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Zaheer, Elkalyoubi, Madkour, Al Adham, and Albahar: Birth of a healthy boy after fertilization of a cryopreserved oocyte and testicular spermatozoon followed by preimplantation genetic screening

Introduction

We report the birth of a healthy male infant from a pregnancy resulting from a thawed from frozen oocyte on which intracytoplasmic sperm injection (ICSI) was performed using a thawed from frozen spermatozoon obtained from testicular aspiration. Preimplantation genetic screening (PGS) was performed on the embryo at the cleavage stage (day 3) to determine the sex.

Expressing a preference for a child of a particular sex has existed for years. Greek philosophers and medical workers offered detailed advice on the means to ensure the production of a child of a chosen sex. Aristotle gave advice on positions for sexual intercourse and a desirable diet for the mother whereas Anaxagoras believed that each testicle determined one sex and advocated tying off one testicle before coitus, and Hippocrates deduced that male and female children developed in different parts of the uterus.1

Preimplantation genetic screening for sex selection is an extremely sensitive issue. The application of techniques for sex selection, including the use of preimplantation genetic diagnosis (PGD), creates moral and ethical concerns in the opinion of some, while the advantages of sensible use of selected technologies is favoured by others.

Since PGS is rarely performed in many in vitro fertilization (IVF) centres, no standard treatment protocol has been established, although a number of births have been recorded. Cryopreservation of oocytes combined with ICSI could offer young women undergoing chemotherapy or radiotherapy a reasonable option to preserve and balance their family.2

Although the cryopreservation of oocytes is ethically more acceptable than the freezing of embryos, variable oocyte survival and pregnancy rates have precluded the application of oocyte cryopreservation in assisted reproductive technology. Cytotoxicity of the cryoprotectants, poor survival, low fertilization rates and high rates of polyploidy37 have prevented the widespread use of oocyte cryopreservation, despite reports of healthy and normal babies.3,4,5,8

Resurgence of interest in human oocyte cryopreservation shows that it may be safe in appropriate circumstances and its clinical application has resulted in a number of live births. Development of oocyte cryopreservation is being pursued because embryo cryopreservation is prohibited in some countries, such as Italy and the United Arab Emirates (UAE).

More than 1000 babies have been born as a result of preimplantation genetic testing, attesting to the accuracy and safety of the procedure, which as a result has gained a place as a good alterative among the choices offered to couples at risk of transmitting serious and incurable genetic diseases.

We report the first case of sex selection of an embryo resulting from a frozen oocyte and a frozen spermatozoon obtained from a testicular sperm aspiration (TESA). PGS was then performed for non-genetic social reasons (sex selection).

Case Report

A couple visited Dubai Gynaecology and Fertility Centre for their second complaint of infertility over a 5-year period. Mrs X is a 31-year-old woman in whom four natural pregnancies had resulted in three normal deliveries at full term and one, the last, in a spontaneous miscarriage at 6 weeks' gestation (i.e. P3 + 1). Over time, Mrs X's husband developed non-obstructive azoospermia and he has suffered from diabetes for the past 9 years. His hormonal profile was normal and a TESA was performed and the sperm cryopreserved. Testicular sperm freezing was carried out with the use of a HEPES-buffered freezing medium (SpermFreeze Solution™; Origio MediCult, Jyllinge, Denmark) containing 0.4% (v/v) human serum albumin. After centrifugation of partly disintegrated testicular tissue (2500 g for 10 minutes), the pellet was rinsed with 0.4 ml of universal IVF culture medium, CE marked (Origio MediCult, Jyllinge, Denmark), mixed with SpermFreeze Solution™ in a 1:1 ratio, equilibrated at room temperature for 10 minutes and loaded into vials. The vials were first placed vertically in liquid nitrogen vapour for 15 minutes and then plunged into liquid nitrogen.

Mrs X has regular menstrual periods but also a strong family history of diabetes mellitus. Her hormonal profile was within normal limits; however, a scan showed that she had polycystic ovaries. She had a controlled ovarian hyperstimulation on a long down-regulation protocol with recombinant follicle-stimulating hormone (rFSH) when 35 eggs, 20 metaphase II (MII) mature oocytes, were collected and frozen to prevent ovarian hyperstimulation.

Oocyte cryopreservation was conducted by vitrification. The oocytes were vitrified according to the Cryotop method.9,10 Basic solution (BS) was used as a buffer and equilibration solution (ES) was made up of 7.5% (v/v) ethylene glycol© (EG©) (Kitazato BioPharma Co., Ltd, Fuji, Shizuoka, Japan) and 7.5% (v/v) dimethylsulphoxide© (DMSO©) (Kitazato BioPharma Co., Ltd, Fuji, Shizuoka, Japan). Vitrification solution (VS) was prepared with 15% (v/v) EG©, 15% (v/v) DMSO© and 0.5 mol/l sucrose. One drop of BS was aligned with three drops of ES and two drops of VS in a small Petri dish and these solutions were then allowed to equilibrate to room temperature (25°C) for 15 minutes.

