Birth and clinical pregnancy from fresh and frozen oocytes fertilized with cryopreserved testicular spermatozoa

Abstract

This is the first report showing a second clinical pregnancy of a couple who already have a baby from a previous frozen embryo transfer cycle when the embryos were generated from fresh oocytes that were fertilized by intracytoplasmic sperm injection (ICSI) using frozen testicular spermatozoa (the couple have unsuccessful fresh and frozen embryo transfer cycles). Fifty-two months after the first IVF/ICSI cycle the couple had their second IVF/ICSI cycle, but the collected oocytes (n = 8) were frozen because no spermatozoa was obtained from the frozen testicular tissue samples which were cryopreserved prior to the first IVF/ICSI cycle. New testicular tissue samples were obtained and frozen. Finally, 58 months after the first IVF/ICSI cycle all of the 8 frozen oocytes of the couple were thawed and fertilized by ICSI using frozen testicular spermatozoa obtained from the newly cryopreserved testicular tissue. Three embryos were transferred and the couple has an ongoing pregnancy, which is in the 20^th week of pregnancy. Our case report shows that: 1) developmentally competent embryos can be generated by ICSI of frozen-thawed testicular spermatozoa into both fresh and frozen human oocytes, and 2) clinical pregnancy and a healthy baby can be conceived from both frozen and fresh oocytes fertilized with cryopreserved testicular spermatozoa.

Keywords:

• embryo freezing
• frozen testicular spermatozoa
• ICSI
• intracytoplasmic sperm injection
• oocyte cryopreservation
• testicular sperm extraction

The cryopreservation of human oocytes represents an interesting option in cases of non-obstructive azoospermia treated by intracytoplasmic sperm injection (ICSI). Spare oocytes can be cryopreserved for a future attempt if the number of spermatozoa retrieved from the testis is insufficient to inject all available oocytes [Chian 2010; Song et al. 2011]. Cryopreservation of testicular spermatozoa is already a widely used technique with which the repetition of surgical procedures for future ICSI attempts can be avoided [Carrel 2010; Friedler et al. 1997; Küpker et al. 2000; Revel and Mejia 2010; Wald et al. 2006]. However there are limited data reported on the use of frozen testicular spermatozoa for ICSI into frozen-thawed human oocytes [Liow et al. 2009; Selman et al. 2010]. The objective of our case report is to introduce our clinical results obtained with fresh and frozen oocytes fertilized by ICSI with cryopreserved testicular spermatozoa.

A couple (a 33-y-old man and a 30-y-old woman) with the diagnosis of primary infertility caused by non-obstructive azoospermia had two IVF/ICSI cycles and two frozen embryo transfer cycles within 58 months at our clinic. The couple entered into the IVF/ICSI-ET program with frozen testicular spermatozoa. Two testicular sperm extractions were performed and the obtained testicular tissue samples were cryopreserved for later use.

Testicular sperm freezing was carried out with the use of a HEPES-buffered freezing medium (SpermFreeze solution; Ferti-Pro, Lyon, France) containing 0.4% (vol/vol) human serum albumin. After centrifugation of partly disintegrated testicular tissue (1500 rpm; 10 min), the pellet was rinsed with 0.4 mL HEPES-buffered medium (Gamete 30; Vitrolife, Goteborg, Sweden), mixed (1:0.7) with SpermFreeze solution, equilibrated at room temperature for 10 min, and loaded into straws. The straws were first placed vertically in liquid nitrogen vapors (20 min) and then plunged into liquid nitrogen. After thawing, the samples were washed with HEPES-buffered medium (Gamete 30), resuspended in a small volume of the same medium and incubated at 37°C for 30 min before being used for ICSI.

In the first IVF/ICSI cycle, controlled ovarian stimulation and transvaginal ultrasound-guided follicular aspiration resulted in the recovery of 13 oocytes. A total of 12 MII oocytes were fertilized by ICSI with cryopreserved testicular spermatozoa. From the inseminated fresh oocytes 9 fertilized (9/12; 75%), 8 cleaved (8/9; 89%), and 7 embryos showed good morphology and were suitable for embryo transfer. Finally, on day 3 after ICSI, two eight-cell stage embryos were transferred and 5 embryos were cryopreserved with a slow freezing protocol. After uterine transfer of the two fresh embryos no pregnancy was obtained. Two months later 2 out of the 5 frozen embryos were transferred and a pregnancy was established and led, after uneventful course, to the birth of a healthy boy (3,250 g) at 39 w of gestation. Forty-six months later the remaining frozen embryos (n = 3) were thawed and transferred but no pregnancy resulted.

