I; spermatogonia; telomeres.

Figure 1

The flowchart of the study.

Figure 2

Mitotic metaphase chromosomes from human spermatogonium and meiotic prophase chromosomes from human spermatocyte from an azoospermic patient’s testicular tissue sample. Telomeres and 19p subtelomeres were detected through fluorescent in situ hybridization (FISH), and the chromosomes were stained with DAPI.

Figure 3

A comparison of mean relative telomere length of chromosomes in spermatogonia and spermatocytes I obtained from the testicular biopsy samples of 30 azoospermic patients (Mann–Whitney U test, p = 0.01).

Figure 4

A comparison of intraindividual (intercellular) and interindividual variability of mean relative telomere lengths (TLs) in spermatogonia and spermatocytes I obtained from the testicular tissue samples of 30 azoospermic patients (Mann–Whitney U test).

Figure 5

The correlations between relative telomere length (TL) in spermatogonia (A) and spermatocytes I (B) and azoospermic patients’ age (Spearman’s test, ρ = 0.08, p = 0.68 and ρ = −0.15, p = 0.44, respectively).

Figure 6

The correlations between the competence of embryos obtained through fertilization with testicular sperm from azoospermic patients’ testicular tissue samples for development to the morulae stage and the telomere length (TL) in spermatogonia (A: Spearman’s test, ρ = 0.43, p = 0.008) and spermatocytes I (B: Spearman’s test, ρ = 0.3, p = 0.07).

Figure 7

A comparison of median relative telomere length (TL) in spermatogonia (A) and spermatocytes I (B) from azoospermic patients’ biopsy samples across three groups, formed based on the competence of testicular-sperm-derived embryos from each couple for in vitro development to the blastocyst stage. The competence of the couple’s embryos for development was characterised as high if 50% or more two-cell embryos developed to the blastocyst stage (n = 7), low if less than 50% of the embryos reached that stage (n = 12) and absent if none of the embryos developed to the blastocyst stage (n = 6) (Mann–Whitney U test).

Figure 8

A comparative analysis of telomere lengths (TLs) in spermatogonia (A) (n = 99 and n = 204) and spermatocytes I (B) (n = 96 and n = 163) from the testicular biopsy samples of the patients whose testicular sperm yielded embryos that were transferred to the uterus with subsequent pregnancy and birth, and without subsequent pregnancy (Mann–Whitney U test).

Figure 9

A comparative analysis of the female partner’s age between groups with and without occurrence of birth after the transfer of testicular-sperm-derived embryos to the uterus (Mann–Whitney U test).