Oocytes were then placed briefly in the BS drops and a channel was opened with first drop of ES to allow a gradual increase in the cryoprotective concentration and keep the oocytes on the edge of the BS drop for 3 minutes at room temperature. A new channel was then made with next ES drop to transfer the oocytes to the edge of the first ES drop and keep them there for another 3 minutes at room temperature. These oocytes were then transferred to the third ES drop for 9 minutes at room temperature. Oocytes were then transferred to the first drop of VS for 1 minute and then the second VS drop for 30 seconds and subsequently loaded on the flattened tip of the Cryotop with approximately 2 μl of VS. The Cryotop was then immersed directly in liquid nitrogen and stored in the freezing tanks.

Two months later, Mrs X had a trial of ICSI of frozen oocytes by spermatozoa obtained from a frozen TESA conducted earlier. At the time of thawing, the vials were removed from liquid nitrogen and rapidly placed in warm water (30°C). After expulsion from the vials, the thawed samples were washed with universal IVF culture medium, centrifuged at 2500 g twice for 10 minutes each time and the pellet was then resuspended in a small volume of the same universal IVF culture medium, and incubated at 37°C for 30 minutes before being used for ICSI.

Nine oocytes were thawed and the vitrified oocytes were warmed by swiftly immersing the Cryotop in the thawing solution© (Kitazato BioPharma Co., Ltd, Fuji, Shizuoka, Japan) containing 1 mol/l sucrose for 1 minute at 37°C. The oocytes were then equilibrated in a diluent solution© (Kitazato BioPharma Co., Ltd, Fuji, Shizuoka, Japan) containing 0.5 mol/l sucrose for 3 minutes followed by two 5-minute consecutive flushes in washing solution© (Kitazato BioPharma Co., Ltd, Fuji, Shizuoka, Japan). These oocytes were then placed in the fertilization medium (William A. Cook Australia Pty Ltd, Brisbane, Australia) and incubated at 37°C in 6% CO2 air for 2 hours prior to ICSI.

Eight oocytes survived thawing and were injected. All were fertilized and cleaved and PGS was performed for all embryos on day 3.

Biopsy medium (William A. Cook Australia Pty Ltd, Brisbane, Australia) was warmed to 37°C and supplemented with human albumin solution. Numbered drops of biopsy medium were layered in a Petri dish under oil (William A. Cook Australia Pty. Ltd, Brisbane, Australia) and embryos were loaded in to the numbered drops. A hole was made in the zona pellucida of 20 μm using laser-assisted hatching. The location of the embryo was such that a nucleated blastomere occurred at the 3 o'clock position. The blastomere was slowly aspirated using aspiration pipettes of 42 μm (William A. Cook Australia Pty Ltd, Brisbane, Australia) and then released in to the medium.

The biopsied embryos were then washed thoroughly from the biopsy medium and incubated in culture media (William A. Cook Australia Pty Ltd, Brisbane, Australia) until transfer to the uterus. The blastomere was then spread on frosted slides until it burst and the nucleus became exposed. Fixation of the blastomere was conducted using acetic acid and methanol in a 1:3 ratio. The slides then underwent genetic analysis for sex selection and a single embryo of XY karyotype was transferred in to the uterus. Endometrial preparation was conducted on a down-regulated cycle and Triptorelin™ (IPSEN, Boulogne-Billancourt, France) 3.75 mg was given by deep intramuscular injection on the second day of a spontaneous menstrual period. A scan was conducted and a serum estradiol (14 pg/ml) was obtained to confirm down-regulation. Hormone replacement therapy (HRT) was carried out using oral estradiol valerate (2 mg) three times a day for 9 days, after which a scan was performed to assess endometrial thickness, which was found to be 9.5 mm. Progesterone suppositories of 400 mg twice daily were prescribed as a luteal support that was started on the first day of oocyte thawing. Luteal support and HRT was continued until the 12th week of gestation.

Mrs X became pregnant, which was confirmed by ascertaining a single intrauterine gestational sac containing a live fetus, and the pregnancy progressed uneventfully until 35 weeks, when preterm rupture of membranes induced the vaginal delivery of a live healthy baby boy weighing 1.9 kg and with no congenital abnormalities.

Discussion

Reproductive specialists who provide non-medical sex selection services argue that this technology was initiated and pursued by women and is an expression of reproductive rights as well as a sign of female empowerment. In addition providing for family balancing and fulfilment of culture and religious practice,11 non-medical sex selection allows couples to make informed family planning decisions, prevents unintended pregnancy and abortion, and minimizes intimate partner violence and/or child abuse.12 Opponents question whether women can truly express free choice under family and community pressures,13 and contend that sex selection is incompatible with unconditional parental acceptance of offspring and results in potential distortion of sex ratios11 and sex discrimination.14 In addition, PGD used only for sex selection involves preferential use of embryos and termination of those embryos not chosen, which raises ethical questions regarding abortion. In the Dubai Gynaecology and Fertility Centre, we consider social sex selection only for family balancing and we have not noticed a persistent preference for a particular sex, or social disagreement or religious conflict.