Fifty-two months after the first IVF/ICSI cycle, the couple requested a second IVF/ICSI-ET cycle using the husband's frozen testicular spermatozoa which were cryopreserved before the first IVF/ICSI-ET cycle for fertilization of the newly collected oocytes. Eight oocytes were collected and all were frozen because no spermatozoa could be recovered from the frozen testicular tissue samples. Oocyte freezing and thawing were carried out as described earlier [Konc et al. 2008]. In brief, equilibration of the oocytes was performed at room temperature in freezing medium based on PBS + 20% human serum albumin (HAS) and containing 1.5 M propylene-glycol (PrOH) + 0.3 M sucrose (10 min) (OocyteFreeze^TM, MediCult, Mollehaven, Denmark). Oocytes were frozen in straws (max. 3 oocytes per straw) in Planer III Kryo 10 cell freezer (Planer Products Ltd., Sunbury-on-Thames, UK). After seeding at minus 6°C, oocytes were slowly cooled (−0.3°C/min) to −30°C, then they were cooled at a higher speed (−50°C/min) to –150°C before plunging into liquid nitrogen. The straws were then stored in liquid nitrogen until thawing. After thawing, oocytes were rehydrated and PrOH was removed from the eggs in four steps by passage through mediums based on PBS + 20% HSA supplemented with 0.5 and 0.3 M sucrose (OocyteThaw^TM, MediCult, Mollehaven, Denmark).

Before oocyte thawing new testicular tissue samples were collected and frozen for fertilization of the cryopreserved oocytes.

Fifty-eight months after the first IVF/ICSI cycle, all the cryopreserved oocytes (n = 8) from the second IVF/ICSI cycle were thawed and fertilized by ICSI. Intracytoplasmic sperm injection was performed after thawing and 2–3 h in vitro culture (36 h after hCG injection) with cryopreserved testicular spermatozoa obtained at the second testicular tissue sample collection. Fertilization was assessed 12–16 h later. All of the eight oocytes that were thawed survived (8/8; 100%) and were subjected to ICSI. Seven out of the 8 oocytes were fertilized (7/8; 87%) and 4 of them cleaved (4/7; 57%). Finally, 3 embryos reached the 8-cell stage by day 3 after ICSI and were transferred in a simple stimulated cycle (75 IU hMG s.c./d for 13 days; Merional® 75 IU (hMG; menotropin) IBSA Institut Biochimique S.A. Swiss). Ovulation was induced with 10,000 IU hCG s.c. (Pregnyl® (uhCG); NV Organon Oss, The Netherlands). Laser assisted hatching was performed on all embryos prior to transfer. Clinical pregnancy was defined by the presence of an intrauterine gestational sac and fetal heart beat detected by ultrasound performed at 7 w of gestation. At the time of the manuscript submission the couple has a normal, physiological ongoing pregnancy that is in the 20th week of pregnancy. Luteal phase support was applied in the first 3 months of the pregnancy (Utrogestan® Besins International, Drogenbos, Belgien; 3 × 300 mg/d starting 3 d before embryo transfer until 12 w of pregnancy).

Our data indicates that cryopreservation of oocyte, embryo, and testicular spermatozoa supports the application and efficiency of assisted reproduction and makes flexible and safer the organization of IVF/ICSI-ET cycles. If no gametes are obtained from one of the partners the cryopreservation of the gametes from the other partner can prevent the repetition of the surgical intervention (e.g., ovarian hyperstimulation and ultrasound guided follicular aspiration or testicular sperm extraction) in a future attempt. Our case report shows that developmentally competent embryos can be generated by ICSI with frozen-thawed testicular spermatozoa injected into both fresh and frozen human oocytes. There are limited data reported on the use of frozen testicular spermatozoa for ICSI into frozen-thawed human oocytes [Liow et al. 2009; Selman et al. 2010; Song et al. 2011]. Our results demonstrate also that the embryos produced from frozen oocytes fertilized with frozen testicular spermatozoa are cryotolerant. The birth was obtained from a frozen embryo transfer cycle when the transferred frozen/thawed embryos developed from fresh oocytes that were fertilized by ICSI with cryopreserved testicular spermatozoa. However, the ongoing pregnancy which is in the 20th week of pregnancy was generated with embryos developed from frozen oocytes fertilized by ICSI with cryopreserved testicular spermatozoa.

In conclusion, our case report demonstrates that in spite of the persisting problems with human oocyte cryopreservation oocyte freezing does not compromise oocyte and embryo developmental competence even if the frozen/thawed oocyte is injected with frozen testicular spermatozoa. Our data show that a clinical pregnancy and a healthy baby can be conceived from fresh and frozen embryos produced from frozen and fresh oocytes fertilized with cryopreserved testicular spermatozoa.

Declaration of interest: The authors report no declarations of interest. The authors declare that they do not have any financial or commercial interest in this paper.

Abbreviations

IVF:	=	in vitro fertilization
ICSI:	=	intracytoplasmic sperm injection
ET:	=	embryo transfer
hMG:	=	human menopausal gonadotropin
uhCG:	=	human chorionic gonadotropin obtained from urine.