CL IN I CAL CORNER : COMMUN ICAT ION
Clinical outcome of emergency egg vitrification for women when
spermextraction from the testicular tissues of the male partner is
not successful
Wen-Yan Song1, Ying-Pu Sun1, Hai-Xia Jin1, Zhi-Min Xin1,
Ying-Chun Su1, and Ri-Cheng Chian2
1The Reproductive Medical Center, The First Affiliated Hospital
of Zhengzhou University, Zhengzhou, P. R. of China,2Department of
Obstetrics and Gynecology, McGill University, Montreal, Canada
The development of an effective oocyte cryopreservation sys-tem
will have a significant impact on the clinical practice
ofreproductive medicine. However, the important option ofemergency
oocyte cryopreservation has yet to be well docu-mented. In this
report, we review the cases of 15 womenwith male partners who were
diagnosed with nonobstructiveazoospermia and for whom testicular
sperm extraction on theday of oocyte retrieval failed. Emergency
oocyte vitrificationwas performed and after two months, the
vitrified oocyteswere warmed and the surviving oocytes inseminated
with fro-zen-thawed donor sperm by intracytoplasmic sperm
injection(ICSI). A total of 117 mature oocytes from the 15
womenwere vitrified and warmed. The post-warming survival ratewas
84.6% (99/117), and the fertilization rate following ICSIwas 83.8%
(83/99). We selected 30 embryos for transfer to 15patients, 8 of
whom became pregnant. The clinical pregnancyrate was 53.3% (8/15)
and the implantation rate was 30.0%(9/30). Nine healthy live births
resulted from 8 pregnancies.These results indicate that emergency
oocyte vitrification isan effective rescue technique that can be
applied clinicallywith acceptable pregnancy and live birth rates
when testicularsperm extraction from the male partner failed on the
day ofoocyte retrieval. These results also highlight another
importantoption for oocyte cryopreservation through the use of
vitrifica-tion technology.
Keywords egg, pregnancy, sperm, testicular tissue,
vitrification
Abbreviations ICSI: intracytoplasmic sperm injection;
NOA:nonobstructive azoospermia; FSH: follicle-stimulatinghormone;
LH: luteinizing hormone; E2: estradiol;
COCs:cumulus-oocyte-complexes; ET: embryo transfer; MII:metaphase
II; EG: ethylene glycol; DMSO: dimethyl sulfoxide.
Introduction
The development of an effective oocyte cryopreservationsystem
would have a significant impact on the clinical
practice of reproductive medicine. Such a program couldbe
offered to cancer patients before gonadotoxic treatmentand to
infertile couples with moral or religious objectionsto embryo
cryopreservation. In addition to fertility preser-vation for young
women requiring medical treatment thatwould result in
sterilization, cryobanking of oocytes wouldbenefit a large
population of single women who wish todelay motherhood for
personal, professional, or financialreasons.
Women suffering from premature ovarian failure whowish to
conceive must rely on donor oocytes. Oocytedonation can be
complicated and time consuming, requiringhormonal synchronization
of the donor and recipient men-strual cycles. A successful oocyte
cryopreservation protocolwould eliminate the need for
synchronization and enablethe establishment of egg banks,
facilitating the logistics ofcoordinating egg donors with
recipients. Furthermore, eggcryopreservation allows for temporary
quarantine of donoreggs to test the donors for transmissible
diseases.
Although oocyte cryopreservation provides manybenefits, the
importance of emergency oocyte cryopreserva-tion has not been
emphasized. Many unexpected situationscan occur during infertility
treatment, including a malepartner who is unable or refuses to
produce semen. Surgicaltesticular retrieval of sperm for
intracytoplasmic sperm in-jection (ICSI) is widely used for
treating men diagnosedwith nonobstructive azoospermia (NOA)
[Devroey et al.1995]. However, spermatogenesis in NOA is
usuallylimited, with only small foci spread over limited areas, so
re-trieving sperm can be a challenge. Diagnostic biopsy has
sig-nificant predictive value, but the chance that no sperm willbe
found is 25%-30% [Glina et al. 2005]. If sperm are not ob-tained
then cryopreservation of oocytes or use of a spermdonor is the only
possible alternative. However in somecountries, like China, the use
of donor sperm is impossibleimmediately requiring other options
like, emergencyoocyte cryopreservation until sperm can be recovered
fromthe husband. To date, the information available for
Address correspondence to Dr. Sun, Y-P, The Reproductive Medical
Center, The First Affiliated Hospital of Zhengzhou University,
Zhengzhou,P. R. of China, E-mail: [email protected] or Dr. R-C
Chian, Royal Victoria Hospital, Womens Pavilion F3, 687 Pine Avenue
West, Montreal,Quebec, Canada H3A 1A1, E-mail:
[email protected]
Received 12 November 2010; accepted 22 December 2010.
