If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
Are children born from vitrified-warmed oocytes physically or mentally different from naturally conceived children?
Design
Intracytoplasmic sperm injection (ICSI) of vitrified-warmed oocytes was performed for 282 patients (307 cycles) from August 2000 to March 2020. Long-term follow-up of children born from vitrified-warmed oocytes was performed via a questionnaire that was sent to the parents at regular intervals from 3 to 72 months after the child's birth. Questionnaires were sent 11 times from birth to the age of 6 years. The development of motor function and mental status was evaluated as the primary outcome, based on the reported data. Subsequently, patients were divided into four groups by age at oocyte retrieval (20–29, 30–34, 35–39, and 40 years or older). Clinical outcomes were calculated as a secondary outcome.
Results
For the 282 patients, the birth of 116 babies was reported (110 singletons and three sets of twins), and seven cases are, at the time of writing, unconfirmed. The results of the survey found physical parameters in singletons to be equivalent to the nationally reported average data issued by the Ministry of Health, Labor and Welfare of Japan.
Conclusion
This is the first follow-up report of children born from vitrified-warmed oocytes followed by ICSI. The data suggested that the responses from the study participants on the mental and physical development of children were comparable to the data reported by the government, although more responses from patients should be collected to allow further study.
). In Japan, the first live birth after the transfer of two good-quality 8-cell embryos following intracytoplasmic sperm injection (ICSI) of frozen-thawed oocytes was reported almost 20 years ago (
). At that time, oocyte cryopreservation was still in the experimental phase. Due to improvements in oocyte cryopreservation techniques, vitrified-warmed oocytes can survive in a better condition than before, which has led to embryo development and clinical results equivalent to those for fresh oocytes. In 2013, this team reported the first live birth using vitrified oocytes from a patient with malignant disease (
A live birth from vitrified-warmed oocytes in a Philadelphia chromosome-positive acute lymphoid leukemia patient 5 years following allogenic bone marrow transplantation and after a magnitude 9.0 earthquake in Japan.
Practice Committees of the American Society for Reproductive Medicine and the Society for Assisted Reproductive Technology Mature oocyte cryopreservation: a guideline.
). Nowadays, oocyte cryopreservation is applied for fertility preservation, whether it is elective or medically indicated. It is also a useful method when the spermatozoa of the patient's partner cannot be obtained on the day of oocyte retrieval.
Since the number of babies born by assisted reproductive technology (ART) has been increasing, discussion on the safety of ART has also increased. The cryoprotectant is contained in the cryopreservation solvent used in the vitrification process. It is generally said that organic solvents affect an oocyte with a single cell more than they do a blastocyst consisting of multiple cells. As the survival rate of oocytes is lower than that of blastocysts, these artificial products used for ART could have adverse effects on children conceived by ART.
A previous report stated that frozen embryo transfer (FET) using fresh oocytes led to higher birthweights and increased the rate of premature birth. It also showed higher rates of low birthweight (LBW) and small for gestational age (SGA) babies after FET using fresh oocytes compared with the results of fresh embryo transfer (
). On the other hand, there were no significant differences in gestational age and birthweight between fresh and cryopreserved oocytes when using vitrified-warmed oocytes (
). However, a long-term follow-up of children born from vitrified-warmed oocytes followed by ICSI is yet to be published. In this study, a 6-year follow-up of children born from vitrified oocytes is reported.
Materials and methods
Ethics approval
This study was approved by the Ladies Clinic Kyono Ethics Committee (Submission no. #4501-200501, approval date 1 May 2020). All procedures performed in this study involving human participants were carried out in accordance with the ethical standards of the Institutional Research Committee and the 1964 Declaration of Helsinki and its later amendments or similar ethical standards. All participants provided written informed consent.
Participants
This study included 307 cycles in 282 patients, from August 2000 to March 2020, in which ICSI was performed using vitrified-warmed oocytes. Oocyte cryopreservation was suggested when patients wished for fertility preservation before chemotherapy (medical indication), or wanted to postpone childbearing (non-medical indication). It was also a choice when male partners with severe oligozoospermia or cryptozoospermia needed to undergo testicular sperm extraction. Embryo development and clinical outcome were compared among four groups divided by maternal age (years): 20–29, 30–34, 35–39 and 40 or older.
