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Successful birth of the first frozen oocyte baby in India.
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PMID:  19562074     Owner:  NLM     Status:  PubMed-not-MEDLINE    
We report the first pregnancy and birth in India after the transfer of embryos generated from frozen- thawed oocytes. A 29-year-old woman with previous bad obstetric history and an abnormal karyotype, necessitating donor oocyte programme. Embryos were generated by microinjection of frozen-thawed sperms into thawed human oocytes (intracytoplasmic sperm injection). This resulted in an healthy male baby with a birth weight of 2.54 kg which was born by cesarean section at 35-36 weeks of gestation with normal follow-up. Thus oocyte cryopreservation can be performed with reproducible success leading to a viable offspring.
Priya Selvaraj; Kamala Selvaraj; Kalaichelvi Srinivasan
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Journal of human reproductive sciences     Volume:  2     ISSN:  0974-1208     ISO Abbreviation:  J Hum Reprod Sci     Publication Date:  2009 Jan 
Date Detail:
Created Date:  2009-06-29     Completed Date:  2011-07-14     Revised Date:  2013-05-23    
Medline Journal Info:
Nlm Unique ID:  101473512     Medline TA:  J Hum Reprod Sci     Country:  India    
Other Details:
Languages:  eng     Pagination:  41-4     Citation Subset:  -    
Fertility Research Centre, G G Hospital, Chennai - 600 034, India.
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Journal Information
Journal ID (nlm-ta): J Hum Reprod Sci
Journal ID (publisher-id): JHRS
ISSN: 0974-1208
ISSN: 1998-4766
Publisher: Medknow Publications, India
Article Information
? Journal of Human Reproductive Sciences
Received Day: 21 Month: 11 Year: 2008
Revision Received Day: 10 Month: 2 Year: 2009
Accepted Day: 31 Month: 3 Year: 2009
Print publication date: Season: Jan?Jun Year: 2009
Volume: 2 Issue: 1
First Page: 41 Last Page: 44
ID: 2700691
PubMed Id: 19562074
Publisher Id: JHRS-02-41
DOI: 10.4103/0974-1208.51354

Successful birth of the first frozen oocyte baby in India
Priya SelvarajAF0001
Kamala SelvarajAF0001
Kalaichelvi SrinivasanAF0001
Fertility Research Centre, G G Hospital, Chennai - 600 034, India
Correspondence: Address for correspondence: Dr. Priya Selvaraj, Fertility Research Centre, G G Hospital, Chennai - 600 034, India. E-mail:


The field of Assisted Reproductive Technology (ART) is advancing day by day with newer laboratory techniques being discovered. The area of cryopreservation is one of them. Till date, it has been possible to freeze sperms and embryos for many years, but successful pregnancies from oocytes have been elusive with only few hundred live births worldwide.[1] The world's first birth through oocyte freezing and thawing was pioneered by Prof. Christopher Chen in Australia, in 1986.[2]

Oocyte cryopreservation has gained more attention in ART, especially because synchronization between recipient and donor need not be compulsory in the donor oocyte programme, other indications being pre-chemotherapy preservation of fertility, premature ovarian failure, and postponement of childbearing function. The success rates in oocyte cryopreservation have been cited as low and are debatable, with literature review of 21 studies in peer-reviewed journals revealing a mean survival rate of 47%, fertilization rate of 52.5% and mean pregnancy rate/thawed oocyte being 1.52?1.8%.[3] We report the first successful birth from frozen oocytes in India.


The patient was a 29 year old, with a previous bad obstetric history and an abnormal karyotype (46xx, t[3:9] [p21.3;p24], t [7:10] [p22;q22.1]), which necessitated the use of donor oocytes for the present conception. The reason for the use of frozen oocytes sourced from egg-sharing and egg-donation patients was the non-availability of compatible fresh donors and insistence of the couple to continue the treatment cycle. The couple was then counseled about the use of frozen?thawed oocytes and the expected low success rate for this cycle after obtaining informed consent.