Systems Biology in Reproductive Medicine, 2011, 57:
210213Copyright 2011 Informa Healthcare USA, Inc.ISSN 1939-6368
print/1939-6376 onlineDOI: 10.3109/19396368.2011.566666
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emergency oocyte cryopreservation is very limited [Emeryet al.
2004; Kyono et al. 2005]. The objective of this studyis to report
the pregnancy and obstetric outcomes in 15women following the use
of emergency oocyte vitrification.
Results
Before ovarian stimulation, all female patients underwent
aphysical examination, trans-vaginal basal antral folliclecount,
and a baseline hormonal profile on day 3
measuringfollicle-stimulating hormone (FSH), luteinizing
hormone(LH), and estradiol (E2). The mean age of the women was27.9
2.8 years. The mean duration of infertility was 4.1 2.3 years. The
mean levels on day 3 were 6.1 1.4; 6.2 3.4 IU/L for FSH, 5.6 2.6;
45.1 21.9 pg/L for LH, and43.8 25.3 pg/L for E2, respectively
(Table 1). As shown inTable 2, a total of 135 COCs
(cumulus-oocyte-complexes)were retrieved, and of these 117 oocytes
were mature at theMII stage. Following warming, the oocyte survival
rate was84.6% (99/117). A total of 83 oocytes were fertilized
afterICSI, and the fertilization rate was 83.8% (83/99). Of the
83fertilized oocytes, 75 cleaved resulting in a cleavage rate
of90.4% (75/83). A total of 30 selected embryos were transferredto
15 patients, an average of 2.0 0.8 embryos per patient. Tendays
after ET (embryo transfer), 9 individuals were confirmedpregnant by
beta-hCG followed by ultrasound 4 weeks afterET with a clinical
pregnancy rate of 53.3% (9/15) and animplantation rate of 30.0%
(9/30). After ET, the remaining45 embryos were re-vitrified for
storage, and until nowthose re-vitrified embryos were not yet
warmed.
Obstetric analysis indicated 8 pregnancies with 9 healthyinfants
born (7 singletons and 1 set of twins; Table 3). Of the9 infants, 5
were male and 4 were female; all had normal kar-yotypes. For the
singletons, the mean gestational age was 39weeks + 5 days and the
mean birth weight was 3,807 397.3g, while for the twins the
gestation age was 38 weeks + 6 daysand the mean birth weight was
2,625 530.3 g.
Discussion
These results indicate that acceptable clinical pregnancy
ratesand healthy live-births can be achieved from emergencyoocyte
vitrification followed by the use of donor sperm forinsemination
when it is not possible to extract sperm fromthe testicular tissues
of the male partner. This suggests thatoocyte vitrification
technology is an important option thatcan be applied to this
unexpected situation during treatment.
As mentioned previously, although diagnostic biopsy
hassignificant predictive value, the chance that sperm cannotbe
retrieved on the day of oocyte retrieval is 25% - 30%[Glina et al.
2005]. Some have suggested that the cryopreser-vation of sperm from
TESE before treatment shouldbe implemented to prevent this
situation [Wu et al. 2005].However, the loss of motility of
testicular sperm fromfrozen-thawing procedures may result in a
significantreduction in the fertilization rate. It has been
reported thatin testicular biopsy samples from the patients with
NOA,less than 3% of the sperm observed were motile [Lyrakouet al.
2007]. Therefore, finding sufficiently motile spermfor ICSI in
frozen-thawed testicular tissues in NOA patientsis more difficult,
resulting in no embryos being available fortransfer.
In addition, it has been reported that immature spermfrom
testicular tissues are much more sensitive to freezingand thawing,
possibly leading to a higher rate of aneuploidyamong embryos
conceived from these frozen-thawed sperm[Ravizzini et al. 2008].
Therefore, in our IVF center, we donot routinely freeze immotile
sperm from testicular biopsysamples. It remains to be confirmed
whether or not testiculartissues from diagnostic biopsy should be
frozen as a back upfor NOA patients in case sperm cannot be
retrieved on theday of oocyte retrieval [Kyono et al. 2005; Chen et
al. 2008].