Ovarian stimulation
Ovarian stimulation was performed mainly with a combination of gonadotrophin-releasing hormone (GnRH) agonist, FSH and human menopausal gonadotrophin (HMG) or GnRH antagonist, FSH and HMG. An injection of 5000 IU of human chorionic gonadotrophin (HCG) was administered when the diameter of the dominant follicle reached 18 mm. Transvaginal oocyte retrieval was performed 36 h after the HCG injection.
Vitrification and warming method
Oocyte vitrification protocol was performed as previously described (
Japan Society of Obstetrics and Gynecology Successful pregnancy and delivery after transfer of a single blastocyst derived from a vitrified mature human oocyte.
). Matured oocytes were cryopreserved by the vitrification method using a vitrification solution containing cryoprotectant. ICSI was performed on warmed oocytes. When an oocyte reached the embryo or blastocyst stage, cryopreservation or transfer was performed.
Endometrial preparation
Basal hormone concentrations (LH, FSH, oestradiol and prolactin) were measured to check the patient's cycle using an electro-chemiluminescence immunoassay. To administer oestrogen, Estrana tapes (Hisamitsu Pharmaceutical Co., Japan) and Progynova tablets (Bayer plc, United Kingdom) were applied from day 3. When the concentration of oestrogen and progesterone in the blood attained appropriate values and endometrial thickness reached more than 6 mm, oral progesterone (Lutoral; Fuji Pharma Co., Japan) was administered. ICSI was performed using warmed oocytes on the first day oral Lutoral was taken. On the 3rd to 5th day after progesterone administration, the embryo was transferred at the cleavage or blastocyst stage. Two-step embryo transfer was performed in eight cycles.
Follow-up of children
The current authors previously reported on the physical and mental development of children after IVF and FET (
). Using this report as a reference, an additional questionnaire relating to children up to 6 years old was developed and administered.
A questionnaire was sent to the patients to see how their offspring had developed at certain times (0, 3, 6, 9, 12, 18, 24, 36, 48, 60 and 72 months old). The questionnaire was prepared based on the Maternal and Child Health Handbook in Japan (
). The answers included the children's weight, height, chest measurement and head circumference, which were compared with those published as national averages by the Ministry of Health, Labor and Welfare (3rd, 10th, 25th, 50th, 75th, 90th and 97th percentiles), as well as a range of developmental parameters. Low/high birthweight, preterm birth (PTB) and small/large for gestational age (SGA/LGA) were also analysed based on the definition given by the Japan Society of Obstetrics and Gynecology (Japan Society of Obstetrics and Gynecology, 2017).
High birthweight is defined as 4000 g or more. LBW is less than 2500 g. PTB was recorded when the baby was born alive before 37 weeks’ gestation. SGA and LGA were defined when the birthweight was less or more than 2 standard deviations from the mean reported by Ministry of Health, Labor and Welfare, Japan.
Statistical analysis
Statistical analysis was conducted using the R open-source software environment for statistical computing and graphics (Ihaka et al., 1996). Continuous data, in the case of anti-Müllerian hormone (AMH) concentration, were assessed using the Kruskal–Wallis test because, from the result of a Bartlett test (P = 0.032), the data did not have homogeneity of variance. Count data, presented as numbers and percentages, were assessed using chi-squared analysis and Fisher's exact test. Two-sided P < 0.05 was considered statistically significant.
Results
Clinical outcomes with oocyte vitrification
A total of 116 babies (110 singletons and three sets of twins) were delivered, and seven cases are, at the time of writing, unconfirmed. All of the twins were diagnosed as monochorionic diamniotic, and one pair of twins had congenital malformations including duodenal obstruction, imperforate anus and left ureterocele. All the congenital malformations were diagnosed in one twin. One singleton was also diagnosed with duodenal obstruction.
Patients’ backgrounds and clinical outcomes are summarized in Supplementary Table 1. The number (percentage) of patients by age group (20–29, 30–34, 35–39 and 40 years old or older) was 54 (19.1%), 56 (19.9%), 85 (30.1%) and 87 cycles (30.9%), respectively. Oocyte survival rate and fertilization rate did not show a significant difference among the four groups. Blastocyst formation rate and pregnancy rate in women aged 20–29 years old were statistically higher than those in women aged 40 years old or older (68.2% versus 58.0%, P = 0.023; and 51.1% versus 12.5%, P < 0.001).