The sourced oocytes were cryopreserved using the slow freezing method. Freezing was initiated 1?2 h post-retrieval, after denuding the cumulus with hyaluronidase and confirming metaphase-II maturity status. Cook's oocyte freezing media (Cook Medical, Queensland, Australia) was used after warming the required quantity to room temperature, the active cryoprotectants being propanediol in increasing concentrations from 0.75 to 1.5 mol/l (solutions 1 and 2 ? for 7min and 30 s, respectively) and then transferred to solution 3 containing 1.5 mol/l propanediol and 0.2 mol/l sucrose [Figure 1] for 5 min. The oocytes were loaded into pre-labeled straws (Cryo-Bio Systems, Rocket Medical PLC, Washington, England), plugged and placed into the cryochamber (Planer Kryo 360, Planer Products Ltd, Burnsville, USA). The initial chamber temperature at +20?C was slowly cooled to ?8?C at the rate of ?2?C/min. Seeding was manually performed at ?8?C. After a hold time of 10min at ?8?C, the straws were cooled slowly to ?30?C at a rate of ?0.3?C/min and then rapidly to ?150?C at a rate of ?50?C/min. After 10 min of stabilization, the straws were transferred to a liquid nitrogen tank and stored until thawing.

The oocytes were subjected to cryopreservation for 6 months. Because a suitable fresh donor was unavailable for this particular patient, we were able to use the cryopreserved oocytes for this couple. The endometrium was prepared with incremental doses of estrogen (Estradiol Valerate 2 mg; German Remedies, Mumbai, India) commencing from Day 2 or Day 3 of the cycle to a maximum of 8mg/day with initiation of micronised progesterone 400mg thrice a day, from the day before thawing. The optimum endometrium for transfer was considered to be 10mm, which was observed in mid-cycle. We decided to thaw at least 10 oocytes in order to enhance the chances of achieving enough good-grade embryos for transfer, success having evaded us earlier. For the thawing process, the straws containing the oocytes were removed from the liquid nitrogen, air dried for 30s, and then immersed in a water bath at 30?C for 40s. The oocytes were first passed through solutions 1?2 containing cryobuffer supplemented with decreasing concentration of propanediol (1.0?0.5mol/l) and increasing concentration of sucrose (0.2?0.3mol/l) for 5 min at room temperature, respectively. The oocytes were then transferred to solution 3 (0.3 mol/l sucrose) for 10 min at room temperature followed by 37?C on a warmer plate.

A survival check carried out under the inverted microscope revealed eight oocytes that survived the thawing process [Figure 2]. Oocytes with intact zona, clear perivitelline space, and visible polar body were considered as normal and were further cultured in the Cleavage media (Quinn's, SAGE In vitro fertilization, Trumbull CT, USA) at 37?C and 5% CO2 for 2 h before injection. Subsequently, the oocytes were injected with frozen?thawed and prepared sample of husband's sperms. Intracytoplasmic sperm injection [Figures 3 and 4][4?9] was the preferred method for fertilization, owing to an expected zona hardening following oocyte cryopreservation. The injected oocytes were further cultured in cleavage media in a Trigas incubator until Pronucleus check 16?18h later. Five embryos (two and three cells, grade II?III) [Figures 5 and 6] were observed and transferred on Day 2 using a Labotect catheter (Labotect GmbH, Gottinggen, Germany). The assessment of embryo grading was performed as described by Veeck L.[10] Why five and not the recommended three embryos? Well, considering our previous experience of inadequate embryos for transfer following thawing and negative result for pregnancy, we decided to transfer at least five of this crop, which, as we mentioned, did not have the conventional cleavage or grade as expected (i.e., four cells, grade I?II). Once the serum ?-HCG was positive, an ultrasound on the 38th day revealed a single intrauterine gestational sac. Since then she progressed uneventfully, with early fetal screening tests being normal until the 5th month, when she was diagnosed with mild pregnancy-induced hypertension and was treated with anti-hypertensives. During her level-2 scan at 22 weeks, where the fetal screening was normal, a shortened cervix of 1.5 cm was imaged, suggestive of cervical incompetence. Since then, she was admitted with us for careful surveillance of both the mother and the baby. She did moderate physical activities and her blood pressure was well controlled with anti-hypertensives. An elective cesarean was performed at 35?36 weeks on 27/8/2008 owing to severe oligohydraminos. She delivered a healthy male baby weighing 2.54 kg [Figure 7]. Both the mother and the baby were discharged uneventfully. A general follow-up at 3 months showed normal development for age [Figure 8].


We started oocyte freezing in the month of November 2005 and since then have frozen more than 100 oocytes [Table 1]. The only media available were the Irvine Scientific Vitrification media and Cook media and there was a learning curve to be able to successfully freeze and retrieve oocytes. The oocytes frozen with Cook's media were only metaphase II. However, in the initial phase of vitrification with Irvine scientific media, close to 10 oocytes of different patient were frozen at the germinal vesicle and metaphase I, with three maturing to metaphase II in in vitro maturation, but no fertilization. Subsequent reports of series of pregnancies and live births have confirmed that mature oocyte cryopreservation is now a viable option in appropriate circumstances.[1, 7, 9] Other recent reports show that several babies have been born using oocyte cryopreservation and similar freezing and thawing procedures (slow freezing and rapid thawing).[11, 12] There is still ongoing research with regard to the optimum media and preference of freezing techniques like slow freeze or vitrification, the latter showing promising results. In our experience, the slow freeze method seemed to offer better retrieval rates (74%) and fertilization rates (62%), although vitrification, according to the literature review, seems to offer comparable or even better clinical outcomes.


