Fourteen babies born after round spermatid injection into human oocytes

Abstract

During the human in vitro fertilization procedure in the assisted reproductive technology, intracytoplasmic sperm injection is routinely used to inject a spermatozoon or a less mature elongating spermatid into the oocyte. In some infertile men, round spermatids (haploid male germ cells that have completed meiosis) are the most mature cells visible during testicular biopsy. The microsurgical injection of a round spermatid into an oocyte as a substitute is commonly referred to as round spermatid injection (ROSI). Currently, human ROSI is considered a very inefficient procedure and of no clinical value. Herein, we report the birth and development of 14 children born to 12 women following ROSI of 734 oocytes previously activated by an electric current. The round spermatids came from men who had been diagnosed as not having spermatozoa or elongated spermatids by andrologists at other hospitals after a first Micro-TESE. A key to our success was our ability to identify round spermatids accurately before oocyte injection. As of today, all children born after ROSI in our clinic are without any unusual physical, mental, or epigenetic problems. Thus, for men whose germ cells are unable to develop beyond the round spermatid stage, ROSI can, as a last resort, enable them to have their own genetic offspring.

Keywords: ROSI; azoospermia; human; male infertility; round spermatid.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Various types of spermatogenic cells seen after enzymatic dissociation of seminiferous tubules. (Magnification: 400×.) A few erythrocytes (purple arrowheads) with a distinct shape are also seen (we treated dissected seminiferous tubules with erythrocyte-lysing buffer, but some cells remained nonlysed). Spermatogonia with or without pseudopodia (red arrows and red arrowheads), primary spermatocytes (blue arrowheads), round spermatids (yellow arrowheads), and Sertoli cells (green arrowhead) are seen.

Fig. 2.

Spermatogonia, primary spermatocytes, and round spermatids, (A) seen with interference-contrast optics, and (B) their schematic drawings. (Magnification: A, 400×.) (C) FISH using fluorescent probes specific for chromosomes X, Y, and 18. (Magnification: 400×.) (D) Giemsa-stained chromosome spreads. (Magnification: 200×.) A spermatogonium with diploid (46) chromosomes is characterized by the presence of two to three nucleoli adjacent to a clearly visible nuclear envelope. Some spermatogonia have pseudopodia (red arrow in A). The spermatogonium shown here has two chromosomes 18, and one X and one Y chromosome. Primary spermatocytes are the largest cells, with two to three small nucleoli and tetrad chromosomes characteristic of meiotic cells. Each cell has two spots for chromosome 18 and one X and one Y. The small round spermatid has an indistinct nuclear membrane, and each has a haploid set of chromosomes (23), one chromosome 18 and either one X or one chromosome Y chromosome. Only about 10% of round spermatids examined showed an acrosome vesicle (blue arrow in A) or acrosome cap (green arrow in A).

Fig. 3.

(A) Twelve presumptive round spermatids randomly selected and adherent to a glass slide. (B) The same cells after FISH, each showing a single spot of either chromosome X (green) or Y (orange). (Magnification: A and B, 400×.)

Fig. S1.

More examples of various cell types obtained after enzymatic dissociation of seminiferous tubules. Round spermatids are pointed by yellow arrowheads. (Magnification: 400×.)

Fig. 4.

The procedure of ROSI. (A) Immediately before picking up one spermatid. (B) A spermatid aspirated into the injection pipette, the plasma membrane being broken. The white arrowhead indicates the spermatid nucleus. (C–F) Oocytes before, during and after injection of spermatid. Arrowhead indicates the spermatid nucleus. (Magnification: A–F, 400×.)

Fig. S2.

Intracellular Ca2+ oscillations within in vitro matured oocytes after (A) ooplasm aspiration, (B) electrical stimulation, (C) ROSI alone, and (D) electric stimulation plus ROSI. Note that neither ooplasm aspiration nor electric stimulation alone induced repetitive Ca2+ oscillations consistently. In contrast, electric stimulation plus ROSI (D) induced consistent large, repetitive Ca2+ oscillations.

Fig. S3.

The preimplantation development of electro-activated ROSI oocytes. (A and B) metaphase II (yellow arrow), (C) anaphase II, (D) telophase II, (E) appearance of a small female pronucleus (red arrow), (F–H) female and male pronucleus, (I) syngamy (dotted line), (J) disappearance of syngamy, (K) starting cell division, (L and M) two-cell, (N) four-cell, (O) eight-cell, (P) compacted morula, (Q) early blastocyst, (R) blastocyst, and (S) expanded blastocyst. (Magnification: A–S, 400×.)

Fig. S4.

Histological sections of seminiferous tubules of patient 1 (Table 2). (A) H&E-stained section, showing seminiferous tubes with neither Sertoli cells nor spermatogenic cells; based on this, a pathologist rated the Johnsen score of this patient as 1 (Table 2). (B) Later examination of other seminiferous tubles of this man revealed the presence of spermatogenenic cell-like cells in some other seminiferous tubes (PAS and hematoxylin-stained). See cells within a dotted circle (basement membrane of a seminiferous tubule). (Magnification: A and B, 400×.)