The present study indicates that freezing of either oocytes or spermatozoa can be efficient and can avoid repetitive surgical procedures for future attempts. PGS for sex selection of embryos can be an option for couples, if the law permits. Although Chen8 achieved the first frozen egg pregnancy in 1986, this application was not adopted widely for clinical assistance because survival of oocytes and pregnancy rates were comparatively low. This freezing method was based on a slow cooling and rapid thawing procedure using propanediol and sucrose as cryoprotectants, in line with the conditions originally developed for embryo freezing. Alteration of the ooplasm organelles, owing to the formation of intracellular ice crystals, is one of the main changes inflicted on the oocytes by the freezing process.15 Vitrification was first introduced in order to avoid crystallization damage. Another advantage of this freezing method is that it is very simple and based on direct contact between the vitrification solution containing cryoprotectants and liquid nitrogen.16 Vitrification is an ultra rapid freezing method using high concentration of cryoprotectants (ethylene, glycol and DMSO©) and rapid cooling at −1500°C/minute, which helps to solidify without crystal formation, which reduces the thermal stress of oocytes and decreases injury. Borini et al.17 reported pregnancies and births after oocyte cryopreservation in 68 patients. Since then, several other pregnancies have been reported worldwide.18 Cryopreservation of human oocytes is still considered an experimental technique despite improvements achieved over the past few years;19 however, the cryopreservation of spermatozoa is a widely used and efficient technique that can avoid repetition of surgical procedures for future ICSI attempts.20 The safety of cryopreservation has now been established in the human reproductive field.

Reports on the survival rates of cryopreserved human oocytes using the PROH–sucrose freezing method are highly variable, ranging from 25% to 95%,21 depending on the individual study. The establishment of successful pregnancies from frozen oocytes is highly dependent on the efficacy in preserving the meiotic spindle or microtubule structures within the oocytes after cryopreservation. There has been a consensus that the microtubules within the mammalian oocyte will depolymerize during freezing and therefore early insemination of thawed from frozen oocytes, before full restoration of meiotic spindle, may compromise fertilization outcome and subsequent embryonic development. An incubation of 4 hours before insemination by ICSI may seem to be an appropriate time to allow for the full recovery of normal spindles in cryopreserved human oocytes.22,23 Furthermore, hardening of zona pellucida due to vitrification can be overcome by ICSI24 and applying ICSI to the thawed human oocytes results in reasonable fertilization and blastocyst rates of 50% and 43% respectively.18 More studies should be carried out to explore the optimal conditions for enhancing the developmental potential of thawed from frozen human oocytes. Also, cryopreservation of human oocytes has a role to play in preserving the fertility of young women undergoing chemotherapy or radiotherapy and in cases of IVF when a sperm sample cannot be produced. In such cases, the oocytes can be frozen and then thawed at a later date when a sperm sample has been produced. Additionally, cryopreservation of human oocytes may play a role in circumventing ethical and legal issues associated with the established practice of cryopreservation of embryos.8 A number of babies born in the past few years can be attributed to the changes in the IVF laws, such as occurred in Italy and the UAE in 2010.

The first birth from human oocyte cryopreservation was reported in 1986.22,23 By 2004, approximately 100 children had been born from human oocyte cryopreservation. Pregnancy rate was low at 1–5% due to low oocyte survival at 25–40%, poor fertilization rate after IVF, the high incidence of polyploidy and poor developmental capacity. The incidence of chromosomal abnormalities was not different from that associated with fresh oocytes.24

The European Society of Human Reproduction and Embryology (ESHRE) PGD consortium was set up in 1997 and has been collecting data on PGD and PGS. Since 1997, the consortium has analysed 32 838 PGD and PGS cycles for various indications, of which 786 cycles were conducted for social reasons. Kuleshova et al.25 reported the birth of the first child conceived using oocytes stored by vitrification, and this was successfully followed by other studies and reports of a further 10 pregnancies.26,27 Studies on pregnancies from frozen oocytes inseminated with epididymal and testicular28 spermatozoa have been published, and the birth of a child conceived from frozen oocytes and spermatozoa has been reported.29

Larger studies are needed to quote a realistic figure of live birth rates from frozen oocytes. In addition, it is crucial to follow up children conceived from frozen oocytes.

In conclusion, this case is the first one of its kind in the Gulf region, even, to our knowledge, in the world where PGS was performed on an embryo obtained from a cryopreserved oocyte and a cryopreserved spermatozoon obtained from testicular aspiration on which PGS was conducted for sex selection. This was conducted for social reasons, balancing the family, which was allowed under UAE law at that time. This case also supports the concept that the developmental competence of the oocyte is not affected by vitrification of the oocyte and its biopsy.

Acknowledgement

I am extremely grateful to the staff of Dubai Gynaecology and Fertility Centre for their co-operation.

Notes

Disclosure

No author has any potential conflict of interest.

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