Over the last five years, the new vitrification techniqueshave
significantly improved the survival of cryopreservedoocytes,
indicating that vitrification may be more effectivethan the
slow-freezing method of oocyte cryopreservation[Kuleshova and
Lopata 2002]. Vitrification of oocytes has re-sulted in relatively
high survival rates [Kuleshova et al. 1999;Kuleshova and Lopata
2002; Yoon et al. 2003; Lucena et al.2006; Chian et al. 2008; Chian
et al. 2009; Cao et al. 2009;
Table 1. Patient demographic characteristics.
No. ofpatient Age (yrs)
Duration ofInfertility(yrs)
Day 3FSHlevel(IU/L)
Day 3LH level(IU/L)
Day 3Estradiol
level (pg/L)
1 24 2 5.2 4.5 24.42 29 2 5.2 4.7 66.73 34 11 8.2 5.6 27.04 29 5
7.6 5.8 30.55 28 5 4.0 3.7 35.76 30 5 7.5 3.1 60.07 23 1 4.1 9.5
106.68 28 5 5.3 4.8 42.49 28 3 6.7 3.5 20.210 27 4 6.0 5.2 31.011
30 4 6.8 11.7 37.612 24 3 6.4 5.3 37.813 27 4 5.3 4.9 50.214 29 5
7.0 5.6 45.515 29 3 4.2 15.6 61.2Mean SD 28.2 2.9 4.3 2.6 6.1 1.4
5.6 2.6 43.8 25.3
FSH: follicle-stimulating hormone; LH: luteinizing hormone.
Table 2. Clinical outcomes following transfer of embryos
producedfrom vitrified oocytes.
Group characteristics Value
Patients who underwent thawing and embryo transfer 15Mature
(M-II) oocytes retrieved 117M-II oocytes vitrified and thawed
117Oocytes survived (%) 99 (84.6)Oocytes fertilized (%) 83
(83.8)Oocytes cleaved (%) 75 (90.4)Embryos transferred (meanSD) 30
(2.00.8)Clinical pregnancy rate per cycle started (%) 8
(53.3)Embryos implanted (%) 9 (30.0)Singleton pregnancies 7Twin
pregnancies 1Live-birth rate per cycle thawed (%) 8 (53.3)Newborns
9
Emergency egg vitrification 211
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Cobo et al., 2008; Cobo et al. 2010; Nagy et al. 2009; Rienziet
al. 2010]. The results from this study showed an 84.6%survival
rate, 30.0% implantation rate, and 53.3% clinicalpreg ancy rate
following oocyte vitrification and thawing(Table 2), which are
comparable to previously reported results.
In our experience, we found the cleavage rate after
fertili-zation from vitrified and thawed oocytes is lower than that
offresh oocytes (Table 2, 90.4% in this observation). Further-more,
the time of cleavage after fertilization from vitrifiedand thawed
oocytes is also slightly delayed compared tothat of fresh oocytes.
Therefore, the lower cleavage rateand time delay involved in
cleavage need to be studiedproperly using fresh oocytes as
controls. Interestingly,there were more embryos available after ET,
because weonly transferred 30 embryos out of 75 cleaved
embryos.Although the remaining embryos were not warmed for ET,it is
possible to obtain viable pregnancies and live birthsfrom those
re-vitrified embryos.
It has been reported that pregnancies and infants con-ceived
following oocyte vitrification are not associated withincreased
risk of adverse obstetric and perinatal outcomes[Chian et al. 2008;
Chian et al. 2009]. Although thenumber of pregnancies and
live-births was small, theresults from our observation also support
this finding(Table 3). In conclusion, these results indicate that
emer-gency oocyte vitrification is an effective rescue
techniquethat can be applied clinically with acceptable
pregnancyand live birth rates when testicular sperm extraction is
un-successful in the male partner on the day of oocyte
retrieval.
Materials and Methods
From June 2008 to May 2009, 108 patients with NOA weretreated by
TESE in the first affiliated hospital of ZhengzhouUniversity,
China. Of these, we failed to extract sperm fromthe testicular
tissues in 22 male patients (20.4%) on the dayof oocyte retrieval.
After informed consent, 15 patientsaccepted emergency oocyte
vitrification, due to failure ofsperm extraction from the
testicular tissues on the day ofoocyte retrieval. Institutional
review board approval wasobtained for this retrospective study.
Ovarian stimulation was performed using a GnRHagonist
(Triptorelin, Ferring GmbH, Germany) combinedwith recombinant FSH
(Serono Laboratories, Switzerland)and HMG (Ferring GmbH, Germany),
and then 10,000 IU
of human chorionic gonadotropin (hCG, Serono Labora-tories,
Switzerland) was administered when at least twofollicles reached 18
mm in diameter. Oocyte retrieval wasperformed 36 h later.
All male partners were diagnosized as NOA by hormonalassessment
(FSH, LH, and testosterone), biochemical markerassessment (fructose
and -glucosidase), karyotype analysis,and Yq microdeletion
assessment. Testicular biopsy wasperformed and histopathological
analysis demonstratedthat mature sperm were found in the testicular
tissuesample in advance, but the testicular tissue was not
frozen.
Within 2 h of oocyte retrieval, COCs were exposed to 60IU/mL
hyaluronidase for partial removal of cumulus cells.Only metaphase
II (MII) oocytes were selected for vitrifica-tion. A modified
vitrification method was adopted for cryo-preservation of the
mature oocytes [Chian et al. 2005;Antinori et al. 2007]. Briefly,
the oocytes were placed intoequilibration medium, containing 7.5%
(v/v) ethylene glycol(EG) and 7.5% (v/v) dimethyl sulfoxide (DMSO),
at roomtemperature for 5 min, and then the oocytes were
transferredto vitrification medium, containing 15% (v/v) EG, 15%
(v/v)DMSO, and 0.50 mol/L sucrose at room temperature for 45 to60
s. Two or three oocytes were loaded on a McGill Cryoleaf(Medicult
Company, Denmark) and plunged immediatelyin liquid nitrogen for
vitrification and then for storage.
Two months later, the couples decided to use frozen donorsperm
of the same blood type as the male partner and theoocytes were
thawed by inserting the McGill Cryoleaf directlyinto warming medium
(MediCult Company, Denmark) con-taining 1.00 mol/L sucrose for 1
min at 37C. The warmedoocytes were transferred into diluent
medium-I containing0.50 mol/L sucrose and then into diluent
medium-II contain-ing 0.25 mol/L sucrose for 3 min each. The
oocytes werewashed twice in washing medium for 3 min each time
afterwhich they were incubated in culture medium under 6%CO2 at 37C
for approximately 2 h. Oocyte survival afterwarming was evaluated
microscopically based on the mor-phology of the oocyte membrane
integrity.
Prior to ICSI, frozen donor sperm (Sperm Bank, HunanProvince,
China) were thawed rapidly at 37C for 10 minin water.
Cryoprotectant was removed by centrifugation at500 x g for 15 min
and resuspension in 0.5 mL of freshmedium. A motile spermatozoon
was injected into each sur-viving oocyte, and fertilization was
checked approximately16-18 h after ICSI. Embryo transfer (ET) was
performed
Table 3. Obstetric and perinatal outcomes of each patient
following oocyte vitrification and thawing.
Patient number Singleton (S) or twin (T) Gestational age
(week+days) Birth weight (g) Karyotype Birth defect (yes or no)
2 S 40 4,000 XY no3 S 39+2 4,400 XY no4 S 40 3,250 XY no6 S 39+3
3,500 XX no7 S 40 3,850 XY no10 S 40 4,100 XX no12 S 39+6 3,550 XY
noMeanSD 39+5 3,807397.314 T 35 3,000; 2,250 XX; XX noMeanSD 38 +6
2,625530.3
212 Song et al.
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under trans-abdominal ultrasound guidance on either day 2or 3,
depending on the number and quality of the embryos.Before ET,
assisted hatching was performed with a lasersystem OCTX (Eyeware TM
Company, Germany). Clinicalpregnancy was defined as the presence of
a fetal sac withheartbeats revealed by ultrasonography.
The endometrial lining was prepared for approximately 2w using
E2 transdermal patches until the endometrial thick-ness was 8 mm in
patients naturally menstrual cycle beforeoocyte warming.
Progesterone (100 mg/d) was started whenthe oocytes were warmed,
continuing until the time of -hCG assay. Both E2 and progesterone
were continued forluteal support until the 12th w of pregnancy if
positive clini-cal pregnancy was confirmed.
Acknowledgments
This work was supported by a grant from the National
HighTechnology Research and Development Program of China(863
Program) (No. J2006AA02Z4A4).
Declaration of Interest: The authors, W.Y. Song, Y.P. Sun,H.X.
Jin, Z.M. Xin, and Y.C. Su, have no declarations of in-terest. R.C.
Chian has interest in McGill Cryoleaf, Origio(MediCult) Company,
Denmark.
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