The degree of sperm maturation can affect its fertilization competence when fertilized with oocytes matured in vitro after ICSI, the percentage of IVM oocytes achieving normal fertilization following ICSI was significantly lower using the testicular spermatozoa than the ejaculated spermatozoa [1,2]. The published experience on pregnancies derived from IVM oocytes fertilized by spermatozoa retrieved from the testis remains limited to only some case reports and a very small series of treatments. We report a case of successful pregnancy and healthy birth resulting from in vitro matured oocytes fertilized with testicular spermatozoa.

A couple consulted our centre of reproductive Biology for primary 8 year infertility. A 41-year-old man had no medical or surgical history except non obstructive azoospermia checked in two sperm analysis. The clinical examination of the testes was normal. The follicle stimulating hormone level was 13.1 IU/L (reference values 1.4-11.2 IU/L). The constitutional karyotype was 46, XY. His wife is 32 years, the results of her infertility investigations were normal except the biochemical features of polycystic ovaries (PCO) (FSH = 6, 81 IU/L, LH=14, 27 IU/L, PRL= 14 IU/L). The couple was counselled to undergo therapeutic testicular biopsy on the day of ovum pick up. The cycle treatment with controlled ovarian hyperstimulation was started with 225 IU of recombinant FSH (Gonal-F^®; Serono Nordic, Solna, Sweden) after downregulation with triptorelin LP (Decapeptyl^®, Ipsen Pharma) [3]. Transvaginal oocyte retrieval was performed 36 h after a human chorionic gonadotrophin (Ovitrelle^®; Serono Nordic, Solna,Sweden) administration. We retrieved 11 cumulus-oocyte complexes. We removed a cumulus cells carefully by a brief exposure to hyaluronidase (HyaseTM171, Vitrolife, Sweden) and mechanical denudation with a fine pipette. We assessed the oocytes for their meiotic stage according to the presence or absence of GV or first polar body (PB1). The MI state of the oocytes was defined by the absence of a polar body and GV nucleus. Four oocytes were atretic and 7 had MI state. The immature oocytes were incubated in our routine culture medium (G1; Vitrolife, Sweden), at 37°C, 6% CO2 in air. No oocyte became mature the day of ovarian pick up (day 0). Consequently, the testicular biopsy was not performed. After 24 hours of oocyte incubation (day1), all the MI oocytes extruded their polar body (rescued in vitro-matured metaphase II). We recovered a husband’s spermatozoa by microdissection testicular sperm extraction (micro-TESE) [2]. We divided a sample using 2 needles and we examined them at a magnification of 400X in the inverted microscope for the presence of sperm cells. We found a few immotile spermatozoa. Discontinuous minidensity gradients (containing volumes of only 0.3 mL of different density gradient materials (45% and 90% of Sil Select Stock^® Fertipro, Belgium) were used to separate spermatozoa and spermatids. After centrifugation (300 rpm), the pellet was gently resuspended in HSA medium at 37°C, 5% CO2 until ICSI. All MII oocytes were microinjected with testicular immotile spermatozoa according to standard protocols [4]. Fertilization was confirmed 18 hours later. Five oocytes became fertilized and four of them cleaved but only two of them produces good score embryos which were transferred on day 2 at the 4-cell stage. This embryo transfer resulted in a normal intrauterine singleton pregnancy. After 38 weeks of gestation a healthy girl, weighing 3650 g (Apgar 9, 10, 10), was delivered by caesarean section.

Approximately 5%–20% of human oocytes collected from women after stimulation of the ovaries for IVF fail to resume meiosis in vivo and are thus retrieved in immature stages like metaphase I (MI) or germinal vesicle (GV) [5]. The immature oocytes underwent an in vitro maturation process (IVM) using or not specific culture media. Different incubation periods ranged from 2 to 24 hours seem suitable to enable proper nuclear and cytoplasmic maturation (Metaphase II (MII) stage) and the MI oocytes that extruded their polar body, named as rescued in-vitro matured MII oocytes [6]. Several studies have compared the development potential of these rescued IVM-MII oocytes to matured MII oocytes and their conclusion were as a lower normal fertilization rate with abnormal embryonic development and lower implantation rate than in vivo matured oocytes [1,7,8]. Development to term is limited to rare cases. Therefore, sperm testicular extraction to inject these rescued in-vitro matured MII oocytes was much discussed. In fact, the spermatozoa extracted from the testes were immature and the comparative studies after ICSI with ejaculated and testicular sperm showed a lower percentage of new live births per embryo transferred, and twice as many spontaneous miscarriages as after the use of surgical sperm [4,9]. In addition, the baseline incidence of sperm chromosomal abnormalities is higher in the testis of infertile individuals [10]. But in line with our findings, a several analysis of surgical sperm collected in azoospermic patients concluded that ICSI outcomes were not affected by the sperm origin [11,12]. The age of the female partner and oocyte quality has been shown to exert a substantial influence on the success of ICSI treatment using testicular spermatozoa [13,14]. In our case, the young age and the PCO profile of the patient seem to be a good prognostic factor for a successful attempt despite the double immaturity problem of gametes.