AMH concentration, pregnancy rate and live birth rate declined gradually in parallel with age. A total of 74 female patients each had one child from vitrified oocytes, and 21 patients had two children, including a set of twins. The patients with two children were younger than those with one child at the time of egg retrieval (29.5 ± 4.04 versus 33.0 ± 4.53 years old, P = 0.014).
The clinical outcome of embryo transfer type was also sorted by the developmental stage of the embryo (day 2–4 and day 5–6) and by the embryo transfer method (fresh embryo transfer or FET) (Supplementary Table 2). Day 5–6 FET (clinical pregnancy n = 99) showed a higher birth rate than the other methods (P < 0.001). However, a very limited number of cases was evaluated for some conditions (clinical pregnancy n = 7 for day 2–4 FET and day 5–6 fresh embryo transfer), although there were substantially more cases in the day 2–4 fresh embryo transfer group (clinical pregnancy n = 48). Hence, day 5–6 FET can be recommended when more oocytes are retrieved. On the other hand, the survival rate of vitrified oocytes was around 90% and their blastocyst formation rate was around 60–70%. If few oocytes are retrieved, day 2–4 embryo transfer should be considered because of the increased risk that any blastocyst was not successfully cryopreserved after cultivation of inseminated oocytes until blastocyst stage development was attempted.
Growth curve for 6-year follow-up
There were 110, 49, 43, 38, 38, 33, 30, 27, 21, 16, and 17 respondents to the mailed questionnaire when the children were 0, 3, 6, 9, 12, 18, 24, 36, 48, 60 and 72 months old, respectively. Respondents with twin live births were excluded from this study because the number of questionnaire respondents was too small to evaluate the growth development. The weight, height, chest measurement and head circumference of the children are shown in Table 1. All the data were comparable to the reference data reported by the Ministry of Health, Labor and Welfare, Japan.
Table 1Measurements of newborns in the study group compared with the reference group
Weight and height development up to 6 years of age were particularly studied (Figure 1, Figure 2). Each parameter was calculated, displayed on a line graph and compared with the reported data (3rd, 10th, 25th, 50th, 75th, 90th and 97th percentiles). The rates of LBW, PTB and birthweight more than 4000 g were 11.8% (13/110), 3.6% (4/110) and 0.9% (1/110), respectively. The rate of LGA was 3.6% (4/110), and SGA was not observed (Table 2).
Figure 1Growth curve of height for children born from vitrified oocytes. Each line indicates the reported data (3rd, 10th, 25th, 50th, 75th, 90th and 97th percentiles). Almost all the data are located within the growth curve, although the data for 0, 3, 18 and 24 months indicate higher levels at the 97th percentiles. Data were recorded at 0 (n = 116), 3 (n = 49), 6 (n = 43), 9 (n = 38), 12 (n = 38), 18 (n = 33), 24 (n = 30), 36 (n = 27), 48 (n = 21), 60 (n = 16) and 72 (n = 17) months. Data are shown as box and whisker plots representing the median, interquartile range and range.
Figure 2Growth curve of weight for children born from vitrified oocytes. Each line indicates the reported data (3rd, 10th, 25th, 50th, 75th, 90th and 97th percentiles). Almost all the data are located within the standard growth curve, although the data for 0, 3, 6, 18 and 24 months indicate lower levels at the 3rd and 97th percentiles.
). Questions regarding the physical and mental development of children are listed in Table 3. Although some questionnaires were returned unanswered, almost all responses showed no problems, characterized by more than 90% of ‘Yes’ responses to the children's developmental questions.