Oocyte freezing can be welcomed as an emerging and useful technique, especially in donor programmes and in young women with cancer for fertility preservation options. Worldwide, there have been only few hundred babies born through this technique owing to the sensitive morphology and cryokinetics of the oocyte.[13, 14] While we still conduct comparative research between the two freezing methods, namely slow freezing and vitrification, this little bundle of joy signals yet another landmark achievement for our country while serving as a ray of hope to many young women in need of oocyte freezing technology.


Source of Support: Nil

Conflict of Interest: None declared.

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2. Parmegiani L,Cognigni GE,Bernardi S,Ciampaglia W,Infante F,Pocognoli P,et al. Freezing within 2 h from oocyte retrieval increases the efficiency of human oocyte cryopreservation when using a slow freezing/rapid thawing protocol with high sucrose concentrationHum reprodYear: 2008231771718477574
3. Borini A,Sciajno R,Bianchi V,Sereni E,Flamigni C,Coticchio G. Clinical Outcome of oocyte cryopreservation after slow cooling with a protocol utilizing a high sucrose concentrationHum ReprodYear: 200621512716239316
4. Fabbri R,Porcu E,Marsella T,Rocchetta G,Venturoli S,Falmigni C. Human Oocyte cryopreservation: New perspectives regarding oocyte survivalHum ReprodYear: 200116411611228204
5. Antinori S,Dani G,Selman HA. Pregnancy after sperm injection into cryopreserved human oocytesHum ReprodYear: 1998131578
6. Polak de Fried E,Notrica J,Rubinstein M,Marazzi A,G?mez Gonzalez M. Pregnancy after human donor oocyte cryopreservation and thawing in association with intracytoplasmic sperm injection in a patient with ovarian failureFertil SterilYear: 19986955579531897
7. Porcu E,Fabbri R,Seracchioli R,Ciotti PM,Magrini O,Flamigni C. Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytesFertil SterilYear: 19976872469341619
8. Yang DS,Blohm PL,Winslow L,Cramer L. A twin pregnancy after microinjection of human cryopreserved oocyte with a specially developed oocyte cryopreservation regimeFertil SterilYear: 1998S239357
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10. Veeck LL. Atlas of the Human Oocyte and Early ConceptusYear: 1986Vol 2BaltimoreWilliams and Wilkins121
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12. Levi Setti PE,Albani E,Novara PV,Cesana A,Morreale G. Cryopreservation of supernumerary oocytes in IVF / ICSI cyclesHum ReprodYear: 200621370516239315
13. Kuleshova L,Gianaroli L,Magli C,Ferraretti A,Trounson . A Birth following vitrification of a small number of human oocytes: Case reportHum ReprodYear: 1999143077910601099
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[Figure ID: F0001]
Figure 1 

Frozen oocytes

[Figure ID: F0002]
Figure 2 

Thawed oocytes

[Figure ID: F0003]
Figure 3 

Holding egg

[Figure ID: F0004]
Figure 4 

Intracytoplasmic sperm injection

[Figure ID: F0005]
Figure 5 


[Figure ID: F0006]
Figure 6 


[Figure ID: F0007]
Figure 7 

At birth

[Figure ID: F0008]
Figure 8 

At 3 months

[TableWrap ID: T0001] Table 1 

Statistics from November 2005 to November 2008

Total statistics Total (%) Cook (%) Vitrifi cation (%) Quinn's (%)
No. of oocytes frozen 119 83 29 7
No. of oocytes retrieved 84 (70.58) 62 (74.69) 18 (62.06) 4 (57.14)
No. of oocytes that survived the thawing process 46 (54.76) 29 (46.77) 13 (72.22) 4 (100)
No. of oocytes for which ICSI was performed 46 29 9 4
Total no. of oocytes fertilized 18 (39) 18 (62) - -
No. of cases for embryo transfer 5 5 - -
No. of pregnancies 2 (40) 2 (40) - -
Pre-clinical 1 1 - -
Delivered 1 1 - -

Article Categories:
  • Case Report

Keywords: Donor oocyte programme, embryo transfer, intracytoplasmic sperm injection, oocyte cryopreservation, oocyte survival.

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