We may conclude that the use of rescued-IVM MI oocytes can be beneficial in order to obtain pregnancy in younger stimulated patients without MII oocytes. Despite their microinjection with immature spermatozoa, fertilization of in vitro matured MI oocytes can result in normal embryos, pregnancy, and the delivery of a healthy child.

1. Yun Q, Ting F, Chen J, Cai LB, Liu JY, et al. (2005) Pregnancies and births resulting from in vitro matured oocytes fertilized with testicular spermatozoa. J Assist Reprod Genet 22: 133-136.
2. Son WY, Chung JT, Chan PT, Tan SL (2011) Live birth after ICSI of micro-TESE-retrieved spermatozoa into in vitro-matured oocytes. J Androl 32: 23-25.
3. Khrouf M, Kdous M, Bouyahia M, Chaker A, Zhioua F, et al. (2010) [Clinicals and biological aspects of ICSI cycles on patients with polycystic ovary syndrome]. Tunis Med 88: 152-157.
4. Palermo GD, Schlegel PN, Hariprashad JJ, Ergün B, Mielnik A, et al. (1999) Fertilization and pregnancy outcome with intracytoplasmic sperm injection for azoospermic men. Hum Reprod 14: 741-748.
5. Balakier H, Sojecki A, Motamedi G, Librach C (2004) Time-dependent capability of human oocytes for activation and pronuclear formation during metaphase II arrest. Hum Reprod 19: 982-987.
6. Chian RC, Lim JH, Tan SL (2004) State of the art in in-vitro oocyte maturation. CurrOpinObstetGynecol 16: 211-219.
7. Shu Y, Gebhardt J, Watt J, Lyon J, Dasig D, et al. (2007) Fertilization, embryo development, and clinical outcome of immature oocytes from stimulated intracytoplasmic sperm injection cycles. FertilSteril 87: 1022-1027.
8. Strassburger D, Goldstein A, Friedler S, Raziel A, Kasterstein E, et al. (2010) The cytogenetic constitution of embryos derived from immature (metaphase I) oocytes obtained after ovarian hyperstimulation. FertilSteril 94: 971-978.
9. Vernaeve V, Tournaye H, Osmanagaoglu K, Verheyen G, van Steirteghem A, et al. (2003) Intracytoplasmic sperm injection with testicular spermatozoa is less successful in men with nonobstructiveazoospermia than in men with obstructive azoospermia. FertilSteril 79:529–533.
10. Rodrigo L, Rubio C, Peinado V, Villamón R, Al-Asmar Net al. (2011) Testicular sperm from patients with obstructive and nonobstructiveazoospermia: aneuploidy risk and reproductive prognosis using testicular sperm from fertile donors as control samples. FertilSteril 95:1005-1012.
11. Kanto S, Sugawara J, Masuda H, Sasano H, Arai Y, et al. (2008)Fresh motile testicular sperm retrieved from nonobstructiveazoospermic patients has the same potential to achieve fertilization and pregnancy via ICSI as sperm retrieved from obstructive azoospermic patients. FertilSteril 90: 2010.
12. Jamal W, Vélez MP, Zini A, Phillips S, Hemmings R, et al. (2012) Surgically retrieved spermatozoa versus ejaculated spermatozoa in modified natural IVF-ICSI cycles. Reprod Biomed Online 25: 242-247.
13. Devroey P, Nagy P, Tournaye H, Liu J, Silber S, et al. (1996) Outcome of intracytoplasmic sperm injection with testicular spermatozoa in obstructive and non-obstructive azoospermia. Hum Reprod 11: 1015-1018.
14. Son WY, Chung JT, Henderson S, Reinblatt S, Buckett W,et al. (2013) Fertilization and embryo development with spermatozoa obtained from testicular sperm extraction into oocytes generated from human chorionic gonadotropin-primed in vitro maturation cycles. FertilSteril 100: 989-993.