Table 3Achievement rate of mental and physiological development at each stage
Developmental stage
Developmental milestone
Respondents (n)
Yes
No
Singleton (%)
3 months
Hold his/her head up
49
44
5
90
Laugh when amused
49
49
0
100
Turn his/her head in the direction of the voice
49
48
1
98
Eat soup/vegetables
49
8
41
16
6 months
Roll over
43
40
3
93
Sit up
42
37
5
88
Reach for a toy
43
43
0
100
Make a speaking-like sound
42
42
0
100
React to TV or radio
43
43
0
100
Eat weaning food
41
35
6
85
9 months
Crawl
38
32
6
84
Stand up holding onto something
38
34
4
89
Grab a small object
37
37
0
100
Play alone in a good mood
37
36
1
97
Weaning is going well
38
35
3
92
Turn around when whispered to
38
37
1
97
12 months
Walk with support
38
36
2
95
Imitate simple actions such as waving
38
35
3
92
Move his/her body to the music
38
35
3
92
Understand simple words such as ‘come here’
38
38
0
100
Delighted when played with
38
38
0
100
Eat three meals a day
38
36
2
95
18 months
Walk alone
33
33
0
100
Say a meaningful word, such as ‘mom’
33
32
1
97
Hold a glass of water and drink it
33
27
6
82
Use a baby bottle
33
5
28
15
Unfocused eyes
33
2
31
6
Turn around when called from behind
33
33
0
100
24 months
Run
30
30
0
100
Use a spoon
30
30
0
100
Scribble with a crayon
30
30
0
100
Imitate TV or adult gestures
30
30
0
100
Form a two-word sentence
30
26
4
87
Eat meat/fibre-rich vegetables
29
27
2
93
36 months
Walk up the stairs without using his/her hands
27
27
0
100
Draw circles with crayons
27
27
0
100
Prefer to put on and take off clothes alone
27
24
3
89
Say his/her own name
27
27
0
100
Brush teeth and wash hands on his/her own
27
27
0
100
Regularly chewing and eating well
27
27
0
100
48 months
Jump down a couple of steps of stairs
21
19
2
90
Jump with one foot
21
20
1
95
Talk to the family about his/her experience
21
21
0
100
Use scissors well
21
21
0
100
Playing pretend with friends
21
21
0
100
Brush teeth, rinse mouth, and wash hands
21
21
0
100
Go pee alone
21
21
0
100
60 months
Do a forward roll
16
16
0
100
Draw a picture from memory
16
15
1
94
Tell the difference between colours: red, blue, green, and yellow
This study's aim was the long-term follow-up of children born after oocyte vitrification. Some previous studies and reviews have reported on the follow-up of children born by ART (
). Ludwig and colleagues reported a physical health follow-up of children born after ICSI and of children conceived without ART at the age of 4–6 years (
Neuromotor development and mental health at 5.5 years of age of singletons born at term after intracytoplasmatic sperm injection ICSI: results of a prospective controlled single-blinded study in Germany.
Physical health at 5.5 years of age of term-born singletons after intracytoplasmic sperm injection: results of a prospective, controlled, single-blinded study.
b). They interviewed parents and collected data from the child's examination booklet. They found no significant physical difference between children conceived naturally or by ICSI. Wagenaar and co-workers examined levels of physical activity, social withdrawal and depression in children born by ART (
). In this study, the answers were obtained from the parents and teachers. In a later study, these authors mentioned that there were no significant differences from the answers obtained from children themselves (
). Because the evaluation criteria and evaluator varied between the studies mentioned above, a general theory cannot, however, be inferred.
Evaluation of the long-term physical and mental development of children born from vitrified oocytes followed by ICSI has not been yet reported, and this report is the first follow-up report for children born from vitrified oocytes followed by ICSI treatment.
Before the study, it was expected that there would be a greater influence of organic solvents on vitrified oocytes than on frozen embryos using fresh oocytes, because vitrified-warmed oocytes could be cryopreserved again following ICSI; however, this was not found (data not shown).
Two limitations should, however, be considered. First, the response rate to the questionnaire was low. As the children grew older, the survey response rates of their parents fell. More frequent communication with parents and easier contact methods such as e-mail are needed to receive a greater proportion of answers. Another limiting factor is the fact that the survey was not conducted in a medical institution but was based on responses to questionnaires from parents who had given birth. Hence, there is a certain bias in the answers, as Wagenaar and colleagues have reported (
). This handbook may be a good examplar for constructing uniform questions for the follow-up of motor and socioemotional development.
The current study also looked at the difference in congenital anomalies between ART and natural conception. Previous studies have reported that neonatal outcomes differ between fresh embryo transfer and FET. The reason for this difference may be the interaction of the cryoprotectants used with the main enzyme involved in epigenetic programming (
The incidence of congenital anomalies in this study was 1.7% (2/116). This result is comparable to that previous reports on congenital anomalies from vitrified oocytes (3%, 5/165;
). Additionally, a comparison between ART and natural conception should be considered. Pinborg and Hoorsan reported that children born following ART had a 15–40% higher risk of congenital anomalies compared with naturally conceived children (
; Pinborg et al., 2013), but the recent incidence of congenital anomalies after ART treatment has probably decreased due to improvements in ART technology as the survival rate of vitrified oocytes has dramatically increased. Continued monitoring of birth outcomes will help to assess any increased risks associated with oocyte cryopreservation.
In the present study, vitrified oocytes were cryopreserved for 1.58 ± 1.49 years. The longest preservation term among the oocytes was 13 years. Previous studies have reported the survival rate of frozen-thawed oocytes to be approximately 60–75% (
), which means that the vitrification method is suitable for oocyte cryopreservation. In this study the survival rate seen using the vitrification method was also higher than the reported data from the slow-freezing method, although the success rate slightly declined with age (Supplementary Table 1). Cobo and colleagues studied the survival rate of vitrified-warmed oocytes in terms of patients’ age and reported a noticeable decrease in survival and live birth rate from the youngest category (≤29 years old) to the oldest category (40–44 years old) (Cobo et al., 2016).
The oocyte retrieval age in female patients who successfully had two children was statistically significantly less than that in women who had one child. Moreover, Cobo and colleaues (Cobo et al., 2016) suggested that the age-associated live birth rates per number of oocytes retrieved increased from women aged 20–29 years (7.7%, 36/469) to those 40 years or older (1.5%, 5/344). Doyle and co-workers reported that about 13 oocytes would be needed for a woman of 20–29 years old to have a baby, and about 29 eggs for a woman age 40 years or older (
Successful elective and medically indicated oocyte vitrification and warming for autologous in vitro fertilization, with predicted birth probabilities for fertility preservation according to number of cryopreserved oocytes and age at retrieval.
). Hence, if the patient wishes for at least one child, oocyte retrieval at a younger age should be recommended, as previously suggested by Doyle and colleagues.
Conclusion
To the best of the authors’ knowledge, this study is the first report to evaluate the safety of oocyte vitrification in a 6-year follow-up. The data in this study were compared to percentile data on weight or height reported by the Ministry of Health, Labor and Welfare of Japan. Although statistically significant differences were not calculated, the results suggested that the physical and mental development of babies born from vitrified oocytes is comparable to that of naturally conceived babies.
Acknowledgments
The authors would like to thank all team members at Kyono ART Clinic Sendai and Takanawa for their daily clinical work, especially N. Asano for checking this manuscript.
A live birth from vitrified-warmed oocytes in a Philadelphia chromosome-positive acute lymphoid leukemia patient 5 years following allogenic bone marrow transplantation and after a magnitude 9.0 earthquake in Japan.
Successful elective and medically indicated oocyte vitrification and warming for autologous in vitro fertilization, with predicted birth probabilities for fertility preservation according to number of cryopreserved oocytes and age at retrieval.
Neuromotor development and mental health at 5.5 years of age of singletons born at term after intracytoplasmatic sperm injection ICSI: results of a prospective controlled single-blinded study in Germany.
Physical health at 5.5 years of age of term-born singletons after intracytoplasmic sperm injection: results of a prospective, controlled, single-blinded study.
Yuya Takeshige graduated from the Graduate School of Pharmaceutical Sciences, University of Tokyo, Japan, in 2016, and works as a statistician at Kyono ART clinic, Takanawa, Japan. His research interests include the evaluation of clinical outcomes based on statistical analysis.
Key message
This study is the first report to evaluate, in a 6-year follow-up study, the safety of oocyte vitrification in children born using this technique. The results suggest that the physical and mental development of babies born from vitrified oocytes is comparable to that of naturally conceived babies.
Article info
Publication history
Published online: November 13, 2020
Accepted:
November 6,
2020
Received in revised form:
November 1,
2020
Received:
May 30,
2020
Declaration: The authors report no financial or commercial conflicts of interest.
30619-2&id=gr1.jpg){kind=link}
30619-2&id=gr2.jpg){